CN108210901A - A kind of new drug that is hypoglycemic and improving sugar tolerance - Google Patents

Abstract

The present invention relates to hypoglycemic and raising sugar tolerance a methods newly, and including a effective amount of plasminogen of diabetic subjects is administered, while the present invention relates to for hypoglycemic and raising sugar tolerance drug.

Description

A kind of new drug that is hypoglycemic and improving sugar tolerance

Technical field

It is a effective amount of including administration diabetic subjects the present invention relates to new hypoglycemic and raising sugar tolerance a method Plasminogen, while the present invention relates to for hypoglycemic and raising sugar tolerance drug.

Background technology

Diabetes (diabetes mellitus, DM) are a kind of common abnormal glucose metabolisms with genetic predisposition And endocrine disease, it is as caused by absoluteness or relativity hypoinsulinism.2015, the whole world had 4.15 hundred million diabetics, it is contemplated that the year two thousand forty, diabetes number of patients is up to 6.42 hundred million^[1].Diabetes are to seriously endanger One of major disease of human health.

The metabolic disorder for being mainly shown as the substances such as abnormal carbohydrate metabolism and fat, protein of diabetes, and long-term Hyperglycemia state can lead to serious diabetic complication, including microvascular complication, diabetic nephropathy, diabetic cardiomyopathy, Diabetes nerve system lesion, diabetic dermopathy and diabetes concurrent infection etc..Wherein diabetic nephropathy and diabetes Nervous system lesion is huge on patients ' life quality influence, and harm is serious.

Clinically common diabetes can be divided into four types：Type 1 diabetes (type 1diabetes, T1DM), 2 Patients with type Ⅰ DM (type 2diabetes, T2DM), gestational diabetes, specific type diabetes.Wherein, with T1DM and T2DM Patient is the most common, and gestational diabetes and specific type diabetic are relatively fewer.

T1DM is considered and inherent cause, environmental factor (such as virus infection, causing diabetes chemical substance, dietary factor) It is related to role of autoimmune factors.Research shows that the relevant gene locis of T1DM at least 17, are located in different chromosome. In terms of environmental factor, the influential environmental factor of T1DM morbidities is included virus infection, cause diabetes chemical substance and diet because Element, wherein viral factor is mostly important.Have now been found that parotitis, rubella virus, cytomegalovirus etc. fall ill with T1DM It is related.Its mechanism is that virus can directly destroy beta Cell of islet, and excite autoimmunity after viral damage beta Cell of islet React further impaired isle β cells.Diabetogenic chemical substance such as alloxan, streptozotocin (STZ), pentamidine The effects that in beta Cell of islet, lead to the destruction of beta Cell of islet.Role of autoimmune factors includes humoral immunity and cellular immunity.Body Liquid be immunized show as blood samples of patients cycle in there are a variety of anti-beta Cell of islet autoantibody.Cellular immunity is mainly shown as Insulitis infiltrating cells and beta Cell of islet surface are observed that the unconventionality expression of HLA-DA antigens and 2 receptors of IL- and pancreas The overexpression of island cell surface HLA-1 class antigens, and the CD4+/CD8+ ratios and IL-1 of peripheral blood, TNF-α, INF- γ levels increase.Pathological change caused by these factors concentrates on islet p-cell destruction so that internal insulin level is absolute It reduces, causes T1DM, therefore T1DM is considered to be a kind of autoimmune disease.

T2DM is a kind of multiple-factor inheritance disease, it is considered that it is polyphyly, wherein environmental factor and Inherent cause collective effect leads to insulin resistance, shows as the insulin of identical level concentration because of the resistant function of body And normal level can not be played the role of.And body is in order to reach normal glycemic levels, it will excessive secretion insulin is to alleviate " inefficient " state that insulin uses, it is if things go on like this higher and higher to the requirement of beta Cell of islet, eventually lead to beta Cell of islet " excessive exercise " and self-inflicted injury, progress to insulin and absolutely lack.

The pathogenesis of DM

DM pathogenesis is complicated, mainly with Family inherited inclination, ethnic heterogeneity, insulin receptor defect, insulin by The damage of body substrate, the up-regulation of Protein-tyrosine-phosphatase related gene, excessive immune inflammatory reaction, Fatty toxicity, oxidative stress and The correlations such as injury of mitochondria^[2-3]。

1. free fatty

Free fatty acid levels raising is both one of pathogenic factor of insulin resistance and insulin-resistant states One of important feature.Under the action of inherent cause or environmental factor, the raising of free fatty acid levels in blood, when more than The storage capacity of adipose tissue may result in the generation of insulin resistance.The long-term high fat diet of studies have shown that will lead to pancreas Dysfunction occurs for island β cells, this is because high fat diet can also make fat content in abdominal cavity in addition to causing peripheral insulin resistance Raising and insulin inhibit lipolysis ability to reduce, and so as to which free fatty acid content be promoted to increase, then inhibit insulin The tyrosine site phosphorylation of receptor and its substrate IRS-l, 1RS-2, inhibits the activity of P13K, leads to insulin signal transduction Access is obstructed to form insulin resistance.

2. inflammatory reaction

1) inflammation and insulin resistance

T2DM is a kind of slight nonspecific inflammatory disease.Studies have shown that inflammation in recent years leads to insulin resistance Main mechanism is that inflammatory factor and the signal transduction of insulin receptor substrate intersect, and one side nonspecific inflammation generates Inflammatory factor occur inhibition to IRS/PI3K signal paths, and a series of of another aspect inflammatory factor activation swash Enzyme can induce the phosphorylation in the silk of IRS, threonine site to be hindered so as to be generated to normal tyrosine phosphorylation, final to cause Insulin signal transduction ability, which declines, induces insulin resistance^[2-3]。

In target cell, insulin is combined energy activated receptor with its receptor, and signal transduction pathway intracellular later generates A series of endocellular transduction molecules are completed signal in the transmission step by step of intracellular with enzymatic cascade reaction and are amplified, and signal finally passes A series of biological effect is generated to target organ.Signal transduction pathway mainly has two, and one is IRS-1-PI3K- PKB/AKT approach, the other is mitogen-activated protein kinase (Shc/Raf/MAPK) approach.In first access, first It is that insulin occurs under exogenous insulin and/or glucose stimulation to be combined with its receptor, so as to have activated the endogenous of receptor Property tyrosine kinase.The tyrosine kinase of activation is while the phosphorylation of itself is realized induction of insulin receptor substrate The tyrosine site phosphorylation of IRS.The IRS of activation is migrated to cell membrane, by phosphotyrosine binding domain (PTB) by phosphorus Sour tyrosine is anchored in IRS tyrosine kinase, and the IRS of tyrosine phosphorylation recruits the tune to PI3K by its SH2 structural domain Save subunit P85.P85 is combined with 3 phosphoric acid molecules of phosphoinositide, and one phosphoric acid of phosphatidylinositols (PIP) is converted into phosphatidyl Inositol diphosphate (PIP2) and Phosphatidyl inositol triphosphate (PIP3), they are insulin and the second letter of other growth factors Make, be the protein kinase -1 (PDK1) of downstream signaling molecule phosphoinositide dependence and (or) a certain Asia of protein kinase c (PKC) The anchored site of type.PDK1 can be with activated protein kinase B (PKB, also referred to as Akt) and a certain atypia PKC hypotypes.Activation PKB allows Glycogen synthesis kinases -3 (GSK3) to inactivate by serine/threonine phosphorylation on one side, on the other hand activates mammal Rapamycin target spot (mTOR) protein kinase, so as to induce 70ku-S6 kinases (p70S6K) phosphorylation activation downstream. mTOR Protein kinase can be used as " ATP receptors ", and p70s6K is without passing through Ca for activation₂ ⁺/ cAMP, the synthesis of realization control albumen, The transcription of enchancer promotes beta Cell of islet hypertrophy and other biological effect.PKB can directly induce certain transcription factors Serine/threonine phosphorylation promotes the generation of cell mitogen^[4-5].In Article 2 access, the activation of Ras can pass through two Access is realized.1) the insulin receptor activation IRS-2 albumen of activation, and signal can be passed to adaptor protein by IRS-2 albumen Growth factor receptor binding protein precursor 2 (Grb2), then with signal protein GDP/GTP exchange factors (mSOS) interaction so that energy The Ras-GT that the Ras-GDP of activation inactivation is transformed into is so as to fulfill activation Ras.Insulin receptor directly effect makes signal protein The tyrosine phosphorylation of Shc, then Shc combined with Grb2 through mSOS pathway activations Ras.The Ras-GTP of activation recruits Raf Histidine kinase makes mapk kinase, MAPK phosphorylations successively.The MAPK of activation can activate other protein kinases to participate in induced gene The processes such as transcription, regulating cell apoptosis^[6]。

Having proven to the serine residue of IRS-1 at present can be swashed by inflammation tyrosine phosphorylation, such as c-Jun amino terminals Enzyme (JNK), I kappa b kinase ss (I κ K β) and protein kinase C (PKC)-θ.Radio immunoassay shows that serine307 site is The major site of JNK phosphorylations IRS-1, its mutation can make JNK induce IRS-1 phosphorylations and TNF to caused by insulin The inhibiting effect of IRS-1 tyrosine phosphorylations disappears.JNK by the serine307 of phosphorylation IRS-1, reduce insulin by Body substrate tyrosine phosphorylation, inhibits the transduction of insulin signaling^[7].Hiorsumi etc. has found diet type obesity mouse and ob/ob JNK activity significantly increases in the liver of mouse, muscle, adipose tissue.Gene knockout (JNK1-/-) diet can be made to lure type fat Mouse Insulin resistance weakens, and alleviates obesity, hyperglycemia and the hyperinsulinemia of ob/ob mouse.Fat mouse liver organization The phosphorylation level in IRS-1 serine307s site is higher than thin mouse, but in the fat mouse of gene knockout (JNK1-/-) not See raising, it is seen that the serine307 site of IRS-1 is the target spot that JNK is acted in vivo^[,8].Researches show that TNF α stimulation lures In the model for leading hepatic resistance, jnk inhibitor can block the phosphorylation of serine307 completely.I κ K β can pass through At least two approach influence insulin signal transduction, can be the Ser307 site phosphorylations of direct induction IRS-1, can also By the phosphorylation of I κ B, and then NF- κ B are activated, insulin resistance is caused by the expression for stimulating the inflammation factor indirectly.

Inflammatory reaction is the defense that human immune system fights these damages after infection, tissue damage and stress reaction Reaction, while be also the cause of disease or pathogenesis of diabetes, angiocardiopathy and tumour.

Early in 1993, Hotmamisligil etc.^[9]Proved by zoopery, the obese rat of insulin resistance its Pro-inflammatory cytokine, TNF-α are horizontal high in adipose tissue.From this, numerous researchers start to inquire into inflammation and fat, pancreas islet Relationship between element resistance, and probe into its Molecular pathogenesis.Hotmamisligil in 2006^[10]Metabolic is proposed for the first time Inflammation (metabolic inflammation) this new medical definition emphasizes that this minuent, chronic systemic inflammatorome are main It is as caused by extra nutriment and metabolite.There may be the molecules similar with exemplary inflammatory for metabolic inflammation With the conduction path of signal, unlike previously our exemplary inflammatories that are recognized, metabolic inflammation and be not present it is red, The symptom of swollen, heat, pain and dysfunction.Under normal circumstances, organismic internal environment is in steady-state level, inflammation and metabolism respectively and Keep a kind of dynamic balance state between each other.When metabolic disorder occurs for body, break this equilibrium state of body, Cause the unbalance of immune system, excite inflammatory signals conduction path, body is promoted to discharge a series of inflammatory factors, certain inflammation The factor even amplifies auto-inflammatory reaction, forms inflammation water fall effect, further makes body that insulin resistance occur, so as to lead Cause the generation of metabolic syndrome.

Research has shown that TNF-α and metabolic syndrome have substantial connection.TNF is called cachectin, mainly the macrophage by activating Cell, natural kill (NK) cell and T lymphocytes generate, and the TNF that secretion is generated by macrophage is called TNF-α, by The lymphotoxin that T lymphocytes generate secretion is called TNF-beta.The biological activity of TNF-α account for TNF gross activities 70%~ 95%, therefore the TNF frequently involved at present refers to TNF-α mostly.It is inquired by years of researches, at present clear and definite TNF-α It is related with a variety of diseases such as insulin resistance, autoimmune disease, tumour, chronic hepatitis B.In the hair of insulin resistance TNF-α plays the role of vital during hair tonic exhibition.Swaroop etc.^[11]By the serum for detecting 50 T2DM patients TNF-α is horizontal, show that T2DM patient's TNF-α level increases, and with BMI, Fasting insulin level and steady-state model pancreas islet Element resistance index (HOMA-IR) is significantly correlated, and TNF-α is prompted to play an important role in T2DM pathogenesis.Also research refers to Go out, TNF-α can be suppressed the phosphorylation of insulin receptor, can be with when the phosphorylation of insulin receptor is suppressed The gene expression of glucose transporter is reduced, so as to which the activity for making lipoprotein lipase reduces, point of fat may finally be caused Solution^[12]。

2) apoptosis of inflammation and beta Cell of islet

Chronic low grade inflammation reaction is closely related with islet beta cell function obstacle.Pancreas islet β caused by β cell quantities are reduced Cell dysfunction is another major reason of T2DM morbidities, and the apoptosis of β cells is that the reduction of β cell quantities is most important Reason.Due to heredity or diet, T2DM patient's Yi Fasheng insulin resistances, patient blood glucose's raising, hyperglycemia state is again IL-6 can be promoted to generate, IL-6 can not only reduce GLUT4 expression, reduce transhipment of the adipocyte to glucose, hinder glycogen Synthesis reduces the sensibility of insulin；It can also promote islet cells secrete IL-6 simultaneously, cause vicious circle.Hyperglycemia Induction IL-1 β are largely generated, and by the way that the accesses such as NF- κ B, MAPK, Fas, NO is activated to lead to Intra-islet Apoptosis, inflammation leads to Road intersects promotion, exacerbates the apoptosis of islet cells, eventually leads to the failure of islet function^[13].In addition, IL-1 β may be used also Interaction between mediated leucocytes, and influence each other restriction with other cell factors such as IFN-γ, TNF-α etc., it is thin in β It plays an important role during cellular damage.The dyslipidemia of T2DM can cause hormonal substance such as leptin and the horizontal increasings of IL- 6 Add.The release that leptin can increase IL-1 β carrys out inducing beta cell apoptosis, can also negative regulation and control insulin secretion^[14].ROS is removed and is led It causes outside insulin resistance, also has an effect for the damage of beta Cell of islet, under oxidative stress status, the insulin gene transcription factor Expression and insulin binding site significantly reduce, so as to influence the generation of insulin and secretion.Other Adipocyte Factors Such as TNF-α and the thin function that can also reduce β cells^[15].The synergy of these cell factors, makes islet beta cell function Into more obvious damage.In addition, the segmental inflammation factor may also act to the key position of insulin receptor substrate2, make its ammonia Acid/threonine phosphorylation causes the degradation of insulin receptor substrate2 to be accelerated, promotes the apoptosis of beta Cell of islet.

3. oxidative stress

Research shows that an important factor for oxidative stress is the generation and development for causing T2DM.Oxidative stress refers to active oxygen The generation of (reactive oxygen species, ROS) and active nitrogen (reactive nitrogen species, RNS) with It is unbalance between the removing of Antioxidative Defense System in body, ROS and RNS is caused to generate excessive, cause body tissue cell and egg The damage of the large biological molecules such as white and nucleic acid^[13].Hyperglycemia is the main reason for generating oxidative stress, to pass through Mitochondrial electron Transfer chain^[14], the approach such as glucose auto oxidation and polyalcohol access^[15]Increase the ROS and RNS contents in body, center line grain Body electron transport chain is the main path for generating ROS.Mitochondrial electron transport chain relates generally to multienzyme complex I~IV, cell color Plain c and ubiquinone can continue to generate a small amount of super oxygen product in multienzyme complex I and III, including superoxide anion, peroxidating Hydrogen and hydroxyl radical free radical, and superoxide dismutase, catalase and glutathione peroxidase can urge super oxygen product Change is converted to oxygen and water.But under fat or hyperglycemic conditions, super oxygen product can be significantly increased, when the generation of super oxygen product Rate is more than that it can generate oxidative stress when removing rate.

Multinomial research^[16-18]Show ROS can coup injury β cells, particularly destroy cell mitochondrial structure, promote β Apoptosis；ROS can also inhibit β cell functions indirectly by influencing Insulin signaling pathway, such as activate nuclear factor κ B (nuclear transcription factor κ B, NF- κ B) signal path causes β cellular inflammations to be reacted；Inhibit pancreas ten Two duodenum 12 inhibit line with the caryoplasm transposition of the source capsule factor 1 (pancreatic and duodenal homeobox 1, PDX-1) Mitochondrial energetics are metabolized, and reduce insulin synthesis and secretion etc..Oxidative stress causes β cellular damage NF- κ B by NF- κ B accesses For the dimer of two subunit compositions of p50 and RelA, in resting cell, combined with inhibition protein I κ B, with inactive three Dimer form is present in endochylema, be primarily involved in cell to stress, the stimulations such as cell factor, free radical and bacterial virus answer It answers and instantaneous controlling gene expression etc.^[19].Research shows that the ROS of hyperglycemia inductive formation can be turned by upsetting Intracellular signals Lead activation NF- κ B, inducing beta cell damage^[20].Mariappan etc.^[21]Inhibit fertilizer with pyrrolidines aminodithioformic acid (PDTC) NF- κ B are expressed in fat db/db Mice Bodies, it is found that oxidative stress is substantially reduced to the degree of injury of mouse β cell mitochondrial； Hofmann etc.^[22]Diabetic is treated using anti-oxidation medicine alpha-lipoic acid, finds patient's body NF- kB activities It significantly reduces, conditions of patients also has improvement；Eldor etc.^[23]Specifically inhibit the table of mouse NF- κ B using transgenic technology It reaches, hence it is evident that reduce the diabetes morbidity of mouse after STZ inductions.

NF- κ B participate in cell Proliferation, Apoptosis and inflammation and are immunized as a kind of multidirectional nuclear factor after activation Etc. several genes adjusting^[24].In diabetes body, NF- κ B pass through the regulating cell factor and the gene table of chemotactic factor (CF) It reaches, such as IL-1 (interleukin-1) and (the monocyte/macrophage chemoattractant protein- of MCP- 1 1) factor etc. causes pancreas islet leukocytosis, leads to β cellular damages^[25].In addition many genes product of NF- κ B regulation and control is as swollen Tumor necrosis factor α (tumor necrosis factor α, TNF-α) etc. can further activate NF- κ B again, aggravate β cells damage Wound^[26]。

Mahadev etc.^[27]Studies have shown that ROS has insulin signal transduction regulating and controlling effect, and this effect has multi-panel Property.Under insulin stimulating, body can quickly generate micro ROS, the latter by Nox (NADPH oxidase) dependent mechanism As second messenger, the activity for mainly inhibiting PTP1B by oxidation promotes insulin cascade reaction^[28], and use DPI (diphenyleneiodonium) after inhibiting Nox, the insulin receptor (insulin receptor, InsR) of insulin stimulating Decline 48% with insulin receptor substrate (insulin receptor substrate, IRS) phosphorylation^[29].Loh etc.^[30]'s Researches show that physiological ROS can promote sensibility of the body to insulin.Although under physiological status, produced by insulin stimulating Raw micro ROS can promote the effect of insulin, but long term hyperglycemia can be such that body is generated largely by mitochondria pathway ROS^[31], cause insulin resistance.

InsR and IRS is signal element important in insulin signaling pathways：The former is insulin signal transduction Initiator elements, and IRS is the connection bridge of the former and passage downstream element.Numerous studies show that oxidative stress can be by multiple Approach interferes the phosphorylation reaction of InsR and IRS, hinders insulin signal transduction.IKK is swashing for the inhibition subunit I κ B of NF- κ B Agent living, IKK can promote InsR and IRS to send out as the serine/threonine phosphorylating kinase of InsR and IRS under ROS stimulations Raw serine phosphorylation, normal tyrosine phosphorylation is suppressed, hinders insulin signal transduction^[32]。 Brownlee^[33]Research It has been shown that, IKK can Direct Phosphorylation IRS 307 serine residue, the normal tyrosine phosphorylations of IRS is caused to weaken, are hindered The combination of InsR and IRS, so as to cause insulin resistance.

In addition to IKK, multiple members in MAPK families also have an impact InsR and IRS.JNK, extracellular regulated protein swash Enzyme (extracellularregulated protein kinases, ERK) and p38 mitogen-activated protein kinase (p38MAPK) be MAPK family members, have serine/threonine protein kitase activity, by oxidative stress, cell because It can be activated under the effects that son and G- protein-coupled receptor agonists.Multiple studies have shown that the activation of JNK, ERK and p38MAPK The serine/threonine phosphorylation degree of InsR and IRS can be aggravated, makes protein binding capacity and IRS between InsR and IRS The ability for the signaling molecule that SH-2 structural domains are contained in activation downstream reduces^[34-36]。

Oxidative stress caused by the high sugared state of diabetes be one of key reason that a variety of chronic complicating diseases are formed and An important factor for radiation-indued DNA damage^[37].When diabetes occur, extracellular fluid is visible to continue high sugar.In this state, mitochondria The electronics showed increased that electron transport chain generates, generates excessive ROS, causes intracellular environment and lipid, protein and DNA etc. Large biological molecule damages.The active oxygen that body generates in aerobic metabolism approach, can be by DNA chain as a kind of mutation derivant On guanine be oxidized to 8- hydroxy guanines (8-hydroxy-2'-deoxyguanosine, 8-OHdG).In DNA replication dna In the process, 8-OHdG leads to G easily with adenine mispairing:C to T:A transversional mutations form DNA damage.In addition, ROS can also draw The DNA damage of other forms is played, is pressed down including DNA chain fracture, the distortion of DNA site mutations, DNA double chain and proto-oncogene and tumour Gene mutation processed etc..Meanwhile DNA damage may also aggravate ROS and oxidative stress process, as DNA damage can be by H2AX- also 1 (Nox1)/Rac1 accesses of prototype codehydrogenase Ⅱ oxidizing ferment induction ROS is generated.ROS further promotes a large amount of Ca₂ ⁺Into mitochondria, Cause meronecrosis and apoptosis or coup injury mitochondria, cause mitochondria dysfunction, and then impaired isle β cells, add The pathologic process of acute diabetes^[38]。

ROS also has an effect in addition to insulin resistance is caused, for the damage of beta Cell of islet, under oxidative stress status, pancreas The expression of island plain gene transcription factor and insulin binding site significantly reduce, so as to influence the generation of insulin and secretion. Other Adipocyte Factors such as TNF-α can also reduce the function of β cells^[15].The synergy of these cell factors, to pancreas islet β Cell function causes more obviously to damage.In addition, the segmental inflammation factor may also act to the crucial portion of insulin receptor substrate2 Position, makes its serine/threonine phosphorylation, the degradation of insulin receptor substrate2 is caused to be accelerated, promotes withering for beta Cell of islet It dies.

Viewed from above, effect of the oxidative stress during diabetes occurrence and development is sufficiently complex.ROS is except direct Impaired isle β is extracellular, is alternatively arranged as signaling molecule and activates some stress sensitive accesses, adjusts the expression of correlation factor, cause β Apoptosis or necrosis inhibit insulin secretion, induce insulin resistance, final to cause or aggravate diabetes.

The treatment of DM

Diabetes generally use drug therapy, traditional drug therapy include trypsin class medicine and oral class antidiabetic drug Object.

Insulin early stage mainly extracts from the pancreas of the animals such as pigs and cattle, can occur after human body application apparent Allergic reaction.90 years 20th century are more and more ripe so that insulin analog gradually applies, and this insulin can be bright The aobvious pharmacokinetics for changing traditional para-insulin, there is the advantages such as low, the rapid-action, persistent of hypoglycemic incidence.Mesh Before, as what insulin preparation was explored deepens continuously, some Macrulins have stepped into experimental stage, but because technology On difficulty, effective oral preparation there is no to apply in clinic so far.

Conventional oral class hypoglycemic medicine is more, common are following several：(1) biguanides such as melbine.Melbine There is good cardiovascular protective effect, hypoglycemic effect is also good, has multiple countries at present and is treated as first-line drug T2DM.(2) sulfonylureas：Sulfonylureas belongs to a kind of Insulin secretagogues, stimulates beta Cell of islet, it is made to secret out of insulin, is reached To the effect for improving blood glucose level.At present, China allows the para-insulin of listing mainly to have Glimepiride, glibenclamide, lattice Row pyrazine, gliclazide, gliquidone etc., if but showing that taking such drug for a long time is likely to result in from some researchs Hypoglycemic effect fails, and the complication such as hypoglycemia and weight increase easily occur.(3) thiazolidinedione (thiazolidinedionecompounds, TZD) class：Rosiglitazone and Pioglitazone approval are used in T2DM by FDA in 1999 In, the former may aggravate heart disease risk, be defined as the use of second line treatment drug later thus, while disable in heart failure Illness.In June, 2013, FDA audited Rosiglitazone again, it is indicated that the drug can continue on for clinic in addition loosen or The application of this medicine and its compound preparation is released completely.(4) alpha-glucosidase inhibitor：This para-insulin can inhibit on small intestinal mucosa The glycosidase of chrotoplast, and then alleviate the absorption of carbohydrate, level of postprandial blood sugar is caused to reduce.Such drug is normal There are voglibose, acarbose and Miglitol etc..

The drug for the treatment of diabetes is mainly traditional antidiabetic medicine at this stage, including sulfonylurea, meglitinide, Biguanides, thiazolidinediones (thiazolidinediones, TZD), alpha-glucosidase restrainer and insulin etc., this A little drugs are there are different degrees of adverse reaction, such as cause hypoglycemia, gastrointestinal discomfort, obesity.With to diabetes base Plinth theoretical research is goed deep into, and side effect in order to avoid traditional hypoglycemic medicine brings beta Cell of islet protective effect, Ren Menye Energetically finding new treating diabetes target spot.It has now been found that and mainly includes pancreas with the relevant target spot of pathogenesis of diabetes mellitus Glucagon-like peptide -1 (glucagon-like peptide-1, GLP-1), (dipeptide of dipeptidyl peptidase -4 Peptidase-4, DPP-4), sodium-glucose co-transporter -2 (sodium- glucose cotransporter-2, SGLT-2), glycogen synthase kinase-3 (glycogen synthase kinase-3, GSK-3), Protein-tyrosine-phosphatase (protein tyrosine phosphates, PTP), glucokinase (glucokinase, GK) etc..Wherein based on adjustment The drug of glucagon such as glucagon-like-peptide-1 (glucagon like peptide-1, GLP-1) analog, GLP- 1 receptor stimulating agent and dipeptidyl peptidase-4 (dipeptidyl peptidase-4, DPP-4) inhibitor are considered effectively tieing up Glycaemic homeostasis is held, improves β cell functions, delay diabetes de-velopment or even the reverting diabetes course of disease.

Diabetes can still be cured completely without a kind of effective drug or means at present, current drug therapy collection In by the way that blood glucose is controlled to be reduced in certain range and delays the generation of complication.With deeper to pathogenesis of diabetes mellitus Enter, comprehensively understand, the medicine of diabetes is studied, is also transitioned into from the drug research to traditional mechanism to having The drug research of novel targets and novel mechanism, some of which have listed, as GLP-1 receptor stimulating agents, DPP-4 inhibitor and SGLT-2 inhibitor etc., also some drugs are in the clinical or preclinical study stage, as GPR119 receptor stimulating agents, 11 β- HSD1 inhibitor, PTP1B inhibitor and GK agonists etc., efficacy and saferry need further clinical verification.It is although near The appearance of novel targets antidiabetic medicine provides more choices, but for DM treatments due to the pathogenesis of diabetes over year Complexity, the hormone being related to, enzyme and receptor are numerous, novel drugs research field also exist such as single target drug sphere of action it is relatively narrow, Blood sugar reducing function is weaker, acts on the problems such as whole body system causes adverse reaction up for further studying.Therefore, Ren Menxu It finds and may act on all various, the significantly more efficient medicines of pathogenesis of diabetes mellitus.

Present invention discover that plasminogen can mitigate the damage of diabetic experimental mice pancreatic tissue, control inflammation, reduce Islet beta-cell apoptosis, the secreting function for restoring beta Cell of islet, reduces blood glucose at repairing pancreas tissue, is that one kind is expected to become complete Face is directed to all various completely new drugs of pathogenesis of diabetes mellitus.

Invention summary

The present invention includes following items：

1. a kind of method for reducing diabetic subjects blood glucose, including a effective amount of plasminogen of subject is administered.

2. 1 method, wherein the blood glucose is chosen from the followings one or more：Serum level of glucose, serum fructose Amine level, serum glycated hemoglobin are horizontal.

3. 2 method, wherein the blood glucose is serum level of glucose.

4. the method for any one of 1-3, wherein the diabetes are T1DM or T2DM.

5. a kind of method for improving diabetic subjects sugar tolerance, including a effective amount of plasminogen of subject is administered.

6. 5 method, wherein the diabetes are T2DM.

7. a kind of method that diabetic subjects postprandial blood sugar is promoted to decline, including a effective amount of fibrinolytic of subject is administered Proenzyme.

8. 7 method, wherein the plasminogen is given for 30 minutes to 1.5 hours before the meal in subject.

9. 8 method, wherein the plasminogen is given for 30 minutes to 1 hour before the meal in subject.

10. a kind of promote the method that utilizes of the diabetic subjects to glucose, including a effective amount of fibre of subject is administered Lyase is former.

11. a kind of method for promoting diabetic subjects insulin expression and/or secretion, effective including administration subject The plasminogen of amount.

12. 11 method, wherein the plasminogen promotes the insulin secretion after diabetic subjects feed.

13. 11 method, wherein the plasminogen promotes the insulin point under diabetic subjects fasting state It secretes.

14. a kind of method for reducing the expression of diabetic subjects glucagon and/or secretion, including subject is administered A effective amount of plasminogen.

15. 14 method, wherein the plasminogen reduces the pancreas after diabetic subjects feed or under fasting state Glucagons is secreted.

16. the method for any one of 11-15, wherein the plasminogen is by promoting the secretion of insulin and reducing pancreas The secretion of glucagons makes subject's glucose levels return to normal or close to normal.

17. the method for any one of 1-16, wherein the plasminogen can be with one or more other medicines or treatment side Method is combined.

18. 17 method, wherein the plasminogen can be with one or more drug combinations chosen from the followings：Anti- sugar Urinate medicine, medicament against cardiovascular disease, antithrombotic reagent, drug for hypertension, anti-lipid drug, anticoagulant, anti-sense Contaminate drug.

19. the method for any one of 1-18, wherein the plasminogen has at least with sequence 2,6,8,10 or 12 75%th, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity, and still there is fibrinolysin Former activity.

20. the method for any one of 1-19, the plasminogen is on the basis of sequence 2,6,8,10 or 12, add, It deletes and/or replaces 1-100,1-90,1-80,1-70,1-60,1-50,1-45,1-40,1-35,1-30,1-25,1-20,1- 15th, 1-10,1-5,1-4,1-3,1-2,1 amino acid, and still there is the protein of activities of endothelial tissue plasminogen.

21. the method for any one of 1-20, the plasminogen is comprising activities of endothelial tissue plasminogen segment and still has There is the protein of activities of endothelial tissue plasminogen.

22. the method for any one of 1-21, the plasminogen is selected from Glu- plasminogens, Lys- plasminogens, small fibre Lyase original, Microplasminogen, delta- plasminogens or their variant for retaining activities of endothelial tissue plasminogen.

23. the method for any one of 1-22, the plasminogen for natural or synthetic human plasminogen or its still Retain the variant or segment of activities of endothelial tissue plasminogen.

24. the method for any one of 1-22, the plasminogen is from the people of primate or rodent fibre Lyase original directly to homologue or its still retain the variant of activities of endothelial tissue plasminogen or segment.

25. the method for any one of 1-24, the amino acid of the plasminogen is as shown in sequence 2,6,8,10 or 12.

26. the method for any one of 1-25, wherein the plasminogen is naive plasminogen.

27. the method for any one of 1-26, wherein the subject is people.

28. the method for any one of 1-27, wherein the subject lacks or missing plasminogen.

29. the method for any one of 1-28, the shortage or missing are inborn, secondary and/or local.30. one Kind is used for the plasminogen of the method for any one of item 1-29.

31. a kind of pharmaceutical composition, it includes pharmaceutically acceptable supporting agent and for any one of claim 1-29 The plasminogen of the method.

32. a kind of preventative or therapeutic agent box, it includes：(i) for any one of the claim 1-29 sides The plasminogen of method and (ii) are for delivering the plasminogen to the component (means) of the subject.

33. according to the kit described in item 32, wherein the component is syringe or bottle.

34. 32 or 33 kit, also comprising label or operation instructions, the label or operation instructions instruction The plasminogen is administered into the subject with any one of practical matter 1-29 the methods.

35. a kind of product, it includes：

Container containing label；With

Include the pharmaceutical composition of (i) for the plasminogen of any one of item 1-29 the methods or comprising plasminogen Object, wherein the plasminogen or composition are administered the subject with any one of practical matter 1-29 by label instruction The method.

36. the kit of any one of 32-34 or the product of item 35, also comprising other one or more components or Container contains other drugs in the component or container.

37. 36 kit or product, wherein the other drugs are selected from the group：Antidiabetic medicine, anti-heart and brain blood Pipe disease medicament, antithrombotic reagent, drug for hypertension, anti-lipid drug, anticoagulant, anti-infectives.

On the one hand, the present invention relates to a kind of method for preventing and treating diabetes, including a effective amount of fibre of subject is administered Fibrillarin lyase original or fibrinolysin.

On the other hand, it is effective including administration subject the present invention relates to a kind of method for reducing diabetic subjects blood glucose The plasminogen of amount.The invention further relates to plasminogen for reducing the purposes of diabetic subjects blood glucose.The invention further relates to Plasminogen is used to prepare the purposes for the drug for reducing diabetic subjects blood glucose.Moreover, it relates to for reducing sugar Urinate the plasminogen of disease subject blood glucose.In some embodiments, the blood glucose is chosen from the followings one or more：Serum Glucose level, Serum Fructosamine are horizontal, serum glycated hemoglobin is horizontal.In other embodiments, the blood glucose For serum level of glucose.In the above-described embodiment, the diabetes are T1DM or T2DM.

On the other hand, the present invention relates to a kind of method for improving diabetic subjects sugar tolerance, have including administration subject The plasminogen of effect amount.It is used to improve the purposes of diabetic subjects sugar tolerance the invention further relates to plasminogen.The present invention is also It is related to the purposes that plasminogen is used to prepare the drug for improving diabetic subjects sugar tolerance.Moreover, it relates to it is used for Improve the plasminogen of diabetic subjects sugar tolerance.In some embodiments, the diabetes are T2DM.

On the one hand, the present invention relates to a kind of methods that diabetic subjects postprandial blood sugar is promoted to decline, tested including being administered A effective amount of plasminogen of person.The invention further relates to the use that plasminogen is used to that diabetic subjects postprandial blood sugar to be promoted to decline On the way.The invention further relates to the purposes that plasminogen is used to prepare the drug that diabetic subjects postprandial blood sugar is promoted to decline.This Outside, the invention further relates to the plasminogens for diabetic subjects postprandial blood sugar to be promoted to decline.In some embodiments, The plasminogen is given for 30 minutes to 1.5 hours before the meal in subject.In other embodiments, the plasminogen It is given within 30 minutes to 1 hour before the meal in subject.

On the one hand, promote diabetic subjects to the method utilized of glucose the present invention relates to a kind of, including administration by A effective amount of plasminogen of examination person.The invention further relates to plasminogens for promoting utilization of the diabetic subjects to glucose Purposes.It is used to prepare the invention further relates to plasminogen and promotes use of the diabetic subjects to the drug utilized of glucose On the way.Moreover, it relates to for promoting the plasminogen that utilizes of the diabetic subjects to glucose.On the other hand, originally Invention is related to a kind of method for promoting diabetic subjects insulin secretion, including a effective amount of plasminogen of subject is administered. In some embodiments, the plasminogen also promotes the expression of diabetic subjects insulin.In the embodiment above In, the diabetes are T1DM or T2DM.In some embodiments, the plasminogen promotes diabetic subjects feed Insulin secretion afterwards.In other embodiments, the plasminogen promotes the pancreas under diabetic subjects fasting state Island element secretion.In some embodiments, the plasminogen promotes the pancreas islet of diabetic subjects response blood glucose rise stimulation Element secretion is returned to blood glucose normal or close to normal level.In other embodiments, the plasminogen is promoting While the insulin expression and/or secretion, the expression and/or secretion of subject's glucagon, specifically, institute are reduced While plasminogen is stated by promoting the insulin expression and/or secretion, the expression of subject's glucagon is reduced And/or secretion, it is normal or close to normal level that realization is returned to subject's blood glucose.

On the one hand, the present invention relates to it is a kind of reduce diabetic subjects glucagon secretion method, including administration by A effective amount of plasminogen of examination person.The invention further relates to plasminogen for reducing diabetic subjects glucagon secretion Purposes.The invention further relates to the use that plasminogen is used to prepare the drug for reducing diabetic subjects glucagon secretion On the way.Moreover, it relates to the plasminogen for reducing diabetic subjects glucagon secretion.In some implementations In scheme, the plasminogen also reduces the expression of diabetic subjects glucagon.In the above-described embodiment, it is described Diabetes are T1DM or T2DM.In some embodiments, the plasminogen reduces the pancreas after diabetic subjects feed Glucagons is secreted.In other embodiments, the pancreas that the plasminogen is reduced under diabetic subjects fasting state is high Blood glucose element is secreted.In some embodiments, the plasminogen reduces pancreas height under diabetic subjects blood glucose rise state The secretion of blood glucose element is returned to blood glucose normal or close to normal level.In some embodiments, the plasminogen exists The secretion of glucagon is reduced under diabetic subjects blood glucose rise state, is returned to blood glucose normal or close to normal water It is flat.In other embodiments, the plasminogen reduce subject's glucagon expression and/or secretion it is same When, promote the insulin expression and/or secretion, specifically, the plasminogen is by reducing subject's glucagon While expression and/or secretion, promote the insulin expression and/or secretion, realization make subject's blood glucose be returned to it is normal or Close to normal level.In the above-described embodiment, the plasminogen promotes the expression of insulin receptor substrate2 (IRS-2).

On the one hand, the present invention relates to a kind of method for promoting diabetic subjects islet cells injury repair, including administration A effective amount of plasminogen of subject.The invention further relates to plasminogens for diabetic subjects islet cells to be promoted to damage The purposes of reparation.The medicine for promoting diabetic subjects islet cells injury repair is used to prepare the invention further relates to plasminogen The purposes of object.Moreover, it relates to for promoting the plasminogen of diabetic subjects islet cells injury repair. In some embodiments, the plasminogen promotes the expression of insulin receptor substrate2 (IRS-2).In other embodiment party In case, the plasminogen promotes the expression of cytokine TNF-α.In other embodiments, the plasminogen promotes The expression of the multidirectional Nuclear Factor kappa B of subject.In some embodiments, the islet cells damage is selected from following It is one or more：Beta Cell of islet synthesizes and function damage, islet tissue structural damage, the pancreas islet collagen of excreting insulin sink Product, the fibrosis of pancreas islet, Intra-islet Apoptosis and islet secretion glucagon, the Balance disorders of insulin, islet secretion pancreas The level of glucagons and insulin cannot be adapted with subject's blood glucose level.In some embodiments, the fibrinolytic Proenzyme reduces the diabetic subjects glucagon secretion, and insulin secretion increases, and specifically, the pancreas islet pancreas is high Blood glucose element and the normal equilibrium of insulin secretion are repaired.

On the other hand, the present invention relates to a kind of method for protecting subject's pancreas islet, including a effective amount of fibre of subject is administered Lyase is former.It is used to protect the purposes of subject's pancreas islet the invention further relates to plasminogen.The invention further relates to plasminogens to be used for Prepare the purposes of the drug of protection subject's pancreas islet.Moreover, it relates to for protecting the fibrinolysin of subject's pancreas islet It is former.In some embodiments, the plasminogen reduces pancreas islet collagen deposition.In other embodiments, the fibrinolytic Proenzyme mitigates the fibrosis of pancreas islet.In other embodiments, the plasminogen mitigates Intra-islet Apoptosis.Another In a little embodiments, the plasminogen promotes the expression of pancreatic insulin receptor substrate 2 (IRS-2).In some embodiments In, the plasminogen promotes the reparation of insulitis.In other embodiments, the plasminogen promote cell because The expression of sub- TNF-α.In other embodiments, the plasminogen promotes the multidirectional Nuclear Factor kappa B's of subject Expression.In the above-described embodiment, the subject is diabetic, specifically, the diabetic for T1DM or T2DM.In some embodiments, the T1DM subject is damaged subject for the active normal or PLG activity of PLG.

On the other hand, the present invention relates to it is a kind of promote diabetic subjects insulitis reparation method, including administration by A effective amount of plasminogen of examination person.The invention further relates to plasminogens for promoting diabetic subjects insulitis reparation Purposes.The invention further relates to the purposes that plasminogen is used to prepare the drug for promoting diabetic subjects insulitis reparation.This Outside, the invention further relates to the plasminogens for promoting diabetic subjects insulitis reparation.In some embodiments, it is described Plasminogen promotes the expression of cytokine TNF-α.In other embodiments, the plasminogen promotes subject more Expression to Nuclear Factor kappa B.In other embodiments, the plasminogen reduces pancreas islet collagen deposition.Another In some embodiments, the plasminogen mitigates the fibrosis of pancreas islet.In other embodiments, the plasminogen Inhibit Intra-islet Apoptosis.In the above-described embodiment, the diabetic is T1DM or T2DM, specifically, the T1DM Subject is damaged subject for the active normal or PLG activity of PLG.

On the one hand, the present invention relates to a kind of method for promoting diabetic subjects cytokine TNF-alpha expression, including giving A effective amount of plasminogen of medicine subject.The invention further relates to plasminogens for promoting diabetic subjects cell factor The purposes of TNF-α expression.It is used to prepare the invention further relates to plasminogen and promotes diabetic subjects cytokine TNF-α tables The purposes of the drug reached.Moreover, it relates to for promoting the fibrinolytic of diabetic subjects cytokine TNF-alpha expression Proenzyme.

On the other hand, the present invention relates to the method for promoting the multidirectional Nuclear Factor kappa B expression of diabetic subjects, packets Include administration a effective amount of plasminogen of subject.The invention further relates to plasminogens for promoting the multidirectional core of diabetic subjects The purposes of transcription factor NF-KB expression.It is used to prepare the invention further relates to plasminogen and promotes the multidirectional consideration convey of diabetic subjects Record the purposes of the drug of factor NF- κ B expression.

On the other hand, the present invention relates to it is a kind of promote pancreatic insulin receptor substrate 2 (IRS-2) express method, including A effective amount of plasminogen of subject is administered.The invention further relates to plasminogens for promoting pancreatic insulin receptor substrate 2 (IRS-2) purposes of expression.It is used to prepare the invention further relates to plasminogen and promotes pancreatic insulin receptor substrate 2 (IRS-2) The purposes of the drug of expression.Moreover, it relates to for the fibre that pancreatic insulin receptor substrate 2 (IRS-2) is promoted to express Lyase is former.

On the other hand, it is tested including being administered the present invention relates to a kind of method for promoting diabetic subjects insulin secretion A effective amount of plasminogen of person promotes the expression of insulin receptor substrate2 (IRS-2).The invention further relates to plasminogens to be used for Promote the purposes of diabetic subjects insulin secretion.It is used to prepare the invention further relates to plasminogen and promotes diabetes tested The purposes of the drug of person's insulin secretion.Moreover, it relates to for promoting the fibre of diabetic subjects insulin secretion Lyase is former.

On the other hand, promote the increased method of diabetic subjects beta Cell of islet quantity the present invention relates to a kind of, including A effective amount of plasminogen of subject is administered.The invention further relates to plasminogens for promoting diabetic subjects beta Cell of islet The increased purposes of quantity.Being used to prepare the invention further relates to plasminogen promotes diabetic subjects beta Cell of islet quantity to increase Drug purposes.Moreover, it relates to for promoting the increased fibrinolysin of diabetic subjects beta Cell of islet quantity It is former.In some embodiments, the plasminogen promotes insulin receptor substrate2 (IRS-2) to express.

On the other hand, the present invention relates to a kind of method for reducing islet beta-cell apoptosis, including subject's effective quantity is administered Plasminogen.It is used to reduce the purposes of islet beta-cell apoptosis the invention further relates to plasminogen.The invention further relates to fibrinolytics Proenzyme is used to prepare the purposes for the drug for reducing islet beta-cell apoptosis.Moreover, it relates to for reducing beta Cell of islet The plasminogen of apoptosis.In some embodiments, the plasminogen promotes insulin receptor substrate2 (IRS-2) to express.

On the other hand, the present invention relates to a kind of method for promoting beta Cell of islet injury repair, have including administration subject The plasminogen of effect amount.It is used to promote the purposes of beta Cell of islet injury repair the invention further relates to plasminogen.The present invention is also It is related to the purposes that plasminogen is used to prepare the drug for promoting beta Cell of islet injury repair.The invention further relates to for promoting pancreas The plasminogen of island β cellular damage reparations.In some embodiments, the plasminogen promotes insulin receptor substrate2 (IRS-2) it expresses.

On the other hand, the present invention relates to a kind of methods that islet beta cell function is promoted to restore, and have including administration subject The plasminogen of effect amount.The invention further relates to the purposes that plasminogen is used to that islet beta cell function to be promoted to restore.The present invention is also It is related to the purposes that plasminogen is used to prepare the drug that islet beta cell function is promoted to restore.Moreover, it relates to it is used for The plasminogen that islet beta cell function is promoted to restore.In some embodiments, the plasminogen promotes insulin receptor Substrate 2 (IRS-2) is expressed.

In the above-described embodiment, the plasminogen can be combined with one or more other medicines or therapy.Tool Body, the plasminogen can be with one or more drug combinations chosen from the followings：Antidiabetic medicine, resisting cardiovascular disease Medicine, antithrombotic reagent, drug for hypertension, anti-lipid drug, anticoagulant, anti-infectives.

In the above-described embodiment, the plasminogen and sequence 2,6,8,10 or 12 have at least 75%, 80%, 85%th, 90%, 95%, 96%, 97%, 98% or 99% sequence identity, and still there is activities of endothelial tissue plasminogen.

In the above-described embodiment, the amino acid of the plasminogen is as shown in sequence 2,6,8,10 or 12.In some realities It applies in scheme, the plasminogen is on the basis of sequence 2,6,8,10 or 12, and addition deletes and/or replaces 1-100,1- 90、1-80、1-70、1-60、1-50、1- 45、1-40、1-35、1-30、1-25、1-20、1-15、1-10、1-5、1-4、1-3、 1-2,1 amino acid, and still there is the protein of activities of endothelial tissue plasminogen.

In the above-described embodiment, the plasminogen is comprising activities of endothelial tissue plasminogen segment and still with fibrinolytic The protein of zymogen activity.Specifically, the plasminogen is selected from Glu- plasminogens, Lys- plasminogens, miniPlm Original, Microplasminogen, delta- plasminogens or their variant for retaining activities of endothelial tissue plasminogen.

In the above-described embodiment, plasminogen for natural or synthetic human plasminogen or its still retain fibrinolysin The variant or segment of former activity.In some embodiments, the plasminogen is from primate or rodent Human plasminogen directly to homologue or its still retain the variant of activities of endothelial tissue plasminogen or segment.For example, it is moved from primate Object or the plasminogen of rodent are directly to homologue, such as from gorilla, rhesus macaque, mouse, ox, horse, dog Plasminogen is directly to homologue.Most preferably, the amino acid sequence of plasminogen of the invention such as sequence 2, 6th, shown in 8,10 or 12.

In the above-described embodiment, the subject is people.In some embodiments, wherein the subject lacks Or missing plasminogen.Specifically, the shortage or missing are inborn, secondary and/or local.

In one embodiment, the plasminogen preferably passes through following way by administered either systemically or locally Diameter is applied：Surface, intravenous, intramuscular, subcutaneous, sucking, intraspinal tube, local injection, intra-articular injection pass through rectum.One In a embodiment, the local administration be directly to osteoporosis regional administration, such as pass through the modes such as dressing, conduit come It carries out.

In one embodiment, the plasminogen is applied with appropriate peptide carrier or combination of stabilizers.At one In embodiment, the plasminogen with daily 0.0001-2000mg/kg, 0.001- 800mg/kg, 0.01-600mg/kg, 0.1-400mg/kg, 1-200mg/kg, 1-100mg/kg, 10- 100mg/kg (being calculated with per kilogram of body weight) or 0.0001- 2000mg/cm²、0.001-800 mg/cm²、0.01-600mg/cm²、0.1-400mg/cm²、1-200mg/cm²、1-100 mg/ cm²、10-100mg/cm²The dosage of (being calculated with body surface area every square centimeter) is applied, and is preferably at least repeated once, preferably extremely Few application daily.In the case of local application, above-mentioned dosage according to circumstances can also be adjusted further.On the one hand, it is of the invention It is related to a kind of pharmaceutical composition, it includes pharmaceutically acceptable supporting agent and the plasminogens for the method for the invention.

On the other hand, the present invention relates to a kind of preventative or therapeutic agent box, it includes：(i) for of the present invention The plasminogen of method and (ii) are for delivering the plasminogen to the component (means) of the subject, specifically, institute Component is stated as syringe or bottle.In some embodiments, the kit is also comprising label or operation instructions, the mark The plasminogen is administered the subject to implement method of the present invention by label or operation instructions instruction.

On the other hand, the invention further relates to a kind of product, it includes：Container containing label；(i) for the present invention The plasminogen of the method or the pharmaceutical composition comprising plasminogen, wherein the label is indicated the plasminogen Or composition administers the subject to implement the method for the invention.

In the above-described embodiment, the kit or product, should also comprising other one or more components or container Contain other drugs in component or container.In some embodiments, the other drugs are selected from the group：Antidiabetic medicine, Medicament against cardiovascular disease, antithrombotic reagent, drug for hypertension, anti-lipid drug, anticoagulant, anti-infectives.

Detailed description of the invention

" diabetes " are by inherent cause, immunologic function disorder, microorganism infection and its toxin, free radical toxin, spirit The various virulence factors of factor etc. act on sugar, the albumen that body leads to hypoinsulinism, insulin resistance etc. and causes A series of metabolic disorder syndromes such as matter, fat, water and electrolyte, clinically using hyperglycemia as main feature.

" diabetic complication " is by bad caused other organ or tissues of body of glycemic control in diabetes mellitus Damage or dysfunction, including liver, kidney, heart, retina, the damage of nervous system or dysfunction etc..According to generation Boundary's health organization statistics, diabetic complication are up to more than 100 kinds, are to be currently known a kind of most disease of complication.

" insulin resistance " refers to that a variety of causes makes insulin that glucose uptake and the efficiency utilized be promoted to decline, body The hypersecretion insulin of compensatory generates hyperinsulinemia, to maintain the stabilization of blood glucose.

" fibrinolysin " is a kind of very important enzyme being present in blood, being capable of fibrin degradation polymer.

" plasminogen (plasminogen, plg) " is the zymogen forms of fibrinolysin, according to the sequence in swiss prot Row are calculated by the natural human source plasminogen amino acid sequences (sequence 4) containing signal peptide and are made of 810 amino acid, Molecular weight is about 90kD, and the glycoprotein that mainly synthesizes and can recycle in blood in liver encodes the amino acid sequence CDNA sequence is as shown in sequence 3.The PLG of overall length includes seven structural domains：Serine protease domain, N positioned at C-terminal Pan Apple (PAp) structural domains of end and 5 Kringle structural domains (Kringle1-5).With reference in swiss prot Sequence, signal peptide includes residue Met1-Gly19, and PAp includes residue Glu20-Val98, Kringle1 and includes residue Cys103-Cys181, Kringle2 include residue Glu184-Cys262, Kringle3 and include residue Cys275-Cys352, Kringle4 includes residue Cys377-Cys454, Kringle5 and includes residue Cys481-Cys560.According to NCBI data, silk Serine protease domain includes residue Val581-Arg804.

Glu- plasminogens are the plasminogens of Native full-length, are made of 791 amino acid and (do not contain 19 amino acid Signal peptide), encode the cDNA sequence of the sequence as shown in sequence 1, amino acid sequence is as shown in sequence 2.In vivo, also There are it is a kind of be at the 76-77 amino acids of Glu- plasminogens hydrolysis so as to formed Lys- plasminogens, such as sequence Shown in 6, the cDNA sequence of the amino acid sequence is encoded as shown in sequence 5.δ-plasminogen (δ-plasminogen) are complete Long plasminogen has lacked the segment of Kringle2-Kringle5 structures, only containing Kringle1 and serine protease Domain^[39,40], there is the amino acid sequence (sequence 8) of document report δ-plasminogen^[40], encode the amino acid sequence CDNA sequence such as sequence 7.Mini-plasminogen is made of Kringle5 and serine protease domain, have document report its Including residue Val443-Asn791 (using do not contain signal peptide Glu-plg sequences Glu residues as initial amino acid)^[41], Its amino acid sequence encodes the cDNA sequence of the amino acid sequence as shown in sequence 9 as shown in sequence 10.And Micro- Plasminogen only contains serine protease domain, has its amino acid sequence of document report to include residue A la543- Asn791 (using do not contain signal peptide Glu-plg sequences Glu residues as initial amino acid)^[42], also there is patent CN102154253A reports that its sequence includes residue Lys531-Asn791 (not contain the Glu of the Glu-plg sequences of signal peptide Residue is initial amino acid), this patent sequence reference patent CN102154253A, amino acid sequence is compiled as shown in sequence 12 The cDNA sequence of the code amino acid sequence is as shown in sequence 11.

" fibrinolysin " and " fibrinolysin ", " fibrinoclase " of the present invention is used interchangeably, meaning phase Together；" plasminogen " is used interchangeably with " plasminogen ", " plasminogen ", and meaning is identical.

In this application, content or activity of the meaning of the plasminogen " shortage " for plasminogen in subject's body It is lower than normal person, down to the normal physiological function for being enough to influence the subject；The meaning of the plasminogen " missing " be by The content of plasminogen or activity are substantially less than normal person or even activity in examination person's body or expression is atomic, are only carried by external source For normal physiological function could be maintained.

It will be understood by those skilled in the art that all technical solutions of plasminogen of the present invention are suitable for fibrinolysin, because This, the technical solution that the present invention describes covers plasminogen and fibrinolysin.

In embodiments of the invention, " aging " and " early ageing " may be used interchangeably, and represent same meaning.

In cyclic process, plasminogen is worked as using the nonactive conformation closed and is bound to thrombus or cell surface When, under the mediation of PLG activator (plasminogen activator, PA), it is changed into the activity in open conformation PLM.Fibrin clot further can be hydrolyzed to fibrin degradation product (FDP) and d-dimer by active PLM, and then Thrombus.The PAp structural domains of wherein PLG include the important determinant that plasminogen is maintained to be in nonactive closing conformation, and KR structural domains can then be combined with the lysine residue being present on receptor and substrate.It is known it is a variety of can be used as PLG activate The enzyme of agent, including：Tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), kallikrein and solidifying Blood factor XII (Hageman factor (HF)) etc..

" activities of endothelial tissue plasminogen segment " refers in plasminogen protein, can be combined concurrently with the target sequence in substrate Wave the active fragment of proteolysis function.Technical solution the present invention relates to plasminogen is covered with activities of endothelial tissue plasminogen piece Section replaces the technical solution of plasminogen.Activities of endothelial tissue plasminogen segment of the present invention is the serine comprising plasminogen The protein of protease domain, it is preferable that activities of endothelial tissue plasminogen segment of the present invention includes sequence 14, has with sequence 14 Have at least 80%, 90%, 95%, 96%, 97%, 98%, the protein of the amino acid sequence of 99% homology.Therefore, this hair The bright plasminogen includes containing the activities of endothelial tissue plasminogen segment and still maintains the albumen of the activities of endothelial tissue plasminogen.

At present, plasminogen in blood and its activity determination method are included：To tissue plasminogen It is the detection (t-PAA) of activator activity, the detection (t-PAAg) of plasma tissue plasminogen activator antigen, right The detection (plgA) of plasma tissue activities of endothelial tissue plasminogen, the detection (plgAg) of plasma tissue plasminogen antigen, plasma tissue The detection of Plasminogen activator inhibitor activity, plasma tissue Plasminogen activator inhibitor antigen Detection, plasma fibrin lyase-antiplasmin complex detection (PAP).The detection method of most common of which is Chromogenic assay：To by inspection blood plasma in plus streptokinase (SK) and chromophoric substrate, by inspection blood plasma in PLG under the action of SK, It is transformed into PLM, the latter acts on chromophoric substrate, and then with spectrophotometric determination, absorbance increases and plasminogen Activity is directly proportional.In addition immuno-chemical method, gel electrophoresis, immunoturbidimetry, radioimmunodiffusion etc. can also be used to blood In plasminogen activity be measured.

" ortholog thing or lineal homologue (ortholog) " refers to the homologue between different plant species, both including albumen Homologue also includes DNA homology object, also referred to as ortholog, Paralog object.It is referred specifically in different plant species by same ancestral First gene evolution and come albumen or gene.The plasminogen of the present invention includes the natural plasminogen of people, further includes and derives from Different plant species, there is the plasminogen ortholog thing of activities of endothelial tissue plasminogen or lineal homologue.

" conservative substitution variant " refer to one of them given amino acid residue change but do not change protein or enzyme it is whole Body conformation and function, this include but not limited to similar characteristic (as acid, alkalinity, hydrophobicity, etc.) amino acid substitution parent Amino acid in Amino Acids in Proteins sequence.Amino acid with similarity is well-known.For example, arginine, group Propylhomoserin and lysine are hydrophilic basic amino acids and can be interchanged.Equally, isoleucine is hydrophobic amino acid, then can quilt Leucine, methionine or valine are replaced.Therefore, two albumen of identity function or the similitude of amino acid sequence may It is different.For example, 70% to 99% similarity (homogeneity) based on MEGALIGN algorithms." conservative substitution variant " further includes Polypeptide or enzyme with more than 60% amino acid identities are determined by BLAST or fasta algorithm, if can be up to more than 75% More preferably, it is therefore desirable to up to more than 85% or even up to more than 90% be best, and have compared with natural or parent protein or enzyme There are identical or substantially similar property or function.

" separation " plasminogen refers to the plasminogen protein for detaching and/or recycling from its natural surroundings.In some realities It applies in scheme, the plasminogen can purify (1) to more than the 90%, purity more than 95% or more than 98% (by weight Meter), as determined by by Lowry methods, such as more than 99% (by weight), (2) are to being enough by using rotating cup sequence To homogeney, which is logical for the degree of at least 15 residues of analyzer acquisition N-terminal or internal amino acid sequence or (3) It crosses and uses the sodium dodecyl sulfate-polypropylene acrylamide gel of Coomassie blue or silver staining under reproducibility or non-reducing conditions Electrophoresis (SDS-PAGE) is determining.The plasminogen of separation also includes preparing from recombinant cell by biotechnology, and The plasminogen detached by least one purification step.

Term " polypeptide ", " peptide " and " protein " is used interchangeably herein, and refers to the polymerization of the amino acid of any length Form can include the amino acid of genetic coding and non-genetic coding, chemical or biochemical modification or derivatization Amino acid and the polypeptide with modified peptide backbone.The term includes fusion protein, including but not limited to heterologous ammonia The fusion protein of base acid sequence has heterologous and homologous leader sequences (with and without N-terminal methionine residues) fusions Object；Etc..

It is defined as introducing notch if necessary about " amino acid sequence identity percentage (%) " with reference to peptide sequence It is candidate after realizing largest percentage sequence identity, and when any conservative replacement not being considered as a part for sequence identity The percentage of the amino acid residue identical with the amino acid residue in reference peptide sequence in sequence.To measure percentage amino The comparison of acid sequence identity purpose can be realized, such as use public Ke get with the various ways in the range of art technology The computer software arrived, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.People in the art Member can determine the suitable parameter for aligned sequences, any calculation needed including realizing maximum contrast to compared sequence Method.However, for the purposes of the present invention, amino acid sequence identity percent value is to compare computer program using sequence What ALIGN-2 was generated.

In the case of using ALIGN-2 come comparing amino acid sequence, amino acid sequence A is given relative to given amino Acid sequence B % amino acid sequence identities (or can be expressed as having or comprising relative to, with or for given amino acid The given amino acid sequence A of a certain % amino acid sequence identities of sequence B) it is calculated as below：

Score X/Y multiplies 100

Wherein X is that scoring is the amino of identical match in the A and B of the program are compared by alignment programs ALIGN-2 The number of sour residue, and wherein Y is the sum of the amino acid residue in B.It will be appreciated that in the length and ammonia of amino acid sequence A In the case of the length of base acid sequence B is unequal, A can be not equal to B relative to A relative to the % amino acid sequence identities of B % amino acid sequence identities.Unless expressly stated otherwise, all % amino acid sequence identities values used herein are all It is according to described in the preceding paragraph, is obtained using ALIGN-2 computer programs.

As used in this article, term " treatment " and " prevention ", which refer to, obtains desired pharmacology and/or physiologic effect.It is described Effect can be prevented disease or its symptom completely or partially and/or partially or completely cure disease and/or its symptom, and Including：(a) prevention disease occurs in subject's body, and the subject can have the procatarxis of disease, but not yet be diagnosed as With disease；(b) inhibit disease, that is, block its formation；(c) mitigate disease and/or its symptom, that is, cause disease and/or its Resolution of symptoms.

Term " individual ", " subject " and " patient " is used interchangeably herein, and refers to mammal, including but it is unlimited In mouse (rat, mouse), non-human primates, people, dog, cat, ungulate (such as horse, ox, sheep, pig, goat) etc..

" therapeutically effective amount " or " effective quantity " refers to be enough when applying mammal or other subjects to treat disease Realize the amount to the prevention of disease and/or the plasminogen for the treatment of." therapeutically effective amount " can be according to used fibrinolysin The disease of subject former, to be treated and/or the severity of its symptom and age, weight etc. and change.

2. the preparation of plasminogen of the present invention

Plasminogen can be detached from nature and be purified for further treatment purposes, can also pass through standard Chemical peptide symthesis technology synthesizes.When passing through chemically synthesized polypeptide, can be synthesized through liquid phase or solid phase.Solid-phase polypeptide Synthesis (SPPS) (the C-terminal amino acid of sequence is wherein attached into insoluble support, it is then remaining in sequential addition sequence Amino acid) be the method for being suitble to plasminogen chemical synthesis.Various forms of SPPS, such as Fmoc and Boc can be used for synthesizing Plasminogen.Barany and Solid-Phase Peptide Synthesis are described in for the technology of synthesis in solid state；3- Page 284 are in The Peptides:Analysis, Synthesis, Biology. volume 2：Special Methods in Peptide Synthesis, Part A., Merrifield, wait J.Am.Chem.Soc., and 85:2149-2156(1963)； Stewart etc., Solid Phase Peptide Synthesis, 2nd ed.Pierce Chem.Co., Rockford, Ill. (1984)；With Ganesan A.2006Mini Rev.Med Chem.6:The 2005Protein such as 3-10 and Camarero JA Pept Lett.12:In 723-8.In short, it is handled with the functional element for being built with peptide chain thereon small insoluble porous Pearl.After coupling/de-protected repetitive cycling, the solid phase of attachment is dissociated N-terminal amine and the single amino acid list protected by N Member coupling.Then, this element is deprotected, exposes the new N-terminal amine that can be attached with other amino acid.Peptide remains fixed In solid phase, cut away later.

The plasminogen of the present invention can be produced using Standard recombinant methods.For example, the nucleic acid by encoding plasminogen It is inserted into expression vector, it is made to be operatively connected with the regulating and controlling sequence in expression vector.Expression regulation sequence includes but not limited to Promoter (such as naturally associated or heterologous promoter), signal sequence, enhancer element and transcription terminator.Expression Regulation and control can be eukaryotic promoter system in carrier, and the carrier can convert or transfect eukaryotic host cell (such as COS Or Chinese hamster ovary celI).Once carrier is mixed in suitable host, in the high level expression and fibrinolysin for being suitable for nucleotide sequence Host is maintained under conditions of former collection and purifying.

Suitable integration of the expression vector usually in host organisms as episome or as host chromosome DNA It replicates part.In general, expression vector contains selection marker, (such as amicillin resistance, hygromycin resistance, tetracycline resist Property, kalamycin resistance or neomycin resistance) to help to carry out external source with those cells that desired DNA sequence dna converts Detection.

Escherichia coli (Escherichia coli) can be used for the protokaryon place of clone's theme antibody coding polynucleotides The example of chief cell.The other microbial hosts being adapted for use with include bacillus, such as bacillus subtilis (Bacillus Subtilis) and other enterobacteriaceaes (enterobacteriaceae), such as Salmonella (Salmonella), Sha Lei Bordetella (Serratia) and various pseudomonas (Pseudomonas) species.In these prokaryotic hosts, it can also give birth to Into expression vector, the expression control sequence (such as replication orgin) compatible with host cell would generally be contained.In addition, it can deposit In many well known promoters, such as lactose promoter system, tryptophan (trp) promoter systems, beta- lactamases open Subsystem or the promoter systems from phageλ.Promoter would generally control expression, optionally in operator sequence In the case of, and with ribosome bind site sequence etc., to start and complete transcription and translation.

Other microorganisms, such as yeast can also be used for expressing.Yeast (such as saccharomyces cerevisiae (S. cerevisiae)) and finish Red yeast (Pichia) is the example of suitable yeast host cell, wherein suitable carrier has expression control as needed Sequence (such as promoter), replication orgin, termination sequence etc..Typical promoter includes glycerol 3-phosphate acid kinase and other Carbohydrate-splitting enzyme.Induction type yeast starts in particularly including from alcohol dehydrogenase, different cell pigment C and responsible maltose and gala The promoter for the enzyme that sugar utilizes.

Outside microorganism, mammalian cell (such as the mammalian cell cultivated in cell culture in vitro) It can be used for the plasminogen of the expression present invention.Referring to Winnacker, From Genes to Clones, VCH Publishers,N.Y.,N.Y.(1987).Suitable mammalian host cell includes Chinese hamster ovary celI system, various Cos cells System, HeLa cells, myeloma cell line and inverted B cell or hybridoma.It can be with for the expression vector of these cells Comprising expression control sequence, such as replication orgin, promoter and enhancer (Queen etc., Immnol.Rev.89:49 (1986)), And required machining information site, such as ribosome bind site, RNA splice sites, polyadenylation site and turn Record terminator sequence.The example of suitable expression control sequence is white immunoglobulin gene, SV40, adenovirus, cow teats knurl Promoter derived from virus, cytomegalovirus etc..Referring to Co etc., J. Immunol.148:1149(1992).

Once synthesis (chemistry or recombination form), can be according to the standard schedule of this field, including ammonium sulfate precipitation, parent And column, column chromatography, high performance liquid chroma- tography (HPLC), gel electrophoresis etc. purify plasminogen of the present invention.The fibrinolysin Original is substantially pure, and for example, at least about 80% to 85% is pure, and at least about 85% to 90% is pure, at least about 90% to 95% is pure or 98% to 99% is pure or purer, such as without pollutant, the pollutant such as cell fragment, except theme Macromolecular other than antibody, etc..

3. pharmaceutical formulation

Can be by by plasminogen with the desired purity and optional pharmaceutical carrier, excipient or stabilizer (Remington's Pharmaceutical Sciences, 16 editions, Osol, A.ed. (1980)) it is mixed to form lyophilized preparation Or aqueous solution prepares treatment preparaton.Acceptable carrier, excipient, stabilizer under dosage used and concentration to receptor without Toxicity, and including buffer such as phosphate, citrate and other organic acids；Antioxidant includes ascorbic acid and egg ammonia Acid；Preservative (such as stearyl dimethyl benzyl ammonium chloride；Hexamethonium chloride；Benzalkonium chloride (benzalkonium Chloride), benzethonium chloride；Phenol, butanol or benzyl alcohol；Alkyl parabens such as methyl or propyl p-hydroxy benzene first Acid esters；Catechol；Resorcinol；Cyclohexanol；3- amylalcohols；Metacresol)；Low molecular weight polypeptide (less than about 10 residues)；Egg White matter such as seralbumin, gelatin or immunoglobulin；Hydrophilic polymer such as polyvinylpyrrolidone；Amino acid such as glycine, Glutamine, asparagine, histidine, arginine or lysine；Monosaccharide, disaccharides and other carbohydrate include glucose, Mannose or dextrin；Chelating agent such as EDTA；Carbohydrate such as sucrose, mannitol, fucose or sorbierite；Into salt counter ion such as sodium； Metal composite (such as zinc-albumen composition)；And/or nonionic surfactant, such as TWEENTM, PLURONICSTM Or polyethylene glycol (PEG).It is preferred that the anti-VEGF antibodies preparaton being lyophilized, described in WO 97/04801, it includes herein In as reference.

The preparaton of the present invention also contains more than one the reactive compound needed for the specific illness that need to be treated, excellent Those selected complementary activities and be free from side effects between each other.For example, antihypertensive drug, antiarrhythmic drug, Treat drug of diabetes etc..

The plasminogen of the present invention can be wrapped in the microcapsules prepared by such as condensation technique or interfacial polymerization, For example, colloidal drug delivery systems can be embedded in (for example, liposome, albumin microsphere, microemulsion, nano particle and nanometre glue Capsule) in or hydroxymethyl cellulose or gelatin-microcapsule and poly- (methyl methacrylate) micro- glue in merging macro emulsion In capsule.These technologies are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed.(1980)。

Plasminogen for the present invention of vivo medicine-feeding is necessarily sterile.This can be by freeze-drying and again It is realized easily by degerming membrane filtration before or after new preparation.

The plasminogen of the present invention can prepare sustained release preparation.The appropriate example of sustained release preparation include with definite shape and Half penetrating matrix of solid hydrophobic polymers containing glycoprotein, such as film or microcapsules.Sustained-release matrix example includes polyester, water Gel (such as poly- (2- hydroxyethyl-methacrylates) (Langer, J. Biomed.Mater.Res., 15:167-277 (1981)；Langer,Chem.Tech.,12:98- 105 (1982)) or poly- (vinyl alcohol), polyactide (United States Patent (USP) 3773919, EP 58,481), the copolymer (Sidman, etc. Biopolymers of Pidolidone and ethyl-L-glutamates 22:547 (1983)), nondegradable ethylene vinyl acetate (ethylene-vinyl acetate) (Langer, etc., Ibid) or degradable poly lactic coglycolic acid such as Lupron DepotTM (be copolymerized by lactic-co-glycolic acid The microsphere of the injectable of object and leucyl proline (leuprolide) acetic acid esters composition) and poly- D- (-) -3- hydroxyl fourths Acid.Polymer such as ethane-acetic acid ethyenyl ester and lactic-co-glycolic acid energy sustained release molecule 100 days or more, and some water-settings The time of glue release albumen is shorter.Can protein stabilized rational strategy be made to design according to Related Mechanism.If for example, It was found that cohesion mechanism be that intermolecular S -- S is formed by thio Disulfide interchange, then can by modify sulfhydryl residue, from It is lyophilized in acid solution, controls humidity, realized using suitable additive and the specific polymer matrix composition of exploitation Stablize.

4. administration and dosage

Can be by different modes, such as by intravenous, in peritonaeum, subcutaneously, encephalic is intrathecal, intra-arterial (such as through By arteria carotis), intramuscular, intranasal, surface or intradermal administration or spinal cord or brain deliver to realize applying for pharmaceutical composition of the present invention With.The aqueous or other solution and preservative and isotonic agent of purifying of the aerosol preparation such as nose spray preparation comprising activating agent.It will Such preparation is adjusted to the pH compatible with schneiderian membrane and isotonic state.

In some cases, the plasminogen pharmaceutical composition of the present invention can be modified or prepare in the following manner, so as to carry The ability of blood-brain barrier is passed through for it.

Include sterile aqueous or non-aqueous solution, suspension and emulsion for the prepared product of parenteral administration.It is non-aqueous molten The example of agent is propylene glycol, polyethylene glycol, vegetable oil such as olive oil and injectable organic ester, such as ethyl oleate.Aqueous carrier Including water, alcohol/aqueous solution, emulsion or suspension, including brine and buffer medium.Parenteral medium includes sodium chloride Solution, woods grignard dextrose, dextrose and sodium chloride or fixing oil.Intravenous vehicles include liquid and nutritional supplement, Electrolyte supplements, etc..It is such as, antimicrobial, anti-oxidant there may also be preservative and other additives Agent, chelating agent and inert gas, etc..

In some embodiments, plasminogen of the invention is formulated together with promoting the medicament across blood-brain barrier. In some cases, plasminogen of the invention is directly or through connector with promoting carrier molecule, peptide or egg across blood-brain barrier White matter merges.In some embodiments, the polypeptide of plasminogen of the invention with combining endogenous blood-brain barrier (BBB) receptor Fusion.Plasminogen is connected with combining the polypeptide of endogenous BBB receptors, is promoted across BBB.With reference to the suitable of endogenous BBB receptors Polypeptide includes antibody, such as monoclonal antibody or its antigen-binding fragment, specifically binds endogenous BBB receptors.Suitably Endogenous BBB receptors include but not limited to insulin by some cases, and antibody is encapsulated in liposome.See, for example, U.S. Patent Publication text No.2009/0156498.

Medical worker can determine dosage based on various clinical factors.As well known in medical domain, any patient's Dosage depends on many factors, build, body surface area, age including patient, the particular compound to be applied, gender, application Number and path, the general health and other medicines being administered simultaneously.The agent of pharmaceutical composition of the present invention comprising plasminogen It can be, for example, such as daily about 0.0001 to 2000mg/kg or about 0.001 to 500mg/kg (such as 0.02mg/ to measure range Kg, 0.25 mg/kg, 0.5mg/kg, 0.75mg/kg, 10mg/kg, 50mg/kg etc.) subject's weight.For example, dosage can To be 1mg/kg weight or 50mg/kg weight or the range or at least 1mg/kg in 1-50mg/kg.Higher or lower than this illustration Including the dosage of property range is also covered by, it is especially considering that above-mentioned factor.Intermediate dosage in above range is also contained in this In the range of invention.Subject can daily, every other day, weekly or according to any other schedule determined by empirical analysis Using such dosage.Illustrative dosage schedule includes continuous several days 1-10mg/kg.In the medicament administration process of the present invention Middle therapeutic effect and the safety for needing assessment, periodical evaluation thrombus and thrombus relevant disease in real time.

5. treat effect and Therapeutic safety

One embodiment of the invention is related to, using after plasminogen treatment subject, pacifying treatment effect and treatment The judgement of full property.Common osteoporosis treatment effect monitoring with assessment content include follow-up (adverse reaction, Drug adherence, Basic measure and the risk of bone fracture factor reevaluate), (clinic fracture, height reduce and iconography inspection for new hair fracture assessment Look into), bone density (bone mineral density, BMD) measure and bone turnover markers (bone turnover Markers, BTM) detection and the synthesis based on these data reevaluate.Wherein BMD is current most widely used treatment Effect monitoring and appraisal procedure.For example, Dual-energy X-rays absorptionmetry (dual energy X-ray can be passed through Absorptiometry, DXA), quantitive CT (quantitative computed tomography, QCT), Single Photon Absorption Measuring method (SPA) or ultrasonic measuring method measure BMD.Treatment can detect 1 BMD every year after starting, and reach stable in BMD After can be appropriately extended interval, such as 2 years monitor 1 time.For BTM, the more bon e formation used in serological index at present Index is serum 1 type procollagen N-terminal propetide (procollagen type 1n-terminal propeptide, PINP), bone It is serum 1 type procollagen C-terminal peptide (serum C-terminal telopeptide, S- CTX) to absorb index.It can be according to grinding Study carefully progress, in due course adjustment more reasonably Testing index.Baseline value should be detected before starting to treat, drug therapy is formed using rush 3 months afterwards, using inhibit absorb the drug treatment after 3~6 months when be detected.BTM is capable of providing the multidate information of bone, BMD is being acted on and is being functionally separated from, while also become the monitoring means to complement one another with BMD, the two combines with more High clinical value.Usually, if BMD rises or stablizes after treatment, BTM has performance of expected change, while bone free during treatment Folding occurs, it is believed that therapeutic response is good.Moreover, it relates to subject is carried out using plasminogen and its variant In therapeutic process and after treatment, the judgement of the therapeutic scheme safety, including but not limited to drug in subject's body Serum half-life, treatment half-life period, median toxic dose (TD50), median lethal dose (LD50) counted or to treating The various adverse events such as sensitivity response occurred in the process or after treatment is observed.

6. product or medicine box

One embodiment of the invention is related to a kind of product or medicine box, and it includes plasminogens of the present invention.The product Preferably include a container, label or package insert.Appropriate container has bottle, bottle, syringe etc..Container can be by various Material such as glass or plastics are made.The container contains composition, and the composition can effectively treat the disease or disease of the present invention Disease and with sterile access port (such as the container can be intravenous solution packet or bottle, containing can be penetrated by hypodermic needle Plug).At least one of composition activating agent is plasminogen.On the container or appended label illustrates institute Composition is stated for treating aging of the present invention or aging-related disorders.The product can further include containing pharmaceutically acceptable The brine of the second container of buffer solution, such as phosphate-buffered, Ringer's solution and glucose solution.It can further be wrapped Containing required other materials from the point of view of business and user's angle, including other buffer solutions, diluent, filtrate, needle and injection Device.In addition, the product includes the package insert with operation instruction, the user including for example indicating the composition will Plasminogen composition and the other medicines administered patient for treating adjoint disease.

Brief description

Fig. 1 24-25 week old diabetic mices give plasminogen blood glucose test results after 10,31 days.The results show that it gives The blood glucose of plasminogen group mouse, which is significantly lower than, gives solvent PBS control group, and significantly (* represents P to statistical discrepancy<0.05, * * tables Show P<0.01).In addition, with the extension of administration time, there is raising trend to solvent PBS control group mouse blood sugar, and to fibrinolytic Proenzyme group blood glucose continuously decreases.It is hypoglycemic to illustrate that plasminogen has the function of.

Fig. 2 gives influence of the plasminogen to diabetic mice Serum Fructosamine concentration.Testing result is shown, gives fibrinolytic The concentration of Serum Fructosamine is substantially reduced after proenzyme, and compared with before administration, statistical difference is heteropolar, and significantly (* * represent P<0.01).It says Bright plasminogen can significantly reduce the blood glucose of diabetic mice.

The diabetic mice of 26 week old of Fig. 3 gives plasminogen Serum Fructosamine testing result after 35 days.Testing result Display is significantly lower than to the concentration of plasminogen group Serum Fructosamine and gives solvent PBS control group, the close notable (P of statistical discrepancy =0.06).Illustrate that plasminogen can significantly reduce the blood glucose level of diabetic mice.

26 week old diabetic mices of Fig. 4 give plasminogen blood plasma glycosylated hemoglobin testing result after 35 days.As a result Display is significantly lower than to the OD values of plasminogen group mouse glycosylated hemoglobin and gives solvent PBS control group, and statistical difference is heteropolar Significantly (* * represent P<0.01).Illustrate that plasminogen has the function of to reduce blood glucose in diabetic mice.

26 week old diabetic mices of Fig. 5 give plasminogen IPGTT testing results after 10 days.The results show that abdominal cavity is noted After penetrating glucose, it is less than to plasminogen group mouse blood sugar level and gives solvent PBS control group, and with giving solvent PBS control group phase Than being more nearly normal mouse group to plasminogen group sugar tolerance curve.Illustrate that plasminogen can be obviously improved diabetic mice Sugared tolerance.

Fig. 6 T1DM model PLG activity normal mouses give plasminogen blood glucose test results after fasting after 10 days.As a result It has been shown that, is apparently higher than to solvent PBS control group mouse blood sugar and gives plasminogen group, and statistical difference (* * * expressions P heteropolar significantly< 0.001).Illustrate that plasminogen can significantly reduce blood glucose level of the PLG activity normal mouse in T1DM models.

Fig. 7 T1DM model PLG activity normal mouses give plasminogen IPGTT testing results after 28 days.The results show that Be apparently higher than to blood sugar concentration after solvent PBS control group mouse injectable dextrose monohydrate and give plasminogen group, and with to PBS pairs of solvent It is compared according to group and is more nearly normal mouse to plasminogen group sugar tolerance curve.It is normal to illustrate that plasminogen can improve PLG activity Sugared tolerance of the mouse in T1DM models.

Fig. 8 shows that T1DM model mices give plasminogen blood glucose test results after 20 days.The results show that solvent PBS Control group mice blood glucose, which is apparently higher than, gives plasminogen group mouse, and statistical discrepancy is significantly (P=0.04).Illustrate plasminogen It can promote T1DM mouse glucose capacities of decomposition, so as to reduce blood glucose.

26 week old diabetic mices of Fig. 9 give plasminogen serum insulin testing result after 35 days.The results show that it gives Plasminogen group serum insulin level, which is apparently higher than, gives solvent PBS control group, and significantly (* represents P to statistical discrepancy<0.05). Illustrate that plasminogen can effectively facilitate the secretion of insulin.

Figure 10 24-25 week old diabetic mices give the HE dyeing pictures and islet area of plasminogen pancreas after 31 days Than.A, B is gives solvent PBS control group, and to give plasminogen group, E is islet area quantitative analysis results by C, D.The results show that Atrophy occurs to the most pancreas islet of solvent PBS control group, the islet cells of atrophy is replaced, pancreas islet side by acinus (↓ mark) The acinus hyperplasia of edge causes to demarcate between pancreas islet and acinus unclear；To the most pancreas islet of plasminogen group compared to control group face Product is big, and does not have acinus hyperplasia in pancreas islet, and only remaining has a small amount of acinus in a small number of pancreas islet, side between pancreas islet and acinus Boundary is clear.Compare the area ratio discovery that pancreas is accounted for the pancreas islet of plasminogen group and control group, administration group is bigger than control group almost One times.Illustrate that plasminogen can promote the reparation of 24-25 week old diabetic mice islet damages, so as to by repairing damage Pancreas islet treats diabetes.

Figure 11 24-25 week old diabetic mices give plasminogen after 31 days pancreas islet sirius red stains observe result.A To give solvent PBS control group, for B to give plasminogen group, C is quantitative analysis results.The results show that give plasminogen group mouse Pancreas islet collagen deposition (arrow logo), which is considerably less than, gives solvent PBS control groups, and significantly (* represents P to statistical discrepancy<0.05).It says Bright plasminogen can improve the fibrosis of diabetic animal pancreas islet.

Figure 12 24-25 week old diabetic mices give plasminogen pancreas islet Caspase-3 immunohistochemical stainings after 31 days Observe result.A is gives solvent PBS control group, and B is gives plasminogen group.The results show that plasminogen group Caspase-3's Expression (arrow logo) is significantly lower than giving solvent PBS control group.Illustrate that plasminogen can reduce the apoptosis of islet cells, protect Diabetic mice pancreatic tissues.

18 week old diabetic mices of Figure 13 give plasminogen pancreatic insulin immunohistochemical staining result after 35 days.A To give solvent PBS control group, for B to give plasminogen group, C is quantitative analysis results.The results show that give plasminogen group pancreas islet The expression (arrow logo) of element, which is apparently higher than, gives solvent PBS control group, and statistical discrepancy is close to significantly (P=0.15).Illustrate fibre Lyase proper energy enough promotes islet function reparation, promotes the generation and secretion of insulin.

The insulin immunohistochemical staining that Figure 14 24-25 week old diabetic mices give plasminogen pancreas islet after 35 days is seen Examine result.A is gives solvent PBS control group, and for B to give plasminogen group, C is quantitative analysis results.The results show that fibrinolysin The expression (arrow logo) of original group insulin, which is apparently higher than, gives solvent PBS control group, and significantly (* represents P to statistical discrepancy< 0.05).Illustrate that plasminogen can promote islet function reparation, promote the generation and secretion of insulin.

26 week old diabetic mices of Figure 15 give the insulin immunohistochemical staining result of plasminogen pancreas islet after 35 days. A is gives solvent PBS control group, and for B to give plasminogen group, C is quantitative analysis results.The results show that give plasminogen group pancreas islet The expression (arrow logo) of element, which is apparently higher than, gives solvent PBS control group, and statistical difference (* * expressions P heteropolar significantly<0.01).It says Bright plasminogen can effectively facilitate islet function reparation, promote the generation and secretion of insulin.

Figure 16 24-25 week old diabetic mices give plasminogen pancreatic tissue NF- κ B immunohistochemical stainings after 31 days Observe result.A is Normal group, and B is gives solvent PBS control group, and for C to give plasminogen group, D is quantitative analysis results.Knot Fruit shows, is apparently higher than to the expression (arrow logo) of plasminogen group NF- κ B and gives solvent PBS control group, and statistical discrepancy is shown Write (* expressions P<0.05).Illustrate that plasminogen can promote the expression of multidirectional Nuclear Factor kappa B, so as to promote 24-25 weeks The reparation of age diabetic mice insulitis.

The diabetic mice of 18 week old of Figure 17 gives plasminogen pancreas islet glucagon immunohistochemical observation after 35 days As a result.A is Normal group, and B is gives solvent PBS control group, and for C to give plasminogen group, D is quantitative analysis results.As a result it shows Show, glucagon is expressed in normal control mice in pancreas islet periphery α cell compartments.Compared with to plasminogen group, to molten Matchmaker's PBS control group glucagon positive cell (arrow logo) showed increased, glucagon positive cell infiltration to pancreas islet Middle section, and average optical density quantitative analysis results statistical difference it is heteropolar significantly (* * represent P<0.01)；To plasminogen What group glucagon positive cell was dispersed in is distributed in pancreas islet periphery, and to plasminogen group compared with PBS groups, pancreas islet form is more Close to normal mouse.Illustrate that plasminogen can significantly inhibit the secretion of alpha Cell of islet proliferation and glucagon, correct pancreas Island α cell distributions are disorderly, so as to promote the reparation of islet damage.

The diabetic mice of Figure 18 24-25 week old gives plasminogen pancreas islet glucagon immunohistochemistry sight after 35 days Examine result.A is Normal group, and B is gives solvent PBS control group, and C is gives plasminogen group.The results show that glucagon Expression is in pancreas islet periphery α cell compartments in normal control mice.Compared with to plasminogen group, solvent PBS control group pancreas is given Glucagons positive cell (arrow logo) showed increased, positive cell infiltrate the middle section to pancreas islet；Give plasminogen group What glucagon positive cell was dispersed in is distributed in pancreas islet periphery, and to plasminogen group compared with PBS groups, pancreas islet form more connects Nearly normal mouse.Illustrate that plasminogen can significantly inhibit the secretion of alpha Cell of islet proliferation and glucagon, correct pancreatic islet alpha Cell distribution is disorderly, so as to promote the reparation of islet damage.

The diabetic mice of 26 week old of Figure 19 gives plasminogen pancreas islet glucagon immunohistochemical observation after 35 days As a result.A is Normal group, and B is gives solvent PBS control group, and for C to give plasminogen group, D is quantitative analysis results.As a result it shows Show, glucagon is expressed in normal control mice in pancreas islet periphery α cell compartments.Compared with to plasminogen group, to molten Matchmaker's PBS control group glucagon positive cell (arrow logo) showed increased, positive cell infiltrate the central area to pancreas islet Domain, and average optical density quantitative analysis results have significant difference (* expressions P<0.05)；Give plasminogen group pancreas hyperglycemia What plain positive cell was dispersed in is distributed in pancreas islet periphery, and to plasminogen group compared with PBS groups, form is closer to normal mouse. Illustrate that plasminogen can significantly inhibit the secretion of alpha Cell of islet proliferation and glucagon, it is disorderly to correct alpha Cell of islet distribution Disorderly, so as to promote the reparation of islet damage.

Figure 20 give plasminogen after 28 days PLG activity normal mouse pancreas islet glucagon in T1DM models be immunized Groupization observes result.A is blank control group, and B is gives solvent PBS control group, and for C to give plasminogen group, D is quantitative analysis knot Fruit.The results show that it is significantly more than to solvent PBS control group glucagon positive expression to molten plasminogen group, and average light Significantly (* represents P to density quantitative analysis results statistical discrepancy<0.05).Illustrate that plasminogen can substantially reduce diabetic mice Alpha Cell of islet secretes glucagon, promotes the reparation of islet damage.

The diabetic mice of 18 week old of Figure 21 gives plasminogen pancreas islet IRS-2 immunohistochemical observation results after 35 days.A For Normal group, B is gives solvent PBS control group, and for C to give plasminogen group, D is quantitative analysis results.The results show that it gives Solvent PBS control group mouse islets IRS-2 positive expressions (arrow logo) are considerably less than and give plasminogen group, and statistical discrepancy Extremely significantly (* * represent P<0.01)；Relatively give solvent PBS groups closer Normal group to plasminogen group IRS-2 expressions Mouse.Illustrate that plasminogen can be effectively increased the expression of islet cells IRS-2, improve insulin signal transduction, reduce diabetes Mouse islets β cellular damages.

The diabetic mice of Figure 22 24-25 week old gives plasminogen pancreas islet IRS-2 immunohistochemical observation knots after 31 days Fruit.A is Normal group, and B is gives solvent PBS control group, and for C to give plasminogen group, D is quantitative analysis results.As a result it shows Show, be considerably less than to solvent PBS control group mouse islets IRS-2 positive expressions (arrow logo) and give plasminogen group, and counted (* represents P to significant difference<0.05)；Relatively give solvent PBS groups closer normal control to plasminogen group IRS-2 expressions Group mouse.Illustrate that plasminogen can be effectively increased the expression of islet cells IRS-2, improve insulin signal transduction, reduce glycosuria Sick mouse islets β cellular damages.

The diabetic mice of 26 week old of Figure 23 gives plasminogen pancreas islet IRS-2 immunohistochemical observation results after 35 days.A For Normal group, B is gives solvent PBS control group, and C is gives plasminogen group.The results show that solvent PBS control group mouse Pancreas islet IRS-2 positive expressions (arrow logo) are considerably less than and give plasminogen group；To plasminogen group IRS-2 expressions compared with Give solvent PBS groups closer Normal group mouse.Illustrate that plasminogen can be effectively increased the expression of islet cells IRS-2, change Kind insulin signal transduction reduces the damage of diabetic mice beta Cell of islet.

Figure 24 gives plasminogen normal T1DM mouse islets IRS-2 immunohistochemical observation results of PLG activity after 28 days.A For Normal group, B is gives solvent PBS control group, and C is gives plasminogen group.The results show that solvent PBS control group mouse Pancreas islet IRS-2 positive expressions (arrow logo) are considerably less than and give plasminogen group；To plasminogen group IRS-2 expressions compared with Give solvent PBS groups closer Normal group mouse.Illustrate that plasminogen can be effectively increased the expression of islet cells IRS-2, Improve insulin signal transduction, reduce the normal T1DM mouse islets β cellular damages of PLG activity.

The diabetic mice of 26 week old of Figure 25 gives plasminogen pancreas islet neutrophil leucocyte immunohistochemical observation after 35 days As a result.A is Normal group, and B is gives solvent PBS control group, and C is gives plasminogen group.The results show that give plasminogen group Positive expression cell (arrow logo) is less than giving solvent PBS control group, and closer to solvent PBS groups to plasminogen group ratio Normal group.Illustrate that plasminogen can reduce the infiltration of neutrophil leucocyte.

PLG activity is damaged mouse and gives plasminogen pancreas islet neutrophil leucocyte immune group after 28 days in Figure 26 T1DM models Change observation result.A is blank control group, and B is gives solvent PBS control group, and C is gives plasminogen group.The results show that fibrinolytic Proenzyme group positive expression cell (arrow logo) is less than giving solvent PBS control group, and give solvent PBS groups to plasminogen group ratio Closer to blank control group.Illustrate that plasminogen plasminogen can reduce PLG activity and be damaged mouse pancreas islet in T1DM models The infiltration of neutrophil leucocyte.

Figure 27 PLG activity normal mouses given in T1DM models plasminogen after 28 days pancreas islet neutrophil leucocyte be immunized Groupization observes result.A is blank control group, and B is gives solvent PBS control group, and C is gives plasminogen group.The results show that fibre Lyase original group positive expression cell (arrow logo) is less than giving solvent PBS control group, and give solvent PBS to plasminogen group ratio The closer blank control group of group.Illustrate that plasminogen can promote PLG activity normal mouse pancreas islet neutrality grain in T1DM models thin The infiltration of born of the same parents.

PLG activity is damaged mouse and gives plasminogen pancreatic insulin immunohistochemistry sight after 28 days in Figure 28 T1DM models Examine result.A is blank control group, and B is gives solvent PBS control group, and C is gives plasminogen group.Showed by immune group result is given Plasminogen group insulin positive expression (arrow logo) is significantly more than and gives solvent PBS control group, and give to plasminogen group ratio The closer blank control group of solvent PBS groups.Illustrate that plasminogen can promote the PLG activity in T1DM models to be damaged mouse pancreas The synthesis and secretion of island element.

Figure 29 PLG activity normal mouses give plasminogen pancreatic insulin immunohistochemistry after 28 days in T1DM models Observe result.A is blank control group, and B is gives solvent PBS control group, and C is gives plasminogen group.Showed by immune group result, It is significantly more than to plasminogen group insulin positive expression (arrow logo) and gives solvent PBS control group, and give plasminogen group ratio Give solvent PBS groups closer blank control group.Illustrate that plasminogen promotes PLG activity normal mouse insulin in T1DM models Synthesis and expression.

Figure 30 PLG activity is damaged mouse and plasminogen pancreas islet NF- κ B immunohistochemistry sight after 28 days is given in T1DM models Examine result.A is blank control group, and B is gives solvent PBS control group, and C is gives plasminogen group.The results show that plasminogen The expression (arrow logo) of group NF- κ B, which is apparently higher than, gives solvent PBS control group.Illustrate plasminogen can promote inflammation repair because The expression of sub- NF- κ B, so as to promote the reparation of insulitis.

The diabetic mice of 18 week old of Figure 31 gives plasminogen pancreas islet NF- κ B immunohistochemical observation results after 35 days.A To give solvent PBS control group, B is gives plasminogen group.Experimental result is shown, to the expression (arrow of plasminogen group NF- κ B Mark) it is apparently higher than and gives solvent PBS control group.Illustrate that plasminogen can promote the expression of multidirectional Nuclear Factor kappa B, from And promote the reparation of versus young (18 week old) diabetic mice insulitis.

The diabetic mice of 26 week old of Figure 32 gives plasminogen pancreas islet NF- κ B immunohistochemical observation results after 35 days.A For Normal group, B is gives solvent PBS control group, and C is gives plasminogen group.Experimental result of the present invention is shown, to fibrinolysin The expression (arrow logo) of original group NF- κ B, which is apparently higher than, gives solvent PBS control group.Illustrate that plasminogen can promote multidirectional consideration convey The expression of factor NF- κ B is recorded, so as to promote the reparation of relatively old (26 week old) diabetic mice insulitis.

The diabetic mice of Figure 33 24-25 week old gives plasminogen pancreas islet TNF-α immunohistochemical observation knot after 31 days Fruit.A is Normal group, and B is gives solvent PBS control group, and C is gives plasminogen group.Result of study is shown, to plasminogen The positive expression (arrow logo) of group TNF-α, which is apparently higher than, gives solvent PBS control group, and give solvent PBS to plasminogen group ratio The closer Normal group of group.Illustrate that plasminogen can promote the expression of TNF-α, so as to promote 24-25 week old diabetic mices Islet damage reparation.

The diabetic mice of 26 week old of Figure 34 gives plasminogen pancreas islet TNF-α immunohistochemical observation result after 31 days.A For Normal group, B is gives solvent PBS control group, and C is gives plasminogen group.Result of study is shown, gives plasminogen group The positive expression (arrow logo) of TNF-α, which is apparently higher than, gives solvent PBS control group, and give solvent PBS groups to plasminogen group ratio Closer to Normal group.Illustrate that plasminogen can promote the expression of TNF-α, so as to promote 26 week old diabetic mice pancreas islet Injury repair.

Figure 35 shows that PLG activity is damaged in mouse T1DM models and gives plasminogen pancreas islet TNF-α immunohistochemistry after 28 days Observe result.A is gives solvent PBS control group, and B is gives plasminogen group.Result of study is shown, to plasminogen group TNF-α Positive expression (arrow logo), which is apparently higher than, gives solvent PBS control group.Illustrate that plasminogen can promote the expression of TNF-α, so as to PLG activity is promoted to be damaged islet damage reparation in mouse T1DM models.

Figure 36 shows that PLG activity is damaged T1DM model mices and gives plasminogen pancreas islet IgM immunohistochemical observations after 28 days As a result.A is blank control group, and B is gives solvent PBS control group, and C is gives plasminogen group.This experimental studies results is shown, is given The positive expression (arrow logo) of plasminogen group IgM is significantly lower than giving solvent PBS control group, and give to plasminogen group ratio The closer Normal group of solvent PBS groups.Illustrate that plasminogen can reduce the expression of IgM, so as to reduce PLG activity be damaged it is small Mouse islet damage in T1DM models.

The diabetic mice of Figure 37 24-25 week old give plasminogen after 31 days pancreas islet TUNEL dyeing observation result.A For Normal group, B is gives solvent PBS control group, and C is gives plasminogen group.This experimental result is shown, gives plasminogen group Positive cell number (arrow logo) be considerably less than give solvent PBS control group.Normal group TUNEL positive stainings are extremely low.Just Normal control group apoptosis rate is about 8%, is about 93% to solvent PBS group apoptosis rates, is about 16% to plasminogen group apoptosis rate. Illustrate that plasminogen group can substantially reduce the apoptosis of diabetic mice islet cells.

Figure 38 shows that T1DM model mices give plasminogen serum insulin testing result after 20 days.The results show that it gives Solvent PBS control group mice serum insulin concentration, which is significantly lower than, gives plasminogen group mouse, and the close notable (P of statistical discrepancy =0.08).Illustrate that plasminogen can promote the secretion of T1DM mouse islets element.

Embodiment

1 plasminogen of embodiment reduces blood glucose in diabetic mice

24-25 week old db/db male mices 8, are randomly divided into two groups, to plasminogen group 5 and to PBS pairs of solvent According to group 3.Experiment is denoted as the 0th day and grouping of weighing on the day of starting, and the 1st day starts to plasminogen or PBS, to plasminogen 2mg/0.2ml/ pcs/day of group tail vein injection people source plasminogen, to solvent PBS control group tail vein injection same volume PBS, successive administration 31 days.After fasting in the 10th, 31 day 16 hours, with blood sugar test paper (Roche, Mannheim, Germany) Carry out blood sugar test.

The results show that plasminogen group mouse blood glucose significantly lower than giving solvent PBS control group, and statistical discrepancy is shown Write (* expressions P<0.05, * * represents P<0.01).In addition, with the extension of administration time, solvent PBS control group mouse blood sugar is given There is raising trend, and continuously decreased (Fig. 1) to plasminogen group blood glucose.Illustrating that plasminogen has reduces diabetic animal blood The effect of sugar.

2 plasminogen of embodiment reduces diabetic mice fructose amine level

24-25 week old db/db male mices 5, administration every eyeball of mouse veniplex of the previous day take 50 μ l of blood to examine Serum Fructosamine concentration is surveyed, and is denoted as the 0th day, daystart gives plasminogen, successive administration 31 days.It extracts within 32nd day Eyeball takes blood, detects the concentration of Serum Fructosamine.Fructosamine concentration using fructosamine detection kit, (build up, A037- by Nanjing 2) it is detected.

Fructosamine concentration reflects the average level of blood glucose in 1~3 week.The results show that give serum fructose after plasminogen The concentration of amine is substantially reduced, and statistical difference is heteropolar significantly (Fig. 2) compared with before administration.Illustrate that plasminogen can effectively reduce glycosuria Sick animal blood glucose.

It is horizontal that 3 plasminogen of embodiment reduces by 26 week old diabetic mice Serum Fructosamines

26 week old db/db male mices 9, experiment are denoted as the 0th day and weigh, be randomly divided into according to weight on the day of starting Two groups, to plasminogen group 4, to solvent PBS control group 5.Give plasminogen group tail vein injection people source plasminogen 2mg/0.2mL/ pcs/day, to the PBS of solvent PBS control group tail vein injection same volume.Start within 1st day to plasminogen or PBS, successive administration 35 days.Mouse was put to death at the 36th day, detects the concentration of Serum Fructosamine.Fructosamine concentration uses fructosamine Detection kit (Nanjing is built up, A037-2) is detected.

Testing result is shown, is significantly lower than to the concentration of plasminogen group Serum Fructosamine and is given solvent PBS control group, united Difference is counted close to notable (P=0.06) (Fig. 3).Illustrate that plasminogen can reduce the blood glucose fructosamine of 26 week old diabetic mices.

4 plasminogen of embodiment reduces diabetic mice glycated hemoglobin levels

26 week old db/db male mices 9, experiment starts to be randomly divided into two groups according to weight after same day note is weighed, to fibre Lyase original group 4, to solvent PBS control group 5.Start to plasminogen or PBS within 1st day, noted to plasminogen group tail vein 2mg/0.2ml/ pcs/day of people source plasminogen is penetrated, to the PBS of solvent PBS control group tail vein injection same volumes, successive administration 35 days.In mouse fasting in the 35th day 16 hours, pluck eyeball and take blood within the 36th day, to detect the concentration of blood plasma glycosylated hemoglobin.

Saccharification hemoglobin content can usually reflect patient's glycemic control situation of nearly 8~12 weeks.The results show that it gives The concentration of plasminogen group mouse glycosylated hemoglobin, which is significantly lower than, gives solvent PBS control group, and statistical discrepancy is significantly (Fig. 4). Illustrate that plasminogen can effectively reduce diabetic animal blood glucose level.

5 plasminogen of embodiment improves diabetic mice sugar tolerance

26 week old db/db male mices 9 and db/m mouse 3.Experiment start the same day, db/db mouse weigh after simultaneously Two groups are randomly divided into according to weight, to plasminogen group 4 and to solvent PBS control group 5, db/m mouse are as normal right According to group.Start to plasminogen or PBS within 1st day, give plasminogen group tail vein injection people source plasminogen 2mg/0.2ml/ Pcs/day, to the PBS of solvent PBS control group tail vein injection same volume, successive administration 10 days.Mouse fasting 16 hours in 11st day Afterwards, 5% glucose solution is injected intraperitoneally by 5g/kg weight in every mouse, and blood glucose was used at 0,30,60,90,120,180 minute Test paper (Roche, Mannheim, Germany) detects blood sugar concentration.

Abdominal cavity sugar resistance to examined (Intraperitoneal glucose tolerance test, IPGTT) can detect machine Body is to the tolerance of glucose.Diabetic's sugar tolerance known in the art is to decline.

Experimental result is shown, to be less than to plasminogen group mouse blood sugar level after intraperitoneal injection glucose and be given solvent PBS Control group, and compared with to solvent PBS control group normal mouse group (Fig. 5) is more nearly to plasminogen group sugar tolerance curve. Illustrate that plasminogen can be obviously improved diabetic mice sugar tolerance.

6 plasminogen of embodiment reduces PLG activity normal mouse blood glucose level in T1DM models

9-10 week old PLG activity Normal male mice 10, is randomly divided into two groups, to solvent PBS control group and to fibre Lyase original group, every group each 5.Single intraperitoneal injection 200mg/kg streptozotocins (STZ) after two groups of mouse fasting 4 hours (sigma S0130) induces T1DM^[43].STZ starts to be administered and is denoted as administration the 1st day after injecting 12 days, gives plasminogen group tail 1mg/0.1ml/ pcs/day of intravenous injection human source fibrinolysin, to the PBS of solvent PBS control group tail vein injection same volume, continuously Administration 10 days.After mouse fasting in the 11st day 6 hours, blood is measured with blood sugar test paper (Roche, Mannheim, Germany) Sugar.

Plasminogen group mouse is given, and statistical discrepancy the results show that being apparently higher than to solvent PBS control group mouse blood sugar Extremely significantly (Fig. 6).Illustrate that plasminogen can significantly reduce the blood glucose level of PLG activity normal mouse T1DM models.

7 plasminogen of embodiment improves T1DM model mice sugar tolerance levels

9-10 week old PLG activity normal males mouse 15, is randomly divided into three groups, blank control group gives solvent PBS Control group and plasminogen group is given, every group each 5.To solvent PBS control group and to plasminogen group mouse fasting 4 hours Single intraperitoneal injection 200mg/kg STZ (sigma S0130) induce T1DM afterwards^[43], blank control group do not process.STZ is noted Start to be administered after penetrating 12 days and be denoted as administration the 1st day, give plasminogen group tail vein injection people source fibrinolysin 1mg/0.1ml/ Pcs/day, to the PBS of solvent PBS control group tail vein injection same volume, successive administration 28 days.Mouse fasting 6 hours in 28th day Afterwards, 5% glucose solution is injected intraperitoneally according to 5g/kg weight, uses blood sugar test paper within 0,15,30,60,90 minute after injection (Roche, Mannheim, Germany) detects blood sugar concentration.

Abdominal cavity sugar resistance to examined (Intraperitoneal glucose tolerance test, IPGTT) can detect machine Body is to the tolerance of glucose.Diabetic's sugar tolerance known in the art declines.

The results show that it is apparently higher than to blood sugar concentration after solvent PBS control group mouse injectable dextrose monohydrate to plasminogen Group, and compared with to solvent PBS control group, normal mouse (Fig. 7) is more nearly to plasminogen group sugar tolerance curve.Explanation Plasminogen can improve the sugared tolerance of PLG activity normal mouse T1DM models.

8 plasminogen of embodiment improves T1DM model mice breakdown of glucose abilities

9-10 week old C57 male mices 8, are randomly divided into two groups, to solvent PBS control group and give plasminogen group, Every group each 4.To solvent PBS control group and to single intraperitoneal injection 200mg/kg chains after plasminogen group mouse fasting 4 hours Urea assistant rhzomorph (STZ) (sigma S0130) induces T1DM^[43].STZ starts to be administered and is set to administration the 1st day after injecting 12 days, It is same to solvent PBS control group tail vein injection to 1mg/0.1ml/ pcs/day of plasminogen group tail vein injection people source fibrinolysin The PBS of volume.Successive administration 19 days, after mouse fasting in the 20th day 6 hours, with the glucose of 2g/kg weight gavage 20%, After sixty minutes, orbital venous plexus blood sampling and centrifuging and taking supernatant, are measured with Glucose estimation kit (Shanghai Rong Sheng 361500) Blood glucose.

Plasminogen group mouse blood sugar is given the results show that being apparently higher than to solvent PBS control group mouse blood sugar, and is counted Significant difference (P=0.04) (Fig. 8).Illustrate that plasminogen can improve T1DM mouse glucose capacities of decomposition, so as to reduce blood Sugar.

9 plasminogen of embodiment promotes diabetic mice insulin secretion function

26 week old db/db male mices 9, experiment are denoted as the 0th day on the day of starting, weigh and be randomly divided into according to weight Two groups, to plasminogen group 4, to solvent PBS control group 5.Start to plasminogen or PBS, to plasminogen within 1st day 2mg/0.2ml/ pcs/day of group tail vein injection people source plasminogen, to solvent PBS control group tail vein injection same volume PBS, successive administration 35 days.After mouse fasting in the 35th day 16 hours, eyeball was plucked at the 36th day and takes blood, centrifuging and taking supernatant uses Detection kit for insulin (Mercodia AB) detects serum insulin level according to operation instruction.

Testing result is shown, is apparently higher than to plasminogen group serum insulin level and is given solvent PBS control group, and is united Count significant difference (Fig. 9).Illustrate that plasminogen can significantly improve the secretion of diabetic mice insulin.

Protective effect of 10 plasminogen of embodiment to diabetic mice pancreas

24-25 week old db/db male mices 7, experiment are denoted as the 0th day and weigh, divided at random according to weight on the day of starting It it is two groups, to plasminogen group 4 and to solvent PBS control group 3., the 1st day starts to plasminogen or PBS, to fibrinolytic 2mg/0.2ml/ pcs/day of proenzyme group tail vein injection people source plasminogen, gives solvent PBS control group tail vein injection same volume PBS, successive administration 31 days.Mouse was put to death at the 32nd day, takes pancreas fixed in 4% paraformaldehyde.Pancreas group after fixation It knits and carries out paraffin embedding after alcohol serial dehydration and dimethylbenzene are transparent.Histotomy thickness is 3 μm, and slice dewaxing rehydration is simultaneously With haematoxylin and eosin stains (HE dyeing), the differentiation of 1% hydrochloride alcohol, ammonium hydroxide returns indigo plant, and alcohol serial dehydration mounting, is sliced In 200 and 400 times of optical microphotograph Microscopic observations.

The results show that atrophy occurs to the most pancreas islet of solvent PBS control group (Figure 10 A, 10B), the pancreas islet of atrophy is thin Born of the same parents are replaced by acinus (arrows), the acinus hyperplasia at pancreas islet edge, cause to demarcate between pancreas islet and acinus unclear；To fibrinolytic Most pancreas islet is big compared to control group area, and does not have acinus hyperplasia in pancreas islet for proenzyme group (Figure 10 C, 10D), only few The a small amount of acinus of remaining, sharpness of border between pancreas islet and acinus in several pancreas islet.The pancreas islet for comparing administration group and control group accounts for pancreas For the area of gland than finding, administration group is bigger than control group intimate one times (Figure 10 E).Illustrate that plasminogen can promote diabetic mice The reparation of islet damage prompts reparation of the plasminogen possibly through islet damage is promoted, so as to fundamentally cure glycosuria Disease.

11 plasminogen of embodiment reduces diabetic mice pancreas islet collagen deposition

24-25 week old db/db male mices 16, experiment is denoted as the 0th day and weighs on the day of starting, random according to weight It is divided into two groups, to plasminogen group 10, to solvent PBS control group 6.Start to plasminogen or PBS, to fibrinolytic within 1st day 2mg/0.2ml/ pcs/day of proenzyme group tail vein injection people source plasminogen, gives solvent PBS control group tail vein injection same volume PBS, successive administration 31 days.Mouse was put to death at the 32nd day, takes pancreas fixed in 4% paraformaldehyde.Pancreas group after fixation It knits and carries out paraffin embedding after alcohol serial dehydration and dimethylbenzene are transparent.Histotomy thickness is 3 μm, after slice dewaxing to water Washing 1 time, with 0.1% sirius red stains after sixty minutes, flowing water are rinsed, haematoxylin dyeing 1 minute, and flowing water rinses, 1% salt Indigo plant is returned in sour alcohol and ammonium hydroxide differentiation, and flowing water rinses, and mounting after drying is sliced in 200 times of optical microphotograph Microscopic observations.

Sirius red stains can be such that collagen persistently dyes, and as pathological section specific stain method, sirius red stains can Specifically to show collagen tissue.

Coloration result shows, to plasminogen group mouse (Figure 11 B) pancreas islet collagen deposition (arrow logo) significantly lower than to Solvent PBS control group (Figure 11 A), and statistical discrepancy is significantly (Figure 11 C).Illustrate that plasminogen can reduce diabetic animal pancreas islet Fibrosis.

12 plasminogen of embodiment reduces diabetic mice Intra-islet Apoptosis

24-25 week old db/db male mices 6, experiment are denoted as the 0th day and weigh, divided at random according to weight on the day of starting It it is two groups, to plasminogen group 4 and to solvent PBS control group 2.Start to plasminogen or PBS, to fibrinolysin within 1st day 2mg/0.2ml/ pcs/day of original group tail vein injection people source plasminogen, to solvent PBS control group tail vein injection same volume PBS, successive administration 31 days.Mouse was put to death at the 32nd day, takes pancreas fixed in 4% paraformaldehyde.Pancreatic tissues after fixation Paraffin embedding is carried out after alcohol serial dehydration and dimethylbenzene are transparent.Histotomy thickness is 3 μm, water after slice dewaxing rehydration It washes 1 time.With 3% dioxygen water incubation 15 minutes, wash 2 times, every time 5 minutes.5% normal sheep serum liquid (Vector Laboratories, Inc., USA) it closes 1 hour；Later, reject sheep blood serum liquid irises out tissue with PAP.Rabbit anti-mouse 4 DEG C of overnight incubations of Caspase-3 (Abcam), PBS are washed 2 times, every time 5 minutes.Goat anti-rabbit igg (HRP) antibody (Abcam) two Anti- incubation at room temperature 1 hour, PBS is washed 2 times, every time 5 minutes.By DAB kits (Vector laboratories, Inc., USA) Colour developing, haematoxylin is redyed 30 seconds after washing 3 times, and flowing water rinses 5 minutes.Serial dehydration is transparent and mounting, is sliced in 200 times of light Learn micro- Microscopic observation.

Caspase-3 is that the most important shearing of end eventually enzyme, expression get over multilist daylight in apoptosis shape in apoptosis process The cell of state is more^[44]。

Experimental result show that the expression (arrow logo) to plasminogen group (Figure 12 B) Caspase-3 is bright It shows to be less than and gives solvent PBS control group (Figure 12 A).Illustrate that plasminogen can reduce the apoptosis of islet cells.

13 plasminogen of embodiment promotes expression and secretion of 18 week old into diabetes mouse islets element

18 week old db/db male mices 8, experiment are denoted as the 0th day and weigh, be randomly divided into according to weight on the day of starting Two groups, to plasminogen group and give solvent PBS control group, every group each 4.Start to plasminogen or PBS, to fibrinolytic within 1st day 2mg/0.2ml/ pcs/day of proenzyme group tail vein injection people source plasminogen, gives solvent PBS control group tail vein injection same volume PBS, successive administration 31 days.Mouse was put to death at the 36th day, takes pancreas fixed in 4% paraformaldehyde.Pancreas group after fixation It knits and carries out paraffin embedding after alcohol serial dehydration and dimethylbenzene are transparent.Histotomy thickness is 3 μm, after slice dewaxing rehydration Washing 1 time.With 3% dioxygen water incubation 15 minutes, wash 2 times, every time 5 minutes.5% normal sheep serum liquid (Vector Laboratories, Inc., USA) it closes 1 hour；Later, reject sheep blood serum liquid irises out tissue with PAP.Rabbit anti-mouse pancreas 4 DEG C of overnight incubations of island element antibody (Abcam), PBS are washed 2 times, every time 5 minutes.Goat anti-rabbit igg (HRP) antibody (Abcam) two Anti- incubation at room temperature 1 hour, PBS is washed 2 times, every time 5 minutes.By DAB kits (Vector laboratories, Inc., USA) Colour developing, haematoxylin is redyed 30 seconds after washing 3 times, and flowing water rinses 5 minutes.Serial dehydration is transparent and mounting, and slice is under the microscope It is observed under 200 times.

The results show that the expression (arrow logo) to plasminogen group (Figure 13 B) insulin is apparently higher than and gives solvent PBS Control group (Figure 13 A), and statistical discrepancy is close to significantly (P=0.15) (Figure 13 C).Illustrate that plasminogen can promote pancreas islet work( It can repair, promote the expression and secretion of insulin.

14 plasminogen of embodiment promotes the expression and secretion of 24-25 week old diabetic mice insulin

24-25 week old db/db male mices 8, experiment are denoted as the 0th day and weigh, divided at random according to weight on the day of starting It it is two groups, to plasminogen group 5 and to solvent PBS control group 3.Start to plasminogen or PBS, to fibrinolysin within 1st day 2mg/0.2ml/ pcs/day of original group tail vein injection people source plasminogen, to solvent PBS control group tail vein injection same volume PBS, successive administration 31 days.Mouse was put to death at the 32nd day, takes pancreas fixed in 4% paraformaldehyde.Pancreatic tissues after fixation Paraffin embedding is carried out after alcohol serial dehydration and dimethylbenzene are transparent.Histotomy thickness is 3 μm, water after slice dewaxing rehydration It washes 1 time.With 3% dioxygen water incubation 15 minutes, wash 2 times, every time 5 minutes.5% normal sheep serum liquid (Vector Laboratories, Inc., USA) it closes 1 hour；Later, reject sheep blood serum liquid irises out tissue with PAP.Rabbit anti-mouse pancreas 4 DEG C of overnight incubations of island element antibody (Abcam), PBS are washed 2 times, every time 5 minutes.Goat anti-rabbit igg (HRP) antibody (Abcam) two Anti- incubation at room temperature 1 hour, PBS is washed 2 times, every time 5 minutes.By DAB kits (Vector laboratories, Inc., USA) Colour developing, haematoxylin is redyed 30 seconds after washing 3 times, and flowing water rinses 5 minutes.Serial dehydration is transparent and mounting, and slice is under the microscope It is observed under 200 times.

The results show that the expression (arrow logo) to plasminogen group insulin is apparently higher than and gives solvent PBS control group, And statistical discrepancy is significantly (P=0.02) (Figure 14).Illustrate that plasminogen can effectively repair islet function, promote the table of insulin It reaches and secretes.

15 plasminogen of embodiment promotes the reparation of diabetic mice insulin synthesis secreting function

26 week old db/db male mices 9, experiment are denoted as the 0th day and weigh, be randomly divided into according to weight on the day of starting Two groups, solvent PBS control group 5 is given for 4 to plasminogen group.Start within 1st day, to plasminogen or PBS, to give plasminogen group 2mg/0.2ml/ pcs/day of tail vein injection people source plasminogen, to the PBS of solvent PBS control group tail vein injection same volume, Successive administration 35 days.After mouse fasting in the 35th day 16 hours, mouse was put to death at the 36th day, takes pancreas in 4% paraformaldehyde It is fixed.Pancreatic tissues after fixation carry out paraffin embedding after alcohol serial dehydration and dimethylbenzene are transparent.Histotomy thickness is It 3 μm, is washed 1 time after being sliced dewaxing rehydration.With 3% dioxygen water incubation 15 minutes, wash 2 times, every time 5 minutes.5% it is normal Sheep blood serum liquid (Vector laboratories, Inc., USA) is closed 1 hour；Later, reject sheep blood serum liquid, with PAP circles Go out tissue.4 DEG C of overnight incubations of rabbit anti-mouse insulin antibody (Abcam), PBS are washed 2 times, every time 5 minutes.Goat anti-rabbit igg (HRP) antibody (Abcam) secondary antibody is incubated at room temperature 1 hour, and PBS is washed 2 times, every time 5 minutes.By DAB kits (Vector Laboratories, Inc., USA) colour developing, haematoxylin is redyed 30 seconds after washing 3 times, and flowing water rinses 5 minutes.Serial dehydration is saturating Bright and mounting, slice are observed under 200 times under the microscope.

The results show that the expression (arrow logo) to plasminogen group insulin is apparently higher than and gives solvent PBS control group, And statistical difference is heteropolar significantly (P=0.005) (Figure 15).Illustrate that plasminogen can effectively repair diabetic mice islet function, improve The expression and secretion of insulin.

16 plasminogen of embodiment promotes the expression of the multidirectional nuclear factor NF- κ B of 24-25 week old diabetic mice pancreas islet

24-25 week old db/db male mices 10, experiment is denoted as the 0th day and weighs on the day of starting, random according to weight It is divided into two groups, to plasminogen group 4 and to solvent PBS control group 6, separately takes 4 db/m as Normal group, normally Control group does not process.Start to plasminogen or PBS within 1st day, give plasminogen group tail vein injection people source plasminogen 2mg/0.2ml/ pcs/day, to the PBS of solvent PBS control group tail vein injection same volumes, successive administration 31 days.At the 32nd day Dead mouse takes pancreas fixed in 4% paraformaldehyde.Pancreatic tissues after fixation are transparent through alcohol serial dehydration and dimethylbenzene After carry out paraffin embedding.Histotomy thickness is 3 μm, is washed 1 time after slice dewaxing rehydration.Divided with 3% dioxygen water incubation 15 Clock is washed 2 times, every time 5 minutes.5% normal sheep serum liquid (Vector laboratories, Inc., USA) closing 1 is small When；Later, reject sheep blood serum liquid irises out tissue with PAP.4 DEG C of overnight incubations of rabbit anti-mouse NF- κ B (Abcam), PBS wash 2 It is secondary, 5 minutes every time.Goat anti-rabbit igg (HRP) antibody (Abcam) secondary antibody is incubated at room temperature 1 hour, and PBS is washed 2 times, every time 5 points Clock.It develops the color by DAB kits (Vector laboratories, Inc., USA), haematoxylin is redyed 30 seconds after washing 3 times, is flowed Water rinses 5 minutes.Serial dehydration is transparent and mounting, and slice is observed under 200 times under the microscope.

NF- κ B are transcription factor protein family member, are played an important role in inflammation repair process^[45]。

Experimental result of the present invention is shown, is apparently higher than to the expression (arrow logo) of plasminogen group NF- κ B to solvent PBS control group, and statistical discrepancy is significantly (Figure 16).Illustrate that plasminogen can promote the expression of multidirectional Nuclear Factor kappa B.

17 plasminogen of embodiment reduces the proliferation of 18 week old diabetic mice alpha Cell of islet, restores alpha Cell of islet Normal distribution and the secretion for reducing glucagon

18 week old db/db male mices 8 and db/m male mices 3, experiment are denoted as the 0th day and claim on the day of starting Weight, db/db mouse are randomly divided into two groups according to weight, to plasminogen group and give solvent PBS control group, every group each 4, db/ M mouse are as Normal group.Start to plasminogen or PBS within 1st day.Give plasminogen group tail vein injection people source fibrinolytic 2mg/0.2ml/ pcs/day of proenzyme, to the PBS of solvent PBS control group tail vein injection same volume, successive administration 35 days.The 36th It puts to death mouse, takes pancreas fixed in 4% paraformaldehyde.Pancreatic tissues after fixation are through alcohol serial dehydration and dimethylbenzene Paraffin embedding is carried out after transparent.Histotomy thickness is 3 μm, is washed 1 time after slice dewaxing rehydration.PAP are irised out tissue, with 3% dioxygen water incubation 15 minutes, 0.01MPBS are washed 2 times, every time 5 minutes.5% normal sheep serum liquid (Vector Laboratories, Inc., USA) it closes 30 minutes；After time arrives, rabbit anti-mouse pancreas hyperglycemia is added dropwise in reject sheep blood serum liquid Plain 4 DEG C of overnight incubations of antibody (Abcam), 0.01M PBS are washed 2 times, every time 5 minutes.Goat anti-rabbit igg (HRP) antibody (Abcam) secondary antibody is incubated at room temperature 1 hour, and 0.01M PBS are washed 2 times, every time 5 minutes.By DAB kits (Vector Laboratories, Inc., USA) colour developing, haematoxylin is redyed 30 seconds after washing 3 times, and flowing water rinses 5 minutes.Graded ethanol takes off Water, dimethylbenzene is transparent and neutral gum mounting, is sliced in 200 times of optical microphotograph Microscopic observations.

Alpha Cell of islet synthesis secretion glucagon, is mainly dispersed in and is distributed in pancreas islet neighboring area.

The results show that compared with to plasminogen group (Figure 17 C), solvent PBS control group (Figure 17 B) glucagon is given Positive cell (arrow logo) showed increased, positive cell infiltrate the middle section to pancreas islet, and average optical density quantitative analysis As a result having significant difference, (* * represent P<0.01) (Figure 17 D)；It is dispersed in plasminogen group glucagon positive cell Pancreas islet periphery is distributed in, gives solvent PBS groups closer Normal group (17A) to the pancreas islet form ratio of plasminogen group.It says Bright plasminogen can significantly inhibit the secretion of 18 week old diabetic mice alpha Cell of islet proliferation and glucagon, correct pancreas Island α cell distributions are disorderly, and plasminogen is prompted to promote the reparation of islet damage.

18 plasminogen of embodiment reduces the proliferation of 24-25 week old diabetic mice alpha Cell of islet, restores alpha Cell of islet Normal distribution and reduce glucagon secretion

24-25 week old db/db male mices 11 and db/m male mices 5, experiment are denoted as the 0th day on the day of starting And weigh, db/db mouse are randomly divided into two groups after weighing, to plasminogen group 5, to solvent PBS control group 6, db/m is small Mouse is as Normal group.Start to plasminogen or PBS within 1st day.Give plasminogen group tail vein injection people source plasminogen , PBS to solvent PBS control group tail vein injection same volume or any liquid is not injected, continuously give by 2mg/0.2ml/ pcs/day Medicine 31 days.Mouse was put to death at the 32nd day, takes pancreas fixed in 4% paraformaldehyde.Pancreatic tissues after fixation are through alcohol ladder Paraffin embedding is carried out after degree dehydration and dimethylbenzene are transparent.Histotomy thickness is 3 μm, is washed 1 time after slice dewaxing rehydration.PAP Pen irises out tissue, and with 3% dioxygen water incubation 15 minutes, 0.01MPBS was washed 2 times, every time 5 minutes.5% normal sheep serum liquid (Vector laboratories, Inc., USA) is closed 30 minutes；After time arrives, rabbit anti-mouse is added dropwise in reject sheep blood serum liquid 4 DEG C of overnight incubations of glucagon antibody (Abcam), 0.01M PBS are washed 2 times, every time 5 minutes.Goat anti-rabbit igg (HRP) is anti- Body (Abcam) secondary antibody is incubated at room temperature 1 hour, and 0.01M PBS are washed 2 times, every time 5 minutes.By DAB kits (Vector Laboratories, Inc., USA) colour developing, haematoxylin is redyed 30 seconds after washing 3 times, and flowing water rinses 5 minutes.Graded ethanol takes off Water, dimethylbenzene is transparent and neutral gum mounting, is sliced in 200 times of optical microphotograph Microscopic observations.

Alpha Cell of islet synthesis secretion glucagon, is mainly dispersed in and is distributed in pancreas islet neighboring area.

The results show that with more positive than giving solvent PBS control group (Figure 18 B) glucagon to plasminogen group phase (Figure 18 C) Property cell (arrow logo) showed increased, positive cell infiltrate to pancreas islet middle section；Give plasminogen group glucagon What positive cell was dispersed in is distributed in pancreas islet periphery, closer normal right to solvent PBS groups to the pancreas islet form ratio of plasminogen group According to group (18A).Illustrate that plasminogen can significantly inhibit 24-25 week old diabetic mices alpha Cell of islet proliferation and pancreas hyperglycemia Alpha Cell of islet distribution disorders are corrected in the secretion of element, prompt plasminogen that can promote the reparation of islet damage.

19 plasminogen of embodiment inhibits the proliferation of 26 week old diabetic mice alpha Cell of islet, restores alpha Cell of islet Normal distribution and the secretion for reducing glucagon

26 week old db/db male mices 9 and db/m male mices 3, experiment are denoted as the 0th day and claim on the day of starting Weight, db/db mouse are randomly divided into two groups after weighing, to plasminogen group 4, to solvent PBS control group 5, db/m mouse make For Normal group.Start to plasminogen or PBS within 1st day.Give plasminogen group tail vein injection people source plasminogen 2mg/ 0.2ml/ pcs/day, to the PBS of solvent PBS control group tail vein injection same volume, successive administration 35 days.It was put to death at the 36th day small Mouse takes pancreas fixed in 4% paraformaldehyde.Pancreatic tissues after fixation are transparent laggard through alcohol serial dehydration and dimethylbenzene Row paraffin embedding.Histotomy thickness is 3 μm, is washed 1 time after slice dewaxing rehydration.PAP are irised out tissue, with 3% hydrogen peroxide It is incubated 15 minutes, 0.01MPBS is washed 2 times, every time 5 minutes.5% normal sheep serum liquid (Vector laboratories, Inc., USA) it closes 30 minutes；After time arrives, rabbit anti-mouse glucagon antibody (Abcam) 4 is added dropwise in reject sheep blood serum liquid It DEG C is incubated overnight, 0.01M PBS wash 2 times, every time 5 minutes.Goat anti-rabbit igg (HRP) antibody (Abcam) secondary antibody is incubated at room temperature 1 hour, 0.01M PBS were washed 2 times, every time 5 minutes.It is aobvious by DAB kits (Vector laboratories, Inc., USA) Color, haematoxylin is redyed 30 seconds after washing 3 times, and flowing water rinses 5 minutes.Gradient alcohol dehydration, dimethylbenzene is transparent and neutral gum seals Piece is sliced in 200 times of optical microphotograph Microscopic observations.

Alpha Cell of islet synthesis secretion glucagon, is mainly dispersed in and is distributed in pancreas islet neighboring area.

The results show that with more positive than giving solvent PBS control group (Figure 19 B) glucagon to plasminogen group phase (Figure 19 C) Property cell (arrow logo) showed increased, positive cell infiltrates the middle section to pancreas islet, and average optical density quantitative analysis knot Fruit has significant difference, and (* * represent P<0.01) (Figure 19 D)；Point being dispersed in plasminogen group glucagon positive cell Pancreas islet periphery is distributed in, gives solvent PBS groups closer Normal group (19A) to the pancreas islet form ratio of plasminogen group.Explanation Plasminogen can significantly inhibit the secretion of 26 week old diabetic mice alpha Cell of islet proliferation and glucagon, correct pancreas islet α cell distributions are disorderly, prompt plasminogen that can promote the reparation of islet damage.

20 plasminogen of embodiment reduces the secretion of glucagon in PLG activity normal mouse T1DM models

9-10 week old PLG activity normal males mouse 15, is randomly divided into three groups, blank control group gives solvent PBS Control group and plasminogen group is given, every group each 5.To solvent PBS control group and to plasminogen group mouse fasting 4 hours Single intraperitoneal injection 200mg/kg STZ (Sigma, article No. S0130) induce T1DM models afterwards^[43], blank control group, which is not done, to be located Reason.Injection starts to be administered and is set to administration the 1st day after 12 days, gives plasminogen group tail vein injection people source fibrinolysin 1mg/ 0.1ml/ pcs/day, to the PBS of solvent PBS control group tail vein injection same volume, successive administration 28 days.It was put to death at the 29th day Mouse takes pancreas fixed in 4% paraformaldehyde.Pancreatic tissues after fixation are after alcohol serial dehydration and dimethylbenzene are transparent Carry out paraffin embedding.Histotomy thickness is 3 μm, is washed 1 time after slice dewaxing rehydration.PAP are irised out tissue, with 3% dioxygen Water incubation 15 minutes, 0.01MPBS are washed 2 times, every time 5 minutes.5% normal sheep serum liquid (Vector laboratories, Inc., USA) it closes 30 minutes；After time arrives, rabbit anti-mouse glucagon antibody (Abcam) 4 is added dropwise in reject sheep blood serum liquid It DEG C is incubated overnight, 0.01M PBS wash 2 times, every time 5 minutes.Goat anti-rabbit igg (HRP) antibody (Abcam) secondary antibody incubation at room temperature 1 Hour, 0.01M PBS are washed 2 times, every time 5 minutes.It develops the color by DAB kits (Vector laboratories, Inc., USA), Haematoxylin is redyed 30 seconds after washing 3 times, and flowing water rinses 5 minutes.Gradient alcohol dehydration, dimethylbenzene is transparent and neutral gum seals Piece is sliced in 200 times of optical microphotograph Microscopic observations.

Alpha Cell of islet synthesis secretion glucagon, is distributed mainly on pancreas islet neighboring area.

The results show that it is significantly more than to solvent PBS control group (Figure 20 B) glucagon positive expression to plasminogen Group (Figure 20 C), and average optical density quantitative analysis results statistical discrepancy is significantly (Figure 20 D), and to plasminogen group ratio to solvent The closer blank control group (20A) of PBS groups.Illustrate that plasminogen can substantially reduce the diabetic mice pancreatic islet alpha of STZ inductions Cells secrete glucagon.

21 plasminogen of embodiment promotes the expression of 18 week old diabetic mice pancreatic insulin receptor substrates 2 (IRS-2)

18 week old db/db male mices 7 and db/m male mices 3, experiment are denoted as the 0th day and claim on the day of starting Weight, db/db mouse are randomly divided into two groups according to weight, to plasminogen group 3, to solvent PBS control group 4, db/m mouse As Normal group.Start to plasminogen or PBS within 1st day.Give plasminogen group tail vein injection people source plasminogen 2mg/0.2ml/ pcs/day, to the PBS of solvent PBS control group tail vein injection same volume, successive administration 35 days.At the 36th day Dead mouse takes pancreas fixed in 4% paraformaldehyde.Pancreatic tissues after fixation are transparent through alcohol serial dehydration and dimethylbenzene After carry out paraffin embedding.Histotomy thickness is 3 μm, is washed 1 time after slice dewaxing rehydration.PAP are irised out tissue, with 3% pair Oxygen water incubation 15 minutes, 0.01MPBS are washed 2 times, every time 5 minutes.5% normal sheep serum liquid (Vector Laboratories, Inc., USA) it closes 30 minutes；After time arrives, rabbit anti-mouse IRS-2 antibody is added dropwise in reject sheep blood serum liquid (Abcam) 4 DEG C of overnight incubations, 0.01M PBS are washed 2 times, every time 5 minutes.Goat anti-rabbit igg (HRP) antibody (Abcam) secondary antibody Incubation at room temperature 1 hour, 0.01M PBS are washed 2 times, every time 5 minutes.By DAB kits (Vector laboratories, Inc., USA it) develops the color, haematoxylin is redyed 30 seconds after washing 3 times, and flowing water rinses 5 minutes.Gradient alcohol dehydration, dimethylbenzene are transparent and neutral Gummy mounting is sliced in 200 times of optical microphotograph Microscopic observations.

((Insulin Receptor Substrate-2, IRS-2) is that one kind can be activated to insulin receptor substrate2 Insulin receptor tyrosine kinase effect substrate, be important molecule in insulin signal transduction approach, and thin to pancreas islet β The existence of born of the same parents is extremely important.IRS-2 when beta Cell of islet is expressed and is increased to it with protective effect, and to functional islets β The maintenance of cell is most important^[46,47]。

IRS-2 Showed by immune group result gives solvent PBS control group mouse (Figure 21 B) pancreas islet IRS-2 positive expression (arrows Leader is known) it is considerably less than and gives plasminogen group (Figure 21 C), and statistical difference is heteropolar significantly (Figure 21 D), and gives plasminogen group ratio Give solvent PBS groups closer blank control group (21A).Illustrate that plasminogen can be effectively increased 18 week old diabetic mice pancreas islet The expression of cell IRS-2.

22 plasminogen of embodiment promotes the expression of 24-25 week old diabetic mice pancreas islet IRS-2

24-25 week old db/db male mices 11 and db/m male mices 5, experiment are denoted as the 0th day on the day of starting And weigh, db/db mouse are randomly divided into two groups according to weight, to plasminogen group 5, to solvent PBS control group 6, db/m Mouse is as Normal group.Start to plasminogen or PBS within 1st day.Give plasminogen group tail vein injection people source fibrinolysin , PBS to solvent PBS control group tail vein injection same volume or any liquid is not injected, continuously by former 2mg/0.2ml/ pcs/day Administration 31 days.Mouse was put to death at the 32nd day, takes pancreas fixed in 4% paraformaldehyde.Pancreatic tissues after fixation are through alcohol Paraffin embedding is carried out after serial dehydration and dimethylbenzene are transparent.Histotomy thickness is 3 μm, is washed 1 time after slice dewaxing rehydration. PAP are irised out tissue, and with 3% dioxygen water incubation 15 minutes, 0.01MPBS was washed 2 times, every time 5 minutes.5% normal sheep serum Liquid (Vector laboratories, Inc., USA) is closed 30 minutes；After time arrives, it is small that rabbit-anti is added dropwise in reject sheep blood serum liquid 4 DEG C of overnight incubations of mouse IRS-2 antibody (Abcam), 0.01M PBS are washed 2 times, every time 5 minutes.Goat anti-rabbit igg (HRP) antibody (Abcam) secondary antibody is incubated at room temperature 1 hour, and 0.01M PBS are washed 2 times, every time 5 minutes.By DAB kits (Vector Laboratories, Inc., USA) colour developing, haematoxylin is redyed 30 seconds after washing 3 times, and flowing water rinses 5 minutes.Graded ethanol takes off Water, dimethylbenzene is transparent and neutral gum mounting, is sliced in 200 times of optical microphotograph Microscopic observations.

IRS-2 Showed by immune group result gives solvent PBS control group mouse (Figure 22 B) pancreas islet IRS-2 positive expression (arrows Leader is known) it is considerably less than and gives plasminogen group (Figure 22 C), and statistical discrepancy is significantly (Figure 22 D), and is given to plasminogen group ratio The closer Normal group (22A) of solvent PBS groups.Illustrate that plasminogen can be effectively increased 24-25 week old diabetic mice pancreas islet The expression of cell IRS-2.

23 plasminogen of embodiment promotes the expression of 26 week old diabetic mice pancreas islet IRS-2

26 week old db/db male mices 9 and db/m male mices 3, experiment are denoted as the 0th day and claim on the day of starting Weight, db/db mouse are randomly divided into two groups according to weight, to plasminogen group 4, to solvent PBS control group 5, db/m mouse As Normal group.Start to plasminogen or PBS within 1st day.Give plasminogen group tail vein injection people source plasminogen 2mg/0.2ml/ pcs/day, to the PBS of solvent PBS control group tail vein injection same volume, successive administration 35 days.At the 36th day Dead mouse takes pancreas fixed in 4% paraformaldehyde.Pancreatic tissues after fixation are transparent through alcohol serial dehydration and dimethylbenzene After carry out paraffin embedding.Histotomy thickness is 3 μm, is washed 1 time after slice dewaxing rehydration.PAP are irised out tissue, with 3% pair Oxygen water incubation 15 minutes, 0.01MPBS are washed 2 times, every time 5 minutes.5% normal sheep serum liquid (Vector Laboratories, Inc., USA) it closes 30 minutes；After time arrives, rabbit anti-mouse IRS-2 antibody is added dropwise in reject sheep blood serum liquid (Abcam) 4 DEG C of overnight incubations, 0.01M PBS are washed 2 times, every time 5 minutes.Goat anti-rabbit igg (HRP) antibody (Abcam) secondary antibody Incubation at room temperature 1 hour, 0.01M PBS are washed 2 times, every time 5 minutes.By DAB kits (Vector laboratories, Inc., USA it) develops the color, haematoxylin is redyed 30 seconds after washing 3 times, and flowing water rinses 5 minutes.Gradient alcohol dehydration, dimethylbenzene are transparent and neutral Gummy mounting is sliced in 200 times of optical microphotograph Microscopic observations.

IRS-2 Showed by immune group result gives solvent PBS control group mouse (Figure 23 B) pancreas islet IRS-2 positive expression (arrows Leader is known) it is considerably less than and gives plasminogen group (Figure 23 C)；It is small close to Normal group to plasminogen group IRS-2 expressions Mouse (Figure 23 A).Illustrate that plasminogen can be effectively increased the expression of 26 week old diabetic mice islet cells IRS-2.

24 plasminogen of embodiment promotes the expression of the normal T1DM mouse islets IRS-2 of PLG activity

9-10 week old PLG activity normal males mouse 15, is randomly divided into three groups, blank control group gives solvent PBS Control group and plasminogen group is given, every group each 5.To solvent PBS control group and to plasminogen group mouse fasting 4 hours Single intraperitoneal injection 200mg/kg STZ (Sigma, article No. S0130) inducing type I diabetes afterwards^[43], blank control group, which is not done, to be located Reason.Injection starts to be administered and is set to administration the 1st day after 12 days, gives plasminogen group tail vein injection people source fibrinolysin 1mg/ 0.1ml/ pcs/day, to the PBS of solvent PBS control group tail vein injection same volume, successive administration 28 days.It was put to death at the 29th day Mouse takes pancreas fixed in 4% paraformaldehyde.Pancreatic tissues after fixation are after alcohol serial dehydration and dimethylbenzene are transparent Carry out paraffin embedding.Histotomy thickness is 3 μm, is washed 1 time after slice dewaxing rehydration.PAP are irised out tissue, with 3% dioxygen Water incubation 15 minutes, 0.01MPBS are washed 2 times, every time 5 minutes.5% normal sheep serum liquid (Vector laboratories, Inc., USA) it closes 30 minutes；After time arrives, reject sheep blood serum liquid is added dropwise 4 DEG C of rabbit anti-mouse IRS-2 antibody (Abcam) and incubates It educates overnight, 0.01M PBS are washed 2 times, every time 5 minutes.The incubation at room temperature 1 of goat anti-rabbit igg (HRP) antibody (Abcam) secondary antibody is small When, 0.01M PBS are washed 2 times, every time 5 minutes.It develops the color by DAB kits (Vector laboratories, Inc., USA), water Haematoxylin is redyed 30 seconds after washing 3 times, and flowing water rinses 5 minutes.Gradient alcohol dehydration, dimethylbenzene is transparent and neutral gum mounting, cuts Piece is in 200 times of optical microphotograph Microscopic observations.

IRS-2 Showed by immune group result gives solvent PBS control group mouse (Figure 24 B) pancreas islet IRS-2 positive expression (arrows Leader is known) it is considerably less than and gives plasminogen group (Figure 24 C), and give solvent PBS groups closer blank control to plasminogen group ratio Group (24A).Illustrate that plasminogen can be effectively increased the expression of 9-10 week old PLG activity normal mouse islet cells IRS-2.

25 plasminogen of embodiment reduces the infiltration of 24-26 week old diabetic mice pancreas islet neutrophil leucocytes

24-26 week old db/db male mices 9 and db/m mouse 3, db/db mouse are randomly divided into two groups, to fibre Lyase original group 4, to solvent PBS control group 5, db/m mouse are as Normal group.Experiment is denoted as the 0th day on the day of starting It weighs grouping, experiment starts to plasminogen or PBS and be denoted as the 1st day for second day.Give plasminogen group tail vein injection people source 2mg/0.2ml/ pcs/day of plasminogen, to the PBS of solvent PBS control group tail vein injection same volume, successive administration 35 days. It puts to death mouse within 36th day, takes pancreas fixed in 4% paraformaldehyde.Pancreatic tissues after fixation are through alcohol serial dehydration and two Paraffin embedding is carried out after toluene is transparent.Histotomy thickness is 3 μm, is washed 1 time after slice dewaxing rehydration.PAP are irised out group It knits, with 3% dioxygen water incubation 15 minutes, 0.01MPBS was washed 2 times, every time 5 minutes.5% normal sheep serum liquid (Vector Laboratories, Inc., USA) it closes 30 minutes；After time arrives, it is thin that rabbit anti-mouse neutrality grain is added dropwise in reject sheep blood serum liquid 4 DEG C of overnight incubations of born of the same parents' antibody (Abcam), 0.01M PBS are washed 2 times, every time 5 minutes.Goat anti-rabbit igg (HRP) antibody (Abcam) secondary antibody is incubated at room temperature 1 hour, and 0.01M PBS are washed 2 times, every time 5 minutes.By DAB kits (Vector Laboratories, Inc., USA) colour developing, haematoxylin is redyed 30 seconds after washing 3 times, and flowing water rinses 5 minutes.Graded ethanol takes off Water, dimethylbenzene is transparent and neutral gum mounting, is sliced in 200 times of optical microphotograph Microscopic observations.

Center granulocyte is important member in non-specific cellular immunity system, and when inflammation occurs, they are by chemotaxis Substance is attracted to inflammation part.

Centriole cellular immunity group to plasminogen group (Figure 25 C) positive expression cell the results show that be less than to solvent PBS control group (Figure 25 B), and give solvent PBS groups closer Normal group (25A) to plasminogen group ratio.

26 plasminogen of embodiment reduces the infiltration that PLG activity is damaged mouse pancreas islet neutrophil leucocyte in T1DM models

9-10 week old PLG activity is damaged male mice 10, is randomly divided into three groups, blank control group 3, to PBS control Group 3, to plasminogen group 4.It is noted to solvent PBS control group and to single abdominal cavity after plasminogen group mouse fasting 4 hours Penetrate 200mg/kg STZ (sigma S0130) induction I patients with type Ⅰ DM^[43], blank control group do not process.Injection is opened after 12 days Begin to be administered and be set to administration the 1st day, to 1mg/0.1ml/ pcs/day of plasminogen group tail vein injection people source fibrinolysin, to solvent The PBS of PBS control group tail vein injection same volume, successive administration 28 days.Mouse was put to death at the 29th day, takes pancreas more than 4% It is fixed in polyformaldehyde.Pancreatic tissues after fixation carry out paraffin embedding after alcohol serial dehydration and dimethylbenzene are transparent.Tissue is cut Piece thickness is 3 μm, is washed 1 time after slice dewaxing rehydration.PAP are irised out tissue, with 3% dioxygen water incubation 15 minutes, 0.01MPBS is washed 2 times, every time 5 minutes.5% normal sheep serum liquid (Vector laboratories, Inc., USA) closing 30 minutes；After time arrives, 4 DEG C of overnight incubations of rabbit anti-mouse Antineutrophil antibody (Abcam) are added dropwise in reject sheep blood serum liquid, 0.01M PBS are washed 2 times, every time 5 minutes.Goat anti-rabbit igg (HRP) antibody (Abcam) secondary antibody is incubated at room temperature 1 hour, 0.01M PBS is washed 2 times, every time 5 minutes.It develops the color by DAB kits (Vector laboratories, Inc., USA), revives after washing 3 times Lignin is redyed 30 seconds, and flowing water rinses 5 minutes.Gradient alcohol dehydration, dimethylbenzene is transparent and neutral gum mounting, is sliced at 400 times Optical microphotograph Microscopic observation.

Centriole cellular immunity group is the results show that give plasminogen group (Figure 26 C) positive expression cell (arrow logo) Less than giving solvent PBS control group (Figure 26 B), and solvent PBS groups are given closer to blank control group (26A) to plasminogen group ratio.

27 plasminogen of embodiment reduces the infiltration of PLG activity normal mouse pancreas islet neutrophil leucocyte in T1DM models

9-10 week old PLG activity normal males mouse 11, is randomly divided into three groups, blank control group 3, to solvent PBS control group 4, to plasminogen group 4.To solvent PBS control group and to single after plasminogen group mouse fasting 4 hours Secondary intraperitoneal injection 200mg/kg STZ (sigma S0130) inducing type I diabetes^[43], blank control group do not process.Injection 12 Start to be administered and be set to administration the 1st day after it, to 1mg/0.1ml/ pcs/day of plasminogen group tail vein injection people source fibrinolysin, To the PBS of solvent PBS control group tail vein injection same volume, successive administration 28 days.Mouse was put to death at the 29th day, pancreas is taken to exist It is fixed in 4% paraformaldehyde.Pancreatic tissues after fixation carry out paraffin embedding after alcohol serial dehydration and dimethylbenzene are transparent. Histotomy thickness is 3 μm, is washed 1 time after slice dewaxing rehydration.PAP are irised out tissue, with 3% dioxygen water incubation 15 minutes, 0.01MPBS is washed 2 times, every time 5 minutes.5% normal sheep serum liquid (Vector laboratories, Inc., USA) closing 30 minutes；After time arrives, 4 DEG C of overnight incubations of rabbit anti-mouse centriole cell antibody (Abcam) are added dropwise in reject sheep blood serum liquid, 0.01M PBS are washed 2 times, every time 5 minutes.Goat anti-rabbit igg (HRP) antibody (Abcam) secondary antibody is incubated at room temperature 1 hour, 0.01M PBS is washed 2 times, every time 5 minutes.It develops the color by DAB kits (Vector laboratories, Inc., USA), revives after washing 3 times Lignin is redyed 30 seconds, and flowing water rinses 5 minutes.Gradient alcohol dehydration, dimethylbenzene is transparent and neutral gum mounting, is sliced 400 Times optical microphotograph Microscopic observation.

Centriole cellular immunity group is the results show that give plasminogen group (Figure 27 C) positive expression cell (arrow logo) Less than giving solvent PBS control group (Figure 27 B), and solvent PBS groups are given closer to blank control group (27A) to plasminogen group ratio.

PLG activity of 28 lyase original of the embodiment promotion in T1DM models is damaged the synthesis and secretion of mouse islets element

9-10 week old PLG activity is damaged male mouse 10, is randomly divided into three groups, blank control group 3, to PBS pairs According to group 3, to plasminogen group 4.To solvent PBS control group and to single abdominal cavity after plasminogen group mouse fasting 4 hours Inject 200mg/kg STZ (sigma S0130) inducing type I diabetes^[43], blank control group do not process.After injection 12 days Start to be administered and be set to administration the 1st day, to 1mg/0.1ml/ pcs/day of plasminogen group tail vein injection people source fibrinolysin, to molten The PBS of matchmaker's PBS control group tail vein injection same volume, successive administration 28 days.Mouse was put to death at the 29th day, takes pancreas 4% It is fixed in paraformaldehyde.Pancreatic tissues after fixation carry out paraffin embedding after alcohol serial dehydration and dimethylbenzene are transparent.Tissue Slice thickness is 3 μm, is washed 1 time after slice dewaxing rehydration.PAP are irised out tissue, with 3% dioxygen water incubation 15 minutes, 0.01MPBS is washed 2 times, every time 5 minutes.5% normal sheep serum liquid (Vector laboratories, Inc., USA) closing 30 minutes；After time arrives, 4 DEG C of overnight incubations of rabbit anti-mouse insulin antibody (Abcam), 0.01M is added dropwise in reject sheep blood serum liquid PBS is washed 2 times, every time 5 minutes.Goat anti-rabbit igg (HRP) antibody (Abcam) secondary antibody is incubated at room temperature 1 hour, and 0.01M PBS wash 2 It is secondary, 5 minutes every time.It develops the color by DAB kits (Vector laboratories, Inc., USA), haematoxylin is answered after washing 3 times Dye 30 seconds, flowing water rinse 5 minutes.Gradient alcohol dehydration, dimethylbenzene is transparent and neutral gum mounting, is sliced and is shown in 200 times of optics Micro- Microscopic observation.

Showed by immune group result is significantly more than to plasminogen group (Figure 28 C) insulin positive expression (arrow logo) Give solvent PBS control group (Figure 28 B, and give solvent PBS groups closer blank control group (28A) to plasminogen group ratio).Explanation Plasminogen can promote the PLG activity in T1DM models to be damaged the synthesis and secretion of mouse islets element.

29 plasminogen of embodiment promotes the synthesis and expression of PLG activity normal mouse insulin in T1DM models

9-10 week old PLG activity normal males mouse 11, is randomly divided into three groups, blank control group 3, to solvent PBS control group 4, to plasminogen group 4.To solvent PBS control group and to single after plasminogen group mouse fasting 4 hours Secondary intraperitoneal injection 200mg/kg STZ (sigma S0130) inducing type I diabetes^[43], blank control group do not process.Injection 12 Start to be administered and be set to administration the 1st day after it, to 1mg/0.1ml/ pcs/day of plasminogen group tail vein injection people source fibrinolysin, To the PBS of solvent PBS control group tail vein injection same volume, successive administration 28 days.Mouse was put to death at the 29th day, pancreas is taken to exist It is fixed in 4% paraformaldehyde.Pancreatic tissues after fixation carry out paraffin embedding after alcohol serial dehydration and dimethylbenzene are transparent. Histotomy thickness is 3 μm, is washed 1 time after slice dewaxing rehydration.PAP are irised out tissue, with 3% dioxygen water incubation 15 minutes, 0.01MPBS is washed 2 times, every time 5 minutes.5% normal sheep serum liquid (Vector laboratories, Inc., USA) closing 30 minutes；After time arrives, 4 DEG C of overnight incubations of rabbit anti-mouse insulin antibody (Abcam), 0.01M is added dropwise in reject sheep blood serum liquid PBS is washed 2 times, every time 5 minutes.Goat anti-rabbit igg (HRP) antibody (Abcam) secondary antibody is incubated at room temperature 1 hour, and 0.01M PBS wash 2 It is secondary, 5 minutes every time.It develops the color by DAB kits (Vector laboratories, Inc., USA), haematoxylin is answered after washing 3 times Dye 30 seconds, flowing water rinse 5 minutes.Gradient alcohol dehydration, dimethylbenzene is transparent and neutral gum mounting, is sliced in 200 times of optics Micro- Microscopic observation.

Showed by immune group result is significantly more than to plasminogen group (Figure 29 C) insulin positive expression (arrow logo) Solvent PBS control group (Figure 29 B) is given, and gives solvent PBS groups closer blank control group (29A) to plasminogen group ratio.Explanation Plasminogen promotes the synthesis and expression of PLG activity normal mouse insulin in T1DM models.

30 plasminogen of embodiment promotes PLG activity to be damaged the multidirectional Nuclear Factor kappa B of pancreas islet in mouse T1DM models Expression

9-10 week old PLG activity is damaged male mouse 10, is randomly divided into three groups, blank control group 3, to PBS pairs According to group 3, to plasminogen group 4.To solvent PBS control group and to single abdominal cavity after plasminogen group mouse fasting 4 hours Inject 200mg/kg STZ (sigma S0130) inducing type I diabetes^[43], blank control group do not process.After injection 12 days Start to be administered and be set to administration the 1st day, to 1mg/0.1ml/ pcs/day of plasminogen group tail vein injection people source fibrinolysin, to molten The PBS of matchmaker's PBS control group tail vein injection same volume, successive administration 28 days.Mouse was put to death at the 29th day, takes pancreas 4% It is fixed in paraformaldehyde.Pancreatic tissues after fixation carry out paraffin embedding after alcohol serial dehydration and dimethylbenzene are transparent.Tissue Slice thickness is 3 μm, is washed 1 time after slice dewaxing rehydration.PAP are irised out tissue, with 3% dioxygen water incubation 15 minutes, 0.01MPBS is washed 2 times, every time 5 minutes.5% normal sheep serum liquid (Vector laboratories, Inc., USA) closing 30 minutes；After time arrives, 4 DEG C of overnight incubations of rabbit anti-mouse NF- kappa B antibodies (Cell Signal) are added dropwise in reject sheep blood serum liquid, 0.01M PBS are washed 2 times, every time 5 minutes.Goat anti-rabbit igg (HRP) antibody (Abcam) secondary antibody is incubated at room temperature 1 hour, 0.01M PBS is washed 2 times, every time 5 minutes.It develops the color by DAB kits (Vector laboratories, Inc., USA), revives after washing 3 times Lignin is redyed 30 seconds, and flowing water rinses 5 minutes.Gradient alcohol dehydration, dimethylbenzene is transparent and neutral gum mounting, is sliced at 200 times Optical microphotograph Microscopic observation.

NF- κ B participate in cell Proliferation, Apoptosis and inflammation and are immunized as a kind of multidirectional nuclear factor after activation Etc. several genes adjusting^[24]。

Experimental result is shown, is apparently higher than to the expression (arrow logo) of plasminogen group (Figure 30 C) NF- κ B to solvent PBS control group (Figure 30 B).Illustrate that plasminogen can promote the expression of multidirectional Nuclear Factor kappa B.

31 plasminogen of embodiment promotes the expression of the 18 multidirectional Nuclear Factor kappa B of week old diabetic mice pancreas islet

18 week old db/db male mices 7, experiment are denoted as the 0th day and weigh, be randomly divided into according to weight on the day of starting Two groups, to plasminogen group 3, to solvent PBS control group 4.Start to plasminogen or PBS, to plasminogen within 1st day 2mg/0.2ml/ pcs/day of group tail vein injection people source plasminogen, to solvent PBS control group tail vein injection same volume PBS, successive administration 35 days.Mouse was put to death at the 36th day, takes pancreas fixed in 4% paraformaldehyde.Pancreas group after fixation It knits and carries out paraffin embedding after alcohol serial dehydration and dimethylbenzene are transparent.Histotomy thickness is 3 μm, after slice dewaxing rehydration Washing 1 time.PAP are irised out tissue, and with 3% dioxygen water incubation 15 minutes, 0.01MPBS was washed 2 times, every time 5 minutes.5% just Normal sheep blood serum liquid (Vector laboratories, Inc., USA) is closed 30 minutes；After time arrives, reject sheep blood serum liquid is added dropwise 4 DEG C of overnight incubations of rabbit anti-mouse NF- kappa B antibodies (Cell Signal), 0.01M PBS are washed 2 times, every time 5 minutes.Goat antirabbit IgG (HRP) antibody (Abcam) secondary antibody is incubated at room temperature 1 hour, and 0.01M PBS are washed 2 times, every time 5 minutes.By DAB kits (Vector laboratories, Inc., USA) develops the color, and haematoxylin is redyed 30 seconds after washing 3 times, and flowing water rinses 5 minutes.Ladder Dehydration of alcohol is spent, dimethylbenzene is transparent and neutral gum mounting, is sliced in 200 times of optical microphotograph Microscopic observations.

Experimental result of the present invention is shown, is apparently higher than to the expression (arrow logo) of plasminogen group (Figure 31 B) NF- κ B Give solvent PBS control group (Figure 31 A).Illustrate that plasminogen can promote the expression of multidirectional nuclear factor NF- κ B.

32 plasminogen of embodiment inhibits the expression of the multidirectional Nuclear Factor kappa B of 26 week old diabetic mices

26 week old db/db male mices 9 and db/m male mices 3, experiment are denoted as the 0th day and claim on the day of starting Weight, db/db mouse are randomly divided into two groups according to weight, to plasminogen group 4, to solvent PBS control group 5, db/m mouse As Normal group.Start within 1st day to plasminogen or PBS and be denoted as the 1st day, give plasminogen group tail vein injection people 2mg/0.2ml/ pcs/day of source plasminogen, to the PBS of solvent PBS control group tail vein injection same volume, successive administration 35 My god.Mouse was put to death at the 36th day, takes pancreas fixed in 4% paraformaldehyde.Pancreatic tissues after fixation are through alcohol serial dehydration With the transparent rear progress paraffin embedding of dimethylbenzene.Histotomy thickness is 3 μm, is washed 1 time after slice dewaxing rehydration.PAP are irised out Tissue, with 3% dioxygen water incubation 15 minutes, 0.01MPBS was washed 2 times, every time 5 minutes.5% normal sheep serum liquid (Vector Laboratories, Inc., USA) it closes 30 minutes；After time arrives, rabbit anti-mouse NF- kappa B antibodies are added dropwise in reject sheep blood serum liquid (Cell Signal) 4 DEG C of overnight incubations, 0.01M PBS are washed 2 times, every time 5 minutes.Goat anti-rabbit igg (HRP) antibody (Abcam) secondary antibody is incubated at room temperature 1 hour, and 0.01M PBS are washed 2 times, every time 5 minutes.By DAB kits (Vector Laboratories, Inc., USA) colour developing, haematoxylin is redyed 30 seconds after washing 3 times, and flowing water rinses 5 minutes.Graded ethanol takes off Water, dimethylbenzene is transparent and neutral gum mounting, is sliced in 200 times of optical microphotograph Microscopic observations.

Experimental result is shown, is apparently higher than to the expression (arrow logo) of plasminogen group (Figure 32 C) NF- κ B to solvent PBS control group (Figure 32 B), and give solvent PBS groups closer Normal group (32A) to plasminogen group ratio.Illustrate fibrinolysin Proper energy promotes the expression of the multidirectional Nuclear Factor kappa B of old (26 week old) diabetic mice relatively.

33 plasminogen of embodiment promotes the expression of 24-25 week old diabetic mice pancreas islet TNF-α

24-25 week old db/db male mices 11 and db/m male mices 5, experiment are denoted as the 0th day on the day of starting And weigh, db/db mouse are randomly divided into two groups according to weight, to plasminogen group 5, to solvent PBS control group 6, db/m Mouse is as Normal group.Start to plasminogen or PBS within 1st day, give plasminogen group tail vein injection people source fibrinolysin , PBS to solvent PBS control group tail vein injection same volume or any liquid is not injected, continuously by former 2mg/0.2ml/ pcs/day Administration 31 days.Mouse was put to death at the 32nd day, takes pancreas fixed in 4% paraformaldehyde.Pancreatic tissues after fixation are through alcohol Paraffin embedding is carried out after serial dehydration and dimethylbenzene are transparent.Histotomy thickness is 3 μm, is washed 1 time after slice dewaxing rehydration. PAP are irised out tissue, and with 3% dioxygen water incubation 15 minutes, 0.01MPBS was washed 2 times, every time 5 minutes.5% normal sheep serum Liquid (Vector laboratories, Inc., USA) is closed 30 minutes；After time arrives, it is small that rabbit-anti is added dropwise in reject sheep blood serum liquid 4 DEG C of overnight incubations of mouse TNF-α antibody (Abcam), 0.01M PBS are washed 2 times, every time 5 minutes.Goat anti-rabbit igg (HRP) antibody (Abcam) secondary antibody is incubated at room temperature 1 hour, and 0.01M PBS are washed 2 times, every time 5 minutes.By DAB kits (Vector Laboratories, Inc., USA) colour developing, haematoxylin is redyed 30 seconds after washing 3 times, and flowing water rinses 5 minutes.Graded ethanol takes off Water, dimethylbenzene is transparent and neutral gum mounting, is sliced in 200 times of optical microphotograph Microscopic observations.

Tumor necrosis factor α (Tumor Necrosis Factor- α,TNF- α) it is mainly thin by the monokaryon/macrophage activated Born of the same parents generate, and are a kind of important proinflammatory inflammation factors^[48]。

This experimental studies results is shown, is apparently higher than to the positive expression of plasminogen group (Figure 33 C) TNF-α to solvent PBS control group (Figure 33 B), and give solvent PBS groups closer Normal group (33A) to plasminogen group ratio.Illustrate fibrinolysin Proper energy promotes the expression of 24-25 week old diabetic mice TNF-α.

34 plasminogen of embodiment inhibits the expression of 26 week old diabetic mice pancreas islet TNF-α

26 week old db/db male mices 9 and db/m male mices 3, experiment are denoted as the 0th day and claim on the day of starting Weight, db/db mouse are randomly divided into two groups according to weight, to plasminogen group 4, to solvent PBS control group 5, db/m mouse As Normal group.Start to plasminogen or PBS within 1st day.Give plasminogen group tail vein injection people source plasminogen , PBS to solvent PBS control group tail vein injection same volume or any liquid is not injected, continuously give by 2mg/0.2ml/ pcs/day Medicine 35 days.Mouse was put to death at the 36th day, takes pancreas fixed in 4% paraformaldehyde.Pancreatic tissues after fixation are through alcohol ladder Paraffin embedding is carried out after degree dehydration and dimethylbenzene are transparent.Histotomy thickness is 3 μm, is washed 1 time after slice dewaxing rehydration.PAP Pen irises out tissue, and with 3% dioxygen water incubation 15 minutes, 0.01MPBS was washed 2 times, every time 5 minutes.5% normal sheep serum liquid (Vector laboratories, Inc., USA) is closed 30 minutes；After time arrives, rabbit anti-mouse is added dropwise in reject sheep blood serum liquid 4 DEG C of overnight incubations of TNF-α antibody (Abcam), 0.01M PBS are washed 2 times, every time 5 minutes.Goat anti-rabbit igg (HRP) antibody (Abcam) secondary antibody is incubated at room temperature 1 hour, and 0.01M PBS are washed 2 times, every time 5 minutes.By DAB kits (Vector Laboratories, Inc., USA) colour developing, haematoxylin is redyed 30 seconds after washing 3 times, and flowing water rinses 5 minutes.Graded ethanol takes off Water, dimethylbenzene is transparent and neutral gum mounting, is sliced in 200 times of optical microphotograph Microscopic observations.

Result of study is shown, is apparently higher than to the positive expression of plasminogen group (Figure 34 C) TNF-α and is given solvent PBS control Group (Figure 34 B), and give solvent PBS groups closer Normal group (34A) to plasminogen group ratio.Illustrate plasminogen energy 26 Week old diabetic mice promotes the expression of TNF-α.

35 plasminogen of embodiment promotion PLG activity is damaged the expression of mouse pancreas islet TNF-α in T1DM models

9-10 week old PLG activity is damaged male mouse 7, is randomly divided into two groups, to PBS control group 3, to fibrinolytic Proenzyme group 4.Single intraperitoneal injection 200mg/kg STZ (sigma S0130) inducing type I sugar after two groups of mouse fasting 4 hours Urine disease^[43].Injection starts to be administered and is set to administration the 1st day after 12 days, gives plasminogen group tail vein injection people source fibrinolysin 1mg/0.1ml/ pcs/day, to the PBS of solvent PBS control group tail vein injection same volume, successive administration 28 days.At the 29th day Dead mouse takes pancreas fixed in 4% paraformaldehyde.Pancreatic tissues after fixation are transparent through alcohol serial dehydration and dimethylbenzene After carry out paraffin embedding.Histotomy thickness is 3 μm, is washed 1 time after slice dewaxing rehydration.PAP are irised out tissue, with 3% pair Oxygen water incubation 15 minutes, 0.01MPBS are washed 2 times, every time 5 minutes.5% normal sheep serum liquid (Vector Laboratories, Inc., USA) it closes 30 minutes；After time arrives, rabbit anti-mouse antibody TNF-α is added dropwise in reject sheep blood serum liquid (Abcam) 4 DEG C of overnight incubations, 0.01M PBS are washed 2 times, every time 5 minutes.Goat anti-rabbit igg (HRP) antibody (Abcam) secondary antibody Incubation at room temperature 1 hour, 0.01M PBS are washed 2 times, every time 5 minutes.By DAB kits (Vector laboratories, Inc., USA it) develops the color, haematoxylin is redyed 30 seconds after washing 3 times, and flowing water rinses 5 minutes.Gradient alcohol dehydration, dimethylbenzene are transparent and neutral Gummy mounting is sliced in 200 times of optical microphotograph Microscopic observations.

This experimental studies results is shown, is apparently higher than to the positive expression of plasminogen group (Figure 35 B) TNF-α to solvent PBS control group (Figure 35 A).Illustrate the expression that plasminogen can promote PLG activity to be damaged mouse T1DM model TNF-α.

36 plasminogen of embodiment mitigates PLG activity and is damaged mouse islet damage in T1DM models

9-10 week old PLG activity is damaged male mouse 10, is randomly divided into three groups, blank control group 3, to PBS pairs According to group 3, to plasminogen group 4.To solvent PBS control group and to single abdominal cavity after plasminogen group mouse fasting 4 hours Inject 200mg/kg STZ (sigma S0130) inducing type I diabetes^[43], blank control group do not process.After injection 12 days Start to be administered and be set to administration the 1st day, to 1mg/0.1ml/ pcs/day of plasminogen group tail vein injection people source fibrinolysin, to molten The PBS of matchmaker's PBS control group tail vein injection same volume, successive administration 28 days.Mouse was put to death at the 29th day, takes pancreas 4% It is fixed in paraformaldehyde.Pancreatic tissues after fixation carry out paraffin embedding after alcohol serial dehydration and dimethylbenzene are transparent.Tissue Slice thickness is 3 μm, is washed 1 time after slice dewaxing rehydration.PAP are irised out tissue, with 3% dioxygen water incubation 15 minutes, 0.01MPBS is washed 2 times, every time 5 minutes.5% normal sheep serum liquid (Vector laboratories, Inc., USA) closing 30 minutes；After time arrives, reject sheep blood serum liquid is added dropwise mountain sheep anti mouse IgM (HRP) antibody (Abcam) and is incubated at room temperature 1 hour, 0.01M PBS are washed 2 times, every time 5 minutes.It develops the color by DAB kits (Vector laboratories, Inc., USA), washing 3 Haematoxylin is redyed 30 seconds after secondary, and flowing water rinses 5 minutes.Gradient alcohol dehydration, dimethylbenzene is transparent and neutral gum mounting, slice In 200 times of optical microphotograph Microscopic observations.

IgM antibody plays an important role during apoptosis and non-viable non-apoptotic cell is removed, injuries of tissues and organs local I gM The level of antibody is proportionate with degree of injury^[49,50].Therefore, the level of detection histoorgan local I gM antibody can reflect The degree of impairment of the histoorgan.

Result of study is shown, is significantly lower than to the positive expression of plasminogen group (Figure 36 C) IgM and is given solvent PBS control group (Figure 36 B) gives solvent PBS groups closer blank control group (36A) to plasminogen group ratio.Illustrate that plasminogen can reduce IgM Expression, prompt plasminogen can mitigate the islet damage that PLG activity is damaged in mouse T1DM models.

37 plasminogen of embodiment reduces the apoptosis of 24-25 week old diabetic mice islet cells

24-25 week old db/db male mices 11 and db/m male mices 5, experiment are denoted as the 0th day on the day of starting And weigh, db/db mouse are randomly divided into two groups according to weight, to plasminogen group 5, to solvent PBS control group 6, db/m Mouse is as Normal group.Start to plasminogen or PBS within 1st day, give plasminogen group tail vein injection people source fibrinolysin , PBS to solvent PBS control group tail vein injection same volume or any liquid is not injected, continuously by former 2mg/0.2ml/ pcs/day Administration 31 days.Mouse was put to death at the 32nd day, takes pancreas fixed in 4% paraformaldehyde.Pancreatic tissues after fixation are through alcohol Paraffin embedding is carried out after serial dehydration and dimethylbenzene are transparent.Histotomy thickness is 3 μm, is washed 1 time after slice dewaxing rehydration. PAP are irised out tissue, and Proteinase K working solution covering tissue is added dropwise, is incubated at room temperature 7min, 0.01M PBS are washed 3 times, every time 3 points Clock.2 mixing liquid (5 of TUNEL kits (Roche) reagent 1 and reagent is added dropwise:45), in 37 DEG C of constant-temperature incubation 40min, 0.01M PBS are washed 3 times, every time 3 minutes.3% hydrogen peroxide solution (the hydrogen peroxide that methanol is prepared is added dropwise：Methanol=1:9) room temperature Incubation 20 minutes is protected from light, 0.01M PBS are washed 3 times, every time 3 minutes.Tunel 3,37 DEG C of constant-temperature incubations of kit reagent are added dropwise 30min, 0.01M PBS are washed 3 times, and DAB kits (Vector laboratories, Inc., USA) colour developing is revived after washing 3 times Lignin is redyed 30 seconds, and flowing water rinses 5 minutes.Gradient alcohol dehydration, dimethylbenzene is transparent and neutral gum mounting, is sliced 200 Times optical microphotograph Microscopic observation.

TUNEL dyeing can be used for detecting the crack conditions of histocyte nucleus DNA in apoptosis late processes.

This experimental result is shown, is considerably less than to the positive cell number (arrow logo) of plasminogen group (Figure 37 C) to molten Matchmaker's PBS control group (Figure 37 B).Normal group TUNEL positive stainings are extremely low (Figure 37 A).Normal group apoptosis rate is about 8%, it is about 93% to solvent PBS group apoptosis rates, is about 16% to plasminogen group apoptosis rate.Illustrate that plasminogen group can be shown Write the apoptosis for reducing diabetic mice islet cells.

38 plasminogen of embodiment improves T1DM model mice insulin secretions

9-10 week old C57 male mices 6, are randomly divided into two groups, to solvent PBS control group and to plasminogen group each 3 Only.Single intraperitoneal injection 200mg/kg streptozotocins (STZ) (sigma S0130) induce after two groups of mouse fasting 4 hours T1DM^[43].STZ starts to be administered and is set to administration the 1st day after injecting 12 days, gives plasminogen group tail vein injection people source fibrinolytic 1mg/0.1ml/ pcs/day of enzyme, to the PBS of solvent PBS control group tail vein injection same volume.Successive administration 20 days, at the 21st day After mouse fasting 6 hours, eyeball veniplex takes blood, centrifuging and taking supernatant, with detection kit for insulin (Mercodia AB), Serum insulin concentration is detected according to operation instruction.

Plasminogen group mouse is given the results show that being significantly lower than to solvent PBS control group mouse islets element concentration, and is united Difference is counted close to notable (P=0.08) (Figure 38).Illustrate that plasminogen can promote the secretion of T1DM mouse islets element.

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Sequence table

<110>Shenzhen Co., Ltd of Rui Jian life sciences institute

<120>A kind of new drug that is hypoglycemic and improving sugar tolerance

<130> PDK03588

<160> 14

<170> PatentIn version 3.5

<210> 1

<211> 2376

<212> DNA

<213>Natural plasminogen (Glu-PLG, Glu- plasminogen) nucleic acid sequence of signal peptide is not contained

<400> 1

gagcctctgg atgactatgt gaatacccag ggggcttcac tgttcagtgt cactaagaag 60

cagctgggag caggaagtat agaagaatgt gcagcaaaat gtgaggagga cgaagaattc 120

acctgcaggg cattccaata tcacagtaaa gagcaacaat gtgtgataat ggctgaaaac 180

aggaagtcct ccataatcat taggatgaga gatgtagttt tatttgaaaa gaaagtgtat 240

ctctcagagt gcaagactgg gaatggaaag aactacagag ggacgatgtc caaaacaaaa 300

aatggcatca cctgtcaaaa atggagttcc acttctcccc acagacctag attctcacct 360

gctacacacc cctcagaggg actggaggag aactactgca ggaatccaga caacgatccg 420

caggggccct ggtgctatac tactgatcca gaaaagagat atgactactg cgacattctt 480

gagtgtgaag aggaatgtat gcattgcagt ggagaaaact atgacggcaa aatttccaag 540

accatgtctg gactggaatg ccaggcctgg gactctcaga gcccacacgc tcatggatac 600

attccttcca aatttccaaa caagaacctg aagaagaatt actgtcgtaa ccccgatagg 660

gagctgcggc cttggtgttt caccaccgac cccaacaagc gctgggaact ttgtgacatc 720

ccccgctgca caacacctcc accatcttct ggtcccacct accagtgtct gaagggaaca 780

ggtgaaaact atcgcgggaa tgtggctgtt accgtgtccg ggcacacctg tcagcactgg 840

agtgcacaga cccctcacac acataacagg acaccagaaa acttcccctg caaaaatttg 900

gatgaaaact actgccgcaa tcctgacgga aaaagggccc catggtgcca tacaaccaac 960

agccaagtgc ggtgggagta ctgtaagata ccgtcctgtg actcctcccc agtatccacg 1020

gaacaattgg ctcccacagc accacctgag ctaacccctg tggtccagga ctgctaccat 1080

ggtgatggac agagctaccg aggcacatcc tccaccacca ccacaggaaa gaagtgtcag 1140

tcttggtcat ctatgacacc acaccggcac cagaagaccc cagaaaacta cccaaatgct 1200

ggcctgacaa tgaactactg caggaatcca gatgccgata aaggcccctg gtgttttacc 1260

acagacccca gcgtcaggtg ggagtactgc aacctgaaaa aatgctcagg aacagaagcg 1320

agtgttgtag cacctccgcc tgttgtcctg cttccagatg tagagactcc ttccgaagaa 1380

gactgtatgt ttgggaatgg gaaaggatac cgaggcaaga gggcgaccac tgttactggg 1440

acgccatgcc aggactgggc tgcccaggag ccccatagac acagcatttt cactccagag 1500

acaaatccac gggcgggtct ggaaaaaaat tactgccgta accctgatgg tgatgtaggt 1560

ggtccctggt gctacacgac aaatccaaga aaactttacg actactgtga tgtccctcag 1620

tgtgcggccc cttcatttga ttgtgggaag cctcaagtgg agccgaagaa atgtcctgga 1680

agggttgtag gggggtgtgt ggcccaccca cattcctggc cctggcaagt cagtcttaga 1740

acaaggtttg gaatgcactt ctgtggaggc accttgatat ccccagagtg ggtgttgact 1800

gctgcccact gcttggagaa gtccccaagg ccttcatcct acaaggtcat cctgggtgca 1860

caccaagaag tgaatctcga accgcatgtt caggaaatag aagtgtctag gctgttcttg 1920

gagcccacac gaaaagatat tgccttgcta aagctaagca gtcctgccgt catcactgac 1980

aaagtaatcc cagcttgtct gccatcccca aattatgtgg tcgctgaccg gaccgaatgt 2040

ttcatcactg gctggggaga aacccaaggt acttttggag ctggccttct caaggaagcc 2100

cagctccctg tgattgagaa taaagtgtgc aatcgctatg agtttctgaa tggaagagtc 2160

caatccaccg aactctgtgc tgggcatttg gccggaggca ctgacagttg ccagggtgac 2220

agtggaggtc ctctggtttg cttcgagaag gacaaataca ttttacaagg agtcacttct 2280

tggggtcttg gctgtgcacg ccccaataag cctggtgtct atgttcgtgt ttcaaggttt 2340

gttacttgga ttgagggagt gatgagaaat aattaa 2376

<210> 2

<211> 791

<212> PRT

<213>Natural plasminogen (Glu-PLG, Glu- plasminogen) amino acid sequence of signal peptide is not contained

<400> 2

Glu Pro Leu Asp Asp Tyr Val Asn Thr Gln Gly Ala Ser Leu Phe Ser

1 5 10 15

Val Thr Lys Lys Gln Leu Gly Ala Gly Ser Ile Glu Glu Cys Ala Ala

20 25 30

Lys Cys Glu Glu Asp Glu Glu Phe Thr Cys Arg Ala Phe Gln Tyr His

35 40 45

Ser Lys Glu Gln Gln Cys Val Ile Met Ala Glu Asn Arg Lys Ser Ser

50 55 60

Ile Ile Ile Arg Met Arg Asp Val Val Leu Phe Glu Lys Lys Val Tyr

65 70 75 80

Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg Gly Thr Met

85 90 95

Ser Lys Thr Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser Ser Thr Ser

100 105 110

Pro His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser Glu Gly Leu

115 120 125

Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln Gly Pro Trp

130 135 140

Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys Asp Ile Leu

145 150 155 160

Glu Cys Glu Glu Glu Cys Met His Cys Ser Gly Glu Asn Tyr Asp Gly

165 170 175

Lys Ile Ser Lys Thr Met Ser Gly Leu Glu Cys Gln Ala Trp Asp Ser

180 185 190

Gln Ser Pro His Ala His Gly Tyr Ile Pro Ser Lys Phe Pro Asn Lys

195 200 205

Asn Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu Leu Arg Pro

210 215 220

Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu Cys Asp Ile

225 230 235 240

Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr Tyr Gln Cys

245 250 255

Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly Asn Val Ala Val Thr Val

260 265 270

Ser Gly His Thr Cys Gln His Trp Ser Ala Gln Thr Pro His Thr His

275 280 285

Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp Glu Asn Tyr

290 295 300

Cys Arg Asn Pro Asp Gly Lys Arg Ala Pro Trp Cys His Thr Thr Asn

305 310 315 320

Ser Gln Val Arg Trp Glu Tyr Cys Lys Ile Pro Ser Cys Asp Ser Ser

325 330 335

Pro Val Ser Thr Glu Gln Leu Ala Pro Thr Ala Pro Pro Glu Leu Thr

340 345 350

Pro Val Val Gln Asp Cys Tyr His Gly Asp Gly Gln Ser Tyr Arg Gly

355 360 365

Thr Ser Ser Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser Trp Ser Ser

370 375 380

Met Thr Pro His Arg His Gln Lys Thr Pro Glu Asn Tyr Pro Asn Ala

385 390 395 400

Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp Lys Gly Pro

405 410 415

Trp Cys Phe Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr Cys Asn Leu

420 425 430

Lys Lys Cys Ser Gly Thr Glu Ala Ser Val Val Ala Pro Pro Pro Val

435 440 445

Val Leu Leu Pro Asp Val Glu Thr Pro Ser Glu Glu Asp Cys Met Phe

450 455 460

Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Ala Thr Thr Val Thr Gly

465 470 475 480

Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pro His Arg His Ser Ile

485 490 495

Phe Thr Pro Glu Thr Asn Pro Arg Ala Gly Leu Glu Lys Asn Tyr Cys

500 505 510

Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp Cys Tyr Thr Thr Asn

515 520 525

Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys Ala Ala Pro

530 535 540

Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys Pro Gly

545 550 555 560

Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro Trp Gln

565 570 575

Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly Thr Leu

580 585 590

Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu Lys Ser

595 600 605

Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln Glu Val

610 615 620

Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg Leu Phe Leu

625 630 635 640

Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser Ser Pro Ala

645 650 655

Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro Asn Tyr

660 665 670

Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly Glu Thr

675 680 685

Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu Pro Val

690 695 700

Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly Arg Val

705 710 715 720

Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr Asp Ser

725 730 735

Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys Asp Lys

740 745 750

Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala Arg Pro

755 760 765

Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr Trp Ile

770 775 780

Glu Gly Val Met Arg Asn Asn

785 790

<210> 3

<211> 2433

<212> DNA

<213>Natural plasminogen containing signal peptide（From swiss prot）Nucleic acid sequence

<400> 3

atggaacata aggaagtggt tcttctactt cttttatttc tgaaatcagg tcaaggagag 60

cctctggatg actatgtgaa tacccagggg gcttcactgt tcagtgtcac taagaagcag 120

ctgggagcag gaagtataga agaatgtgca gcaaaatgtg aggaggacga agaattcacc 180

tgcagggcat tccaatatca cagtaaagag caacaatgtg tgataatggc tgaaaacagg 240

aagtcctcca taatcattag gatgagagat gtagttttat ttgaaaagaa agtgtatctc 300

tcagagtgca agactgggaa tggaaagaac tacagaggga cgatgtccaa aacaaaaaat 360

ggcatcacct gtcaaaaatg gagttccact tctccccaca gacctagatt ctcacctgct 420

acacacccct cagagggact ggaggagaac tactgcagga atccagacaa cgatccgcag 480

gggccctggt gctatactac tgatccagaa aagagatatg actactgcga cattcttgag 540

tgtgaagagg aatgtatgca ttgcagtgga gaaaactatg acggcaaaat ttccaagacc 600

atgtctggac tggaatgcca ggcctgggac tctcagagcc cacacgctca tggatacatt 660

ccttccaaat ttccaaacaa gaacctgaag aagaattact gtcgtaaccc cgatagggag 720

ctgcggcctt ggtgtttcac caccgacccc aacaagcgct gggaactttg tgacatcccc 780

cgctgcacaa cacctccacc atcttctggt cccacctacc agtgtctgaa gggaacaggt 840

gaaaactatc gcgggaatgt ggctgttacc gtgtccgggc acacctgtca gcactggagt 900

gcacagaccc ctcacacaca taacaggaca ccagaaaact tcccctgcaa aaatttggat 960

gaaaactact gccgcaatcc tgacggaaaa agggccccat ggtgccatac aaccaacagc 1020

caagtgcggt gggagtactg taagataccg tcctgtgact cctccccagt atccacggaa 1080

caattggctc ccacagcacc acctgagcta acccctgtgg tccaggactg ctaccatggt 1140

gatggacaga gctaccgagg cacatcctcc accaccacca caggaaagaa gtgtcagtct 1200

tggtcatcta tgacaccaca ccggcaccag aagaccccag aaaactaccc aaatgctggc 1260

ctgacaatga actactgcag gaatccagat gccgataaag gcccctggtg ttttaccaca 1320

gaccccagcg tcaggtggga gtactgcaac ctgaaaaaat gctcaggaac agaagcgagt 1380

gttgtagcac ctccgcctgt tgtcctgctt ccagatgtag agactccttc cgaagaagac 1440

tgtatgtttg ggaatgggaa aggataccga ggcaagaggg cgaccactgt tactgggacg 1500

ccatgccagg actgggctgc ccaggagccc catagacaca gcattttcac tccagagaca 1560

aatccacggg cgggtctgga aaaaaattac tgccgtaacc ctgatggtga tgtaggtggt 1620

ccctggtgct acacgacaaa tccaagaaaa ctttacgact actgtgatgt ccctcagtgt 1680

gcggcccctt catttgattg tgggaagcct caagtggagc cgaagaaatg tcctggaagg 1740

gttgtagggg ggtgtgtggc ccacccacat tcctggccct ggcaagtcag tcttagaaca 1800

aggtttggaa tgcacttctg tggaggcacc ttgatatccc cagagtgggt gttgactgct 1860

gcccactgct tggagaagtc cccaaggcct tcatcctaca aggtcatcct gggtgcacac 1920

caagaagtga atctcgaacc gcatgttcag gaaatagaag tgtctaggct gttcttggag 1980

cccacacgaa aagatattgc cttgctaaag ctaagcagtc ctgccgtcat cactgacaaa 2040

gtaatcccag cttgtctgcc atccccaaat tatgtggtcg ctgaccggac cgaatgtttc 2100

atcactggct ggggagaaac ccaaggtact tttggagctg gccttctcaa ggaagcccag 2160

ctccctgtga ttgagaataa agtgtgcaat cgctatgagt ttctgaatgg aagagtccaa 2220

tccaccgaac tctgtgctgg gcatttggcc ggaggcactg acagttgcca gggtgacagt 2280

ggaggtcctc tggtttgctt cgagaaggac aaatacattt tacaaggagt cacttcttgg 2340

ggtcttggct gtgcacgccc caataagcct ggtgtctatg ttcgtgtttc aaggtttgtt 2400

acttggattg agggagtgat gagaaataat taa 2433

<210> 4

<211> 810

<212> PRT

<213>Natural plasminogen containing signal peptide（From swiss prot）Amino acid sequence

<400> 4

Met Glu His Lys Glu Val Val Leu Leu Leu Leu Leu Phe Leu Lys Ser

1 5 10 15

Gly Gln Gly Glu Pro Leu Asp Asp Tyr Val Asn Thr Gln Gly Ala Ser

20 25 30

Leu Phe Ser Val Thr Lys Lys Gln Leu Gly Ala Gly Ser Ile Glu Glu

35 40 45

Cys Ala Ala Lys Cys Glu Glu Asp Glu Glu Phe Thr Cys Arg Ala Phe

50 55 60

Gln Tyr His Ser Lys Glu Gln Gln Cys Val Ile Met Ala Glu Asn Arg

65 70 75 80

Lys Ser Ser Ile Ile Ile Arg Met Arg Asp Val Val Leu Phe Glu Lys

85 90 95

Lys Val Tyr Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg

100 105 110

Gly Thr Met Ser Lys Thr Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser

115 120 125

Ser Thr Ser Pro His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser

130 135 140

Glu Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln

145 150 155 160

Gly Pro Trp Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys

165 170 175

Asp Ile Leu Glu Cys Glu Glu Glu Cys Met His Cys Ser Gly Glu Asn

180 185 190

Tyr Asp Gly Lys Ile Ser Lys Thr Met Ser Gly Leu Glu Cys Gln Ala

195 200 205

Trp Asp Ser Gln Ser Pro His Ala His Gly Tyr Ile Pro Ser Lys Phe

210 215 220

Pro Asn Lys Asn Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu

225 230 235 240

Leu Arg Pro Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu

245 250 255

Cys Asp Ile Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr

260 265 270

Tyr Gln Cys Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly Asn Val Ala

275 280 285

Val Thr Val Ser Gly His Thr Cys Gln His Trp Ser Ala Gln Thr Pro

290 295 300

His Thr His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp

305 310 315 320

Glu Asn Tyr Cys Arg Asn Pro Asp Gly Lys Arg Ala Pro Trp Cys His

325 330 335

Thr Thr Asn Ser Gln Val Arg Trp Glu Tyr Cys Lys Ile Pro Ser Cys

340 345 350

Asp Ser Ser Pro Val Ser Thr Glu Gln Leu Ala Pro Thr Ala Pro Pro

355 360 365

Glu Leu Thr Pro Val Val Gln Asp Cys Tyr His Gly Asp Gly Gln Ser

370 375 380

Tyr Arg Gly Thr Ser Ser Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser

385 390 395 400

Trp Ser Ser Met Thr Pro His Arg His Gln Lys Thr Pro Glu Asn Tyr

405 410 415

Pro Asn Ala Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp

420 425 430

Lys Gly Pro Trp Cys Phe Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr

435 440 445

Cys Asn Leu Lys Lys Cys Ser Gly Thr Glu Ala Ser Val Val Ala Pro

450 455 460

Pro Pro Val Val Leu Leu Pro Asp Val Glu Thr Pro Ser Glu Glu Asp

465 470 475 480

Cys Met Phe Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Ala Thr Thr

485 490 495

Val Thr Gly Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pro His Arg

500 505 510

His Ser Ile Phe Thr Pro Glu Thr Asn Pro Arg Ala Gly Leu Glu Lys

515 520 525

Asn Tyr Cys Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp Cys Tyr

530 535 540

Thr Thr Asn Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys

545 550 555 560

Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys

565 570 575

Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp

580 585 590

Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly

595 600 605

Gly Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu

610 615 620

Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His

625 630 635 640

Gln Glu Val Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg

645 650 655

Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser

660 665 670

Ser Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser

675 680 685

Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp

690 695 700

Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln

705 710 715 720

Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn

725 730 735

Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly

740 745 750

Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu

755 760 765

Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys

770 775 780

Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val

785 790 795 800

Thr Trp Ile Glu Gly Val Met Arg Asn Asn

805 810

<210> 5

<211> 2145

<212> DNA

<213> LYS77-PLG（Lys- plasminogens）Nucleic acid sequence

<400> 5

aaagtgtatc tctcagagtg caagactggg aatggaaaga actacagagg gacgatgtcc 60

aaaacaaaaa atggcatcac ctgtcaaaaa tggagttcca cttctcccca cagacctaga 120

ttctcacctg ctacacaccc ctcagaggga ctggaggaga actactgcag gaatccagac 180

aacgatccgc aggggccctg gtgctatact actgatccag aaaagagata tgactactgc 240

gacattcttg agtgtgaaga ggaatgtatg cattgcagtg gagaaaacta tgacggcaaa 300

atttccaaga ccatgtctgg actggaatgc caggcctggg actctcagag cccacacgct 360

catggataca ttccttccaa atttccaaac aagaacctga agaagaatta ctgtcgtaac 420

cccgataggg agctgcggcc ttggtgtttc accaccgacc ccaacaagcg ctgggaactt 480

tgtgacatcc cccgctgcac aacacctcca ccatcttctg gtcccaccta ccagtgtctg 540

aagggaacag gtgaaaacta tcgcgggaat gtggctgtta ccgtgtccgg gcacacctgt 600

cagcactgga gtgcacagac ccctcacaca cataacagga caccagaaaa cttcccctgc 660

aaaaatttgg atgaaaacta ctgccgcaat cctgacggaa aaagggcccc atggtgccat 720

acaaccaaca gccaagtgcg gtgggagtac tgtaagatac cgtcctgtga ctcctcccca 780

gtatccacgg aacaattggc tcccacagca ccacctgagc taacccctgt ggtccaggac 840

tgctaccatg gtgatggaca gagctaccga ggcacatcct ccaccaccac cacaggaaag 900

aagtgtcagt cttggtcatc tatgacacca caccggcacc agaagacccc agaaaactac 960

ccaaatgctg gcctgacaat gaactactgc aggaatccag atgccgataa aggcccctgg 1020

tgttttacca cagaccccag cgtcaggtgg gagtactgca acctgaaaaa atgctcagga 1080

acagaagcga gtgttgtagc acctccgcct gttgtcctgc ttccagatgt agagactcct 1140

tccgaagaag actgtatgtt tgggaatggg aaaggatacc gaggcaagag ggcgaccact 1200

gttactggga cgccatgcca ggactgggct gcccaggagc cccatagaca cagcattttc 1260

actccagaga caaatccacg ggcgggtctg gaaaaaaatt actgccgtaa ccctgatggt 1320

gatgtaggtg gtccctggtg ctacacgaca aatccaagaa aactttacga ctactgtgat 1380

gtccctcagt gtgcggcccc ttcatttgat tgtgggaagc ctcaagtgga gccgaagaaa 1440

tgtcctggaa gggttgtagg ggggtgtgtg gcccacccac attcctggcc ctggcaagtc 1500

agtcttagaa caaggtttgg aatgcacttc tgtggaggca ccttgatatc cccagagtgg 1560

gtgttgactg ctgcccactg cttggagaag tccccaaggc cttcatccta caaggtcatc 1620

ctgggtgcac accaagaagt gaatctcgaa ccgcatgttc aggaaataga agtgtctagg 1680

ctgttcttgg agcccacacg aaaagatatt gccttgctaa agctaagcag tcctgccgtc 1740

atcactgaca aagtaatccc agcttgtctg ccatccccaa attatgtggt cgctgaccgg 1800

accgaatgtt tcatcactgg ctggggagaa acccaaggta cttttggagc tggccttctc 1860

aaggaagccc agctccctgt gattgagaat aaagtgtgca atcgctatga gtttctgaat 1920

ggaagagtcc aatccaccga actctgtgct gggcatttgg ccggaggcac tgacagttgc 1980

cagggtgaca gtggaggtcc tctggtttgc ttcgagaagg acaaatacat tttacaagga 2040

gtcacttctt ggggtcttgg ctgtgcacgc cccaataagc ctggtgtcta tgttcgtgtt 2100

tcaaggtttg ttacttggat tgagggagtg atgagaaata attaa 2145

<210> 6

<211> 714

<212> PRT

<213> LYS77-PLG（Lys- plasminogens）Amino acid sequence

<400> 6

Lys Val Tyr Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg

1 5 10 15

Gly Thr Met Ser Lys Thr Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser

20 25 30

Ser Thr Ser Pro His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser

35 40 45

Glu Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln

50 55 60

Gly Pro Trp Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys

65 70 75 80

Asp Ile Leu Glu Cys Glu Glu Glu Cys Met His Cys Ser Gly Glu Asn

85 90 95

Tyr Asp Gly Lys Ile Ser Lys Thr Met Ser Gly Leu Glu Cys Gln Ala

100 105 110

Trp Asp Ser Gln Ser Pro His Ala His Gly Tyr Ile Pro Ser Lys Phe

115 120 125

Pro Asn Lys Asn Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu

130 135 140

Leu Arg Pro Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu

145 150 155 160

Cys Asp Ile Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr

165 170 175

Tyr Gln Cys Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly Asn Val Ala

180 185 190

Val Thr Val Ser Gly His Thr Cys Gln His Trp Ser Ala Gln Thr Pro

195 200 205

His Thr His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp

210 215 220

Glu Asn Tyr Cys Arg Asn Pro Asp Gly Lys Arg Ala Pro Trp Cys His

225 230 235 240

Thr Thr Asn Ser Gln Val Arg Trp Glu Tyr Cys Lys Ile Pro Ser Cys

245 250 255

Asp Ser Ser Pro Val Ser Thr Glu Gln Leu Ala Pro Thr Ala Pro Pro

260 265 270

Glu Leu Thr Pro Val Val Gln Asp Cys Tyr His Gly Asp Gly Gln Ser

275 280 285

Tyr Arg Gly Thr Ser Ser Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser

290 295 300

Trp Ser Ser Met Thr Pro His Arg His Gln Lys Thr Pro Glu Asn Tyr

305 310 315 320

Pro Asn Ala Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp

325 330 335

Lys Gly Pro Trp Cys Phe Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr

340 345 350

Cys Asn Leu Lys Lys Cys Ser Gly Thr Glu Ala Ser Val Val Ala Pro

355 360 365

Pro Pro Val Val Leu Leu Pro Asp Val Glu Thr Pro Ser Glu Glu Asp

370 375 380

Cys Met Phe Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Ala Thr Thr

385 390 395 400

Val Thr Gly Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pro His Arg

405 410 415

His Ser Ile Phe Thr Pro Glu Thr Asn Pro Arg Ala Gly Leu Glu Lys

420 425 430

Asn Tyr Cys Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp Cys Tyr

435 440 445

Thr Thr Asn Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys

450 455 460

Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys

465 470 475 480

Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp

485 490 495

Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly

500 505 510

Gly Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu

515 520 525

Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His

530 535 540

Gln Glu Val Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg

545 550 555 560

Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser

565 570 575

Ser Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser

580 585 590

Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp

595 600 605

Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln

610 615 620

Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn

625 630 635 640

Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly

645 650 655

Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu

660 665 670

Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys

675 680 685

Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val

690 695 700

Thr Trp Ile Glu Gly Val Met Arg Asn Asn

705 710

<210> 7

<211> 1245

<212> DNA

<213> delta-plg（Delta- plasminogens）Nucleic acid sequence

<400> 7

gagcctctgg atgactatgt gaatacccag ggggcttcac tgttcagtgt cactaagaag 60

cagctgggag caggaagtat agaagaatgt gcagcaaaat gtgaggagga cgaagaattc 120

acctgcaggg cattccaata tcacagtaaa gagcaacaat gtgtgataat ggctgaaaac 180

aggaagtcct ccataatcat taggatgaga gatgtagttt tatttgaaaa gaaagtgtat 240

ctctcagagt gcaagactgg gaatggaaag aactacagag ggacgatgtc caaaacaaaa 300

aatggcatca cctgtcaaaa atggagttcc acttctcccc acagacctag attctcacct 360

gctacacacc cctcagaggg actggaggag aactactgca ggaatccaga caacgatccg 420

caggggccct ggtgctatac tactgatcca gaaaagagat atgactactg cgacattctt 480

gagtgtgaag aggcggcccc ttcatttgat tgtgggaagc ctcaagtgga gccgaagaaa 540

tgtcctggaa gggttgtagg ggggtgtgtg gcccacccac attcctggcc ctggcaagtc 600

agtcttagaa caaggtttgg aatgcacttc tgtggaggca ccttgatatc cccagagtgg 660

gtgttgactg ctgcccactg cttggagaag tccccaaggc cttcatccta caaggtcatc 720

ctgggtgcac accaagaagt gaatctcgaa ccgcatgttc aggaaataga agtgtctagg 780

ctgttcttgg agcccacacg aaaagatatt gccttgctaa agctaagcag tcctgccgtc 840

atcactgaca aagtaatccc agcttgtctg ccatccccaa attatgtggt cgctgaccgg 900

accgaatgtt tcatcactgg ctggggagaa acccaaggta cttttggagc tggccttctc 960

aaggaagccc agctccctgt gattgagaat aaagtgtgca atcgctatga gtttctgaat 1020

ggaagagtcc aatccaccga actctgtgct gggcatttgg ccggaggcac tgacagttgc 1080

cagggtgaca gtggaggtcc tctggtttgc ttcgagaagg acaaatacat tttacaagga 1140

gtcacttctt ggggtcttgg ctgtgcacgc cccaataagc ctggtgtcta tgttcgtgtt 1200

tcaaggtttg ttacttggat tgagggagtg atgagaaata attaa 1245

<210> 8

<211> 414

<212> PRT

<213> delta-plg（Delta- plasminogens）Amino acid sequence

<400> 8

Glu Pro Leu Asp Asp Tyr Val Asn Thr Gln Gly Ala Ser Leu Phe Ser

1 5 10 15

Val Thr Lys Lys Gln Leu Gly Ala Gly Ser Ile Glu Glu Cys Ala Ala

20 25 30

Lys Cys Glu Glu Asp Glu Glu Phe Thr Cys Arg Ala Phe Gln Tyr His

35 40 45

Ser Lys Glu Gln Gln Cys Val Ile Met Ala Glu Asn Arg Lys Ser Ser

50 55 60

Ile Ile Ile Arg Met Arg Asp Val Val Leu Phe Glu Lys Lys Val Tyr

65 70 75 80

Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg Gly Thr Met

85 90 95

Ser Lys Thr Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser Ser Thr Ser

100 105 110

Pro His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser Glu Gly Leu

115 120 125

Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln Gly Pro Trp

130 135 140

Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys Asp Ile Leu

145 150 155 160

Glu Cys Glu Glu Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val

165 170 175

Glu Pro Lys Lys Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala His

180 185 190

Pro His Ser Trp Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met

195 200 205

His Phe Cys Gly Gly Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala

210 215 220

Ala His Cys Leu Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile

225 230 235 240

Leu Gly Ala His Gln Glu Val Asn Leu Glu Pro His Val Gln Glu Ile

245 250 255

Glu Val Ser Arg Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu

260 265 270

Leu Lys Leu Ser Ser Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala

275 280 285

Cys Leu Pro Ser Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe

290 295 300

Ile Thr Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu

305 310 315 320

Lys Glu Ala Gln Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr

325 330 335

Glu Phe Leu Asn Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His

340 345 350

Leu Ala Gly Gly Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu

355 360 365

Val Cys Phe Glu Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp

370 375 380

Gly Leu Gly Cys Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val

385 390 395 400

Ser Arg Phe Val Thr Trp Ile Glu Gly Val Met Arg Asn Asn

405 410

<210> 9

<211> 1104

<212> DNA

<213> Mini-plg（Small plasminogen）Nucleic acid sequence

<400> 9

gtcaggtggg agtactgcaa cctgaaaaaa tgctcaggaa cagaagcgag tgttgtagca 60

cctccgcctg ttgtcctgct tccagatgta gagactcctt ccgaagaaga ctgtatgttt 120

gggaatggga aaggataccg aggcaagagg gcgaccactg ttactgggac gccatgccag 180

gactgggctg cccaggagcc ccatagacac agcattttca ctccagagac aaatccacgg 240

gcgggtctgg aaaaaaatta ctgccgtaac cctgatggtg atgtaggtgg tccctggtgc 300

tacacgacaa atccaagaaa actttacgac tactgtgatg tccctcagtg tgcggcccct 360

tcatttgatt gtgggaagcc tcaagtggag ccgaagaaat gtcctggaag ggttgtaggg 420

gggtgtgtgg cccacccaca ttcctggccc tggcaagtca gtcttagaac aaggtttgga 480

atgcacttct gtggaggcac cttgatatcc ccagagtggg tgttgactgc tgcccactgc 540

ttggagaagt ccccaaggcc ttcatcctac aaggtcatcc tgggtgcaca ccaagaagtg 600

aatctcgaac cgcatgttca ggaaatagaa gtgtctaggc tgttcttgga gcccacacga 660

aaagatattg ccttgctaaa gctaagcagt cctgccgtca tcactgacaa agtaatccca 720

gcttgtctgc catccccaaa ttatgtggtc gctgaccgga ccgaatgttt catcactggc 780

tggggagaaa cccaaggtac ttttggagct ggccttctca aggaagccca gctccctgtg 840

attgagaata aagtgtgcaa tcgctatgag tttctgaatg gaagagtcca atccaccgaa 900

ctctgtgctg ggcatttggc cggaggcact gacagttgcc agggtgacag tggaggtcct 960

ctggtttgct tcgagaagga caaatacatt ttacaaggag tcacttcttg gggtcttggc 1020

tgtgcacgcc ccaataagcc tggtgtctat gttcgtgttt caaggtttgt tacttggatt 1080

gagggagtga tgagaaataa ttaa 1104

<210> 10

<211> 367

<212> PRT

<213> Mini-plg（Small plasminogen）Amino acid sequence

<400> 10

Val Arg Trp Glu Tyr Cys Asn Leu Lys Lys Cys Ser Gly Thr Glu Ala

1 5 10 15

Ser Val Val Ala Pro Pro Pro Val Val Leu Leu Pro Asp Val Glu Thr

20 25 30

Pro Ser Glu Glu Asp Cys Met Phe Gly Asn Gly Lys Gly Tyr Arg Gly

35 40 45

Lys Arg Ala Thr Thr Val Thr Gly Thr Pro Cys Gln Asp Trp Ala Ala

50 55 60

Gln Glu Pro His Arg His Ser Ile Phe Thr Pro Glu Thr Asn Pro Arg

65 70 75 80

Ala Gly Leu Glu Lys Asn Tyr Cys Arg Asn Pro Asp Gly Asp Val Gly

85 90 95

Gly Pro Trp Cys Tyr Thr Thr Asn Pro Arg Lys Leu Tyr Asp Tyr Cys

100 105 110

Asp Val Pro Gln Cys Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln

115 120 125

Val Glu Pro Lys Lys Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala

130 135 140

His Pro His Ser Trp Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly

145 150 155 160

Met His Phe Cys Gly Gly Thr Leu Ile Ser Pro Glu Trp Val Leu Thr

165 170 175

Ala Ala His Cys Leu Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val

180 185 190

Ile Leu Gly Ala His Gln Glu Val Asn Leu Glu Pro His Val Gln Glu

195 200 205

Ile Glu Val Ser Arg Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala

210 215 220

Leu Leu Lys Leu Ser Ser Pro Ala Val Ile Thr Asp Lys Val Ile Pro

225 230 235 240

Ala Cys Leu Pro Ser Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys

245 250 255

Phe Ile Thr Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu

260 265 270

Leu Lys Glu Ala Gln Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg

275 280 285

Tyr Glu Phe Leu Asn Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly

290 295 300

His Leu Ala Gly Gly Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro

305 310 315 320

Leu Val Cys Phe Glu Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser

325 330 335

Trp Gly Leu Gly Cys Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg

340 345 350

Val Ser Arg Phe Val Thr Trp Ile Glu Gly Val Met Arg Asn Asn

355 360 365

<210> 11

<211> 750

<212> DNA

<213> Micro-plg（Fibrillin lyase is former）Nucleic acid sequence

<400> 11

gccccttcat ttgattgtgg gaagcctcaa gtggagccga agaaatgtcc tggaagggtt 60

gtaggggggt gtgtggccca cccacattcc tggccctggc aagtcagtct tagaacaagg 120

tttggaatgc acttctgtgg aggcaccttg atatccccag agtgggtgtt gactgctgcc 180

cactgcttgg agaagtcccc aaggccttca tcctacaagg tcatcctggg tgcacaccaa 240

gaagtgaatc tcgaaccgca tgttcaggaa atagaagtgt ctaggctgtt cttggagccc 300

acacgaaaag atattgcctt gctaaagcta agcagtcctg ccgtcatcac tgacaaagta 360

atcccagctt gtctgccatc cccaaattat gtggtcgctg accggaccga atgtttcatc 420

actggctggg gagaaaccca aggtactttt ggagctggcc ttctcaagga agcccagctc 480

cctgtgattg agaataaagt gtgcaatcgc tatgagtttc tgaatggaag agtccaatcc 540

accgaactct gtgctgggca tttggccgga ggcactgaca gttgccaggg tgacagtgga 600

ggtcctctgg tttgcttcga gaaggacaaa tacattttac aaggagtcac ttcttggggt 660

cttggctgtg cacgccccaa taagcctggt gtctatgttc gtgtttcaag gtttgttact 720

tggattgagg gagtgatgag aaataattaa 750

<210> 12

<211> 249

<212> PRT

<213> Micro-plg（Fibrillin lyase is former）Amino acid sequence

<400> 12

Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys

1 5 10 15

Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro

20 25 30

Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly

35 40 45

Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu

50 55 60

Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln

65 70 75 80

Glu Val Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg Leu

85 90 95

Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser Ser

100 105 110

Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro

115 120 125

Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly

130 135 140

Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu

145 150 155 160

Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly

165 170 175

Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr

180 185 190

Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys

195 200 205

Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala

210 215 220

Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr

225 230 235 240

Trp Ile Glu Gly Val Met Arg Asn Asn

245

<210> 13

<211> 684

<212> DNA

<213>Serine protease（Structure）The nucleic acid sequence in domain

<400> 13

gttgtagggg ggtgtgtggc ccacccacat tcctggccct ggcaagtcag tcttagaaca 60

aggtttggaa tgcacttctg tggaggcacc ttgatatccc cagagtgggt gttgactgct 120

gcccactgct tggagaagtc cccaaggcct tcatcctaca aggtcatcct gggtgcacac 180

caagaagtga atctcgaacc gcatgttcag gaaatagaag tgtctaggct gttcttggag 240

cccacacgaa aagatattgc cttgctaaag ctaagcagtc ctgccgtcat cactgacaaa 300

gtaatcccag cttgtctgcc atccccaaat tatgtggtcg ctgaccggac cgaatgtttc 360

atcactggct ggggagaaac ccaaggtact tttggagctg gccttctcaa ggaagcccag 420

ctccctgtga ttgagaataa agtgtgcaat cgctatgagt ttctgaatgg aagagtccaa 480

tccaccgaac tctgtgctgg gcatttggcc ggaggcactg acagttgcca gggtgacagt 540

ggaggtcctc tggtttgctt cgagaaggac aaatacattt tacaaggagt cacttcttgg 600

ggtcttggct gtgcacgccc caataagcct ggtgtctatg ttcgtgtttc aaggtttgtt 660

acttggattg agggagtgat gaga 684

<210> 14

<211> 228

<212> PRT

<213>Serine protease（Structure）The amino acid sequence in domain

<400> 14

Val Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro Trp Gln Val

1 5 10 15

Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly Thr Leu Ile

20 25 30

Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu Lys Ser Pro

35 40 45

Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln Glu Val Asn

50 55 60

Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg Leu Phe Leu Glu

65 70 75 80

Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser Ser Pro Ala Val

85 90 95

Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro Asn Tyr Val

100 105 110

Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly Glu Thr Gln

115 120 125

Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu Pro Val Ile

130 135 140

Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly Arg Val Gln

145 150 155 160

Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr Asp Ser Cys

165 170 175

Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys Asp Lys Tyr

180 185 190

Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala Arg Pro Asn

195 200 205

Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr Trp Ile Glu

210 215 220

Gly Val Met Arg

225

Claims (10)

1. a kind of method for reducing diabetic subjects blood glucose, including a effective amount of plasminogen of subject is administered.

2. the method for claim 1 wherein the blood glucose is chosen from the followings one or more：Serum level of glucose, serum fruit Fructosamine level, serum glycated hemoglobin are horizontal.

3. the method for claim 2, wherein the blood glucose is serum level of glucose.

4. the method for any one of claim 1-3, wherein the diabetes are T1DM or T2DM.

5. a kind of method for improving diabetic subjects sugar tolerance, including a effective amount of plasminogen of subject is administered.

6. the method for claim 5, wherein the diabetes are T2DM.

7. a kind of method that diabetic subjects postprandial blood sugar is promoted to decline, including a effective amount of plasminogen of subject is administered.

8. the method for claim 7, wherein the plasminogen is given for 30 minutes to 1.5 hours before the meal in subject.

9. the method for claim 8, wherein the plasminogen is given for 30 minutes to 1 hour before the meal in subject.

10. a kind of promote the method that utilizes of the diabetic subjects to glucose, including a effective amount of fibrinolysin of subject is administered It is former.