WO2018107706A1 - 一种使胰高血糖素、胰岛素恢复正常平衡的方法 - Google Patents

一种使胰高血糖素、胰岛素恢复正常平衡的方法 Download PDF

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WO2018107706A1
WO2018107706A1 PCT/CN2017/089066 CN2017089066W WO2018107706A1 WO 2018107706 A1 WO2018107706 A1 WO 2018107706A1 CN 2017089066 W CN2017089066 W CN 2017089066W WO 2018107706 A1 WO2018107706 A1 WO 2018107706A1
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plasminogen
subject
group
insulin
mice
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PCT/CN2017/089066
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English (en)
French (fr)
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李季男
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深圳瑞健生命科学研究院有限公司
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Priority to JP2019532017A priority Critical patent/JP7175270B2/ja
Priority to CN201780078124.3A priority patent/CN110139667A/zh
Priority to EP17880563.6A priority patent/EP3556384B1/en
Priority to US16/469,817 priority patent/US11311607B2/en
Priority to CA3047176A priority patent/CA3047176C/en
Publication of WO2018107706A1 publication Critical patent/WO2018107706A1/zh
Priority to JP2022119543A priority patent/JP2022166026A/ja

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • A61K38/484—Plasmin (3.4.21.7)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21007—Plasmin (3.4.21.7), i.e. fibrinolysin

Definitions

  • the present invention relates to a method for restoring normal balance of glucagon and insulin, comprising administering an effective amount of plasminogen to a subject, and the present invention relates to a medicament for restoring glucagon and insulin to a normal balance .
  • Diabetes mellitus is a common genetically predisposed glucose metabolism disorder and endocrine disorder disease caused by insufficient absolute or relative insulin secretion.
  • diabetes is one of the major diseases that seriously endanger human health.
  • Diabetes is mainly characterized by abnormal glucose metabolism and metabolic disorders of fats, proteins, etc., while long-term hyperglycemia can lead to serious diabetic complications, including microvascular complications, diabetic nephropathy, diabetic cardiomyopathy, diabetic neuropathy, and diabetes. Skin lesions and diabetes combined with infection. Among them, diabetic nephropathy and diabetic neurological diseases have a great impact on the quality of life of patients, and the harm is serious.
  • T1DM type 1 diabetes
  • T2DM type 2 diabetes
  • gestational diabetes and special type diabetes.
  • T1DM and T2DM patients are the most common, and gestational diabetes and special type of diabetes patients are relatively few.
  • T1DM is thought to be associated with genetic factors, environmental factors (such as viral infections, diabetes-causing chemicals, dietary factors) and autoimmune factors. Studies have shown that there are at least 17 gene loci associated with T1DM, which are located on different chromosomes. In terms of environmental factors, environmental factors that affect the onset of T1DM include viral infections, diabetes-causing chemicals, and dietary factors, with viral factors being the most important. Mumps, rubella virus, cytomegalovirus, etc. have been found to be associated with the pathogenesis of T1DM. The mechanism is that the virus can directly destroy the islet ⁇ cells, and stimulate the autoimmune reaction to further damage the islet ⁇ cells after the virus damages the islet ⁇ cells.
  • Diabetic-causing chemicals such as alloxan, streptozotocin (STZ), and pentamidine act on islet beta cells, leading to destruction of islet beta cells.
  • Autoimmune factors include humoral immunity and cellular immunity.
  • Humoral immunity manifests itself in the presence of multiple autoantibodies against pancreatic islet beta cells in the patient's blood circulation.
  • Cellular immunity mainly manifests as abnormal expression of HLA-DA antigen and IL- in the surface of islet inflammatory cells and islet ⁇ cells. 2 receptors and overexpression of HLA class 1 antigen on the surface of islet cells, while the ratio of CD4+/CD8+ in peripheral blood and IL-1, TNF- ⁇ , INF- ⁇ levels were elevated.
  • the pathological changes caused by these factors focus on the destruction of islet ⁇ -cells, resulting in an absolute decrease in insulin levels in the body, causing T1DM, so T1DM is considered to be an autoimmune disease.
  • T2DM is a multi-gene hereditary disease. It is generally considered to be multi-sourced. Environmental factors and genetic factors work together to cause insulin resistance. It is manifested that the same level of insulin cannot function normally due to the body's resistance. The role of the level. In order to achieve normal blood sugar levels, the body will secrete insulin excessively to alleviate the "inefficient" state of insulin use. In the long run, the requirements for islet ⁇ cells are getting higher and higher, eventually leading to the "overwork" of islet ⁇ cells and their own damage. absolutely lacking for insulin.
  • DM pathogenesis of DM is complex, mainly related to family genetic predisposition, ethnic heterogeneity, insulin receptor deficiency, insulin receptor substrate damage, protein tyrosine phosphatase-related gene up-regulation, hyperimmune inflammatory response, lipotoxicity, oxidative stress And mitochondrial damage and other related [2-3] .
  • Elevated levels of free fatty acids are one of the causes of insulin resistance and one of the important features of insulin resistance. Under the influence of genetic factors or environmental factors, the level of free fatty acids in the blood rises, and when it exceeds the storage capacity of adipose tissue, insulin resistance occurs. Studies have shown that long-term high-fat diets will lead to dysfunction of islet beta cells, because high-fat diets, in addition to triggering peripheral insulin resistance, also increase abdominal fat content and insulin-suppressed lipolytic capacity, thereby promoting free fatty acid content. Elevation, which in turn inhibits the phosphorylation of the tyrosine site of the insulin receptor and its substrates IRS-1 and 1RS-2, inhibits the activity of P13K, leading to inhibition of insulin signaling pathway to form insulin resistance.
  • T2DM is a mild, non-specific inflammatory disease. Recent studies have shown that the main mechanism of inflammation leading to insulin resistance is the crossover of inflammatory factors and insulin receptor substrate signaling. On the one hand, inflammatory factors produced by non-specific inflammation impede the IRS/PI3K signaling pathway. On the other hand, a series of kinases activated by inflammatory factors induce phosphorylation of the silk and threonine sites of IRS, which hinders normal tyrosine phosphorylation, and ultimately leads to decreased insulin signaling capacity and induces insulin resistance. [2-3] .
  • the activated tyrosine kinase induces tyrosine site phosphorylation of the insulin receptor substrate IRS while achieving its own phosphorylation.
  • Activated IRS migrates to the cell membrane, anchors phosphotyrosine to the IRS tyrosine kinase via the phosphotyrosine binding domain (PTB), and tyrosine phosphorylated IRS recruits to regulate PI3K through its SH2 domain Subunit P85.
  • P85 binds to the phospho-inositol 3 phosphate molecule and converts phosphatidylinositol monophosphate (PIP) to phosphatidylinositol diphosphate (PIP2) and phosphatidylinositol triphosphate (PIP3), which are insulin and other growth factors.
  • the second messenger is the anchor site of a subtype of the downstream signaling molecule phosphoinositide-dependent protein kinase-1 (PDK1) and/or protein kinase c (PKC).
  • PDK1 activates protein kinase B (PKB, also known as Akt) and an atypical PKC isoform.
  • Activated PKB inactivates glycogen synthase kinase-3 (GSK3) by silk/threonine phosphorylation and activates mammalian rapamycin target (mTOR) protein kinase on the other hand, thereby inducing its downstream 70 ku- S6 kinase (p70S6K) phosphorylation is activated.
  • mTOR protein kinase can act as an "ATP receptor", activate p70s6K without Ca2+/cAMP, control protein synthesis, enhance gene transcription, promote islet beta cell hypertrophy and other biological effects.
  • PKB can directly induce the phosphorylation of certain transcription factors by silk/threonine to promote the occurrence of cell mitosis [4-5] .
  • activation of Ras can be achieved through two pathways.
  • the activated insulin receptor activates the IRS-2 protein, and the IRS-2 protein transmits a signal to the adaptor growth factor receptor binding protein 2 (Grb2), which in turn interacts with the signaling protein GDP/GTP exchange factor (mSOS).
  • the action in turn activates the Ras-GT into which the inactive Ras-GDP is converted to achieve activation of Ras.
  • Direct action of the insulin receptor phosphorylates the tyrosine of the signaling protein Shc, and then Shc binds to Grb2 to activate Ras via the mSOS pathway.
  • Activated Ras-GTP recruits Raf serine kinase, which in turn phosphorylates MAPK kinase and MAPK.
  • Activated MAPK activates other protein kinases involved in the process of inducing gene transcription and regulating apoptosis [6] .
  • IRS-1 can be phosphorylated by various inflammatory kinases such as c-Jun amino terminal kinase (JNK), I ⁇ B kinase ⁇ (I ⁇ K ⁇ ) and protein kinase C (PKC)- ⁇ .
  • JNK c-Jun amino terminal kinase
  • I ⁇ K ⁇ I ⁇ B kinase ⁇
  • PKC protein kinase C
  • JNK1-/- Gene knockout
  • JNK1-/- Gene knockout
  • the phosphorylation level of IRS-1 serine 307 in liver tissue of obese rats was higher than that of lean mice, but there was no increase in the knockout (JNK1-/-) obese mice, and the serine 307 site of IRS-1 was observed. It is the target of JNK in vivo [, 8] .
  • I ⁇ K ⁇ can affect insulin signaling through at least two pathways, either by directly inducing Ser307 phosphorylation of IRS-1, or by phosphorylation of I ⁇ B, thereby activating NF- ⁇ B, indirectly by stimulating the expression of various inflammatory factors. Insulin resistance.
  • the inflammatory response is the defensive response of the human immune system against these injuries after infection, tissue damage and stress response, as well as the etiology or pathogenesis of diabetes, cardiovascular disease and tumors.
  • Hotmamisligil et al [9] demonstrated through animal experiments that insulin-resistant obese rats have high levels of pro-inflammatory cytokines and TNF- ⁇ in adipose tissue. Since then, many researchers have begun to explore the relationship between inflammation and obesity, insulin resistance, and explore its molecular pathogenesis.
  • Hotmamisligil [10] first proposed a new medical definition of metabolic inflammation, emphasizing that this low-grade, chronic systemic inflammation is mainly caused by excess nutrients and metabolites. Metabolic inflammation may have molecular and signal transduction pathways similar to typical inflammation. Unlike the typical inflammation we have known in the past, metabolic inflammation does not present symptoms of redness, swelling, heat, pain, and dysfunction.
  • the internal environment of the body is at a steady state, and inflammation and metabolism maintain a dynamic equilibrium state.
  • the body breaks the balance of the body, causing imbalance of the immune system, stimulating the inflammatory signaling pathway, prompting the body to release a series of inflammatory factors, and some inflammatory factors even amplify their own inflammatory response, forming an inflammatory waterfall effect.
  • the body develops insulin resistance, which leads to the occurrence of metabolic syndrome.
  • TNF- ⁇ is closely related to metabolic syndrome.
  • TNF also known as cachexia
  • NK natural killer
  • TNF- ⁇ The TNF secreted by macrophages
  • TNF- ⁇ The TNF secreted by T lymphocytes.
  • the lymphotoxin is called TNF- ⁇ .
  • TNF- ⁇ The biological activity of TNF- ⁇ accounts for 70% to 95% of the total activity of TNF. Therefore, TNF which is often involved at present is mostly referred to as TNF- ⁇ .
  • TNF- ⁇ is associated with various diseases such as insulin resistance, autoimmune diseases, tumors, and chronic hepatitis B. TNF- ⁇ plays a crucial role in the development of insulin resistance.
  • TNF- ⁇ levels were elevated in T2DM patients by detecting serum TNF- ⁇ levels in 50 patients with T2DM, and were significantly associated with BMI, fasting insulin levels, and homeostasis model insulin resistance index (HOMA-IR). It suggests that TNF- ⁇ plays an important role in the pathogenesis of T2DM. It has also been pointed out that TNF- ⁇ can inhibit the phosphorylation of insulin receptors, and when the phosphorylation of insulin receptors is inhibited, the gene expression of glucose transporters can be reduced, thereby reducing the activity of lipoprotein lipase. causes the decomposition of fat [12] .
  • Islet beta cell dysfunction caused by a decrease in the number of beta cells is another important cause of the pathogenesis of T2DM, and apoptosis of beta cells is the most important cause of the decrease in the number of beta cells.
  • T2DM patients are prone to insulin resistance, patients with elevated blood glucose, hyperglycemia can promote IL-6 production, IL-6 can not only reduce GLUT4 expression, reduce the transport of glucose by fat cells, hinder glycogen Synthetic, reduce the sensitivity of insulin; can also promote the secretion of IL-6 by islet cells, causing a vicious circle.
  • Hyperglycemia induces a large amount of IL-1 ⁇ production, and activates NF- ⁇ B, MAPK, Fas, NO and other pathways leading to apoptosis of islet cells, and various inflammatory pathways cross each other, which aggravates apoptosis of islet cells and eventually leads to islet function failure.
  • IL-1 ⁇ can also mediate interactions between leukocytes and interact with other cytokines such as IFN- ⁇ , TNF- ⁇ , etc., and play an important role in the process of ⁇ cell injury.
  • Dyslipidemia in T2DM causes an increase in levels of hormonal substances such as leptin and IL-6.
  • Leptin can increase the release of IL-1 ⁇ to induce ⁇ -cell apoptosis, and also negatively regulate insulin secretion [14] .
  • ROS also has an effect on the damage of islet ⁇ cells.
  • Other adipocytokines such as TNF- ⁇ and lean can also reduce the function of beta cells [15] .
  • the combined action of these cytokines causes more pronounced damage to islet beta cell function.
  • some inflammatory factors can also act on the key part of insulin receptor substrate 2, which phosphorylates serine/threonine, which leads to accelerated degradation of insulin receptor substrate 2 and promotes apoptosis of islet ⁇ cells.
  • Oxidative stress refers to the imbalance between the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) and the elimination of antioxidant defense systems in the body, resulting in excessive production of ROS and RNS, resulting in body tissues. Damage to biological macromolecules such as cells and proteins and nucleic acids [13] . Hyperglycemia is the main cause of oxidative stress. It increases ROS and RNS levels in the body through mitochondrial electron transport chains [14] , glucose autooxidation and polyol pathways [15] . The mitochondrial electron transport chain produces ROS. The main route.
  • ROS reactive oxygen species
  • RNS reactive nitrogen species
  • the mitochondrial electron transport chain mainly involves enzyme complexes I to IV, cytochrome c and coenzyme Q.
  • enzyme complexes I and III a small amount of superoxide products, including superoxide anion, hydrogen peroxide and hydroxyl radicals, are continuously produced.
  • superoxide dismutase, catalase and glutathione peroxidase catalyze the conversion of superoxide products to oxygen and water.
  • the superoxide product is greatly increased, and oxidative stress is generated when the rate of production of the superoxide product exceeds its removal rate.
  • ROS can directly damage ⁇ cells, especially destroy the mitochondrial structure of cells and promote ⁇ cell apoptosis; ROS can also inhibit ⁇ cell function indirectly by affecting insulin signaling pathway, such as activation of nuclear transcription.
  • Factor ⁇ B (NF- ⁇ B) signaling pathway induces ⁇ -cell inflammatory response; inhibits nuclear translocation of pancreatic and duodenal homeobox 1, PDX-1, and inhibits mitochondria Energy metabolism, reducing insulin synthesis and secretion. Oxidative stress causes ⁇ -cell damage through NF- ⁇ B pathway.
  • NF- ⁇ B is a dimer composed of two subunits, p50 and RelA.
  • NF- ⁇ B As a multi-directional nuclear transcription factor, NF- ⁇ B is involved in the regulation of various genes such as cell proliferation, apoptosis, inflammation and immunity after activation [24] .
  • NF- ⁇ B causes islet leukocytosis by regulating gene expression of cytokines and chemokines, such as IL-1 (interleukin-1) and MCP-1 (monocyte/macrophage chemoattractant protein-1) factors. Lead to beta cell damage [25] .
  • IL-1 interleukin-1
  • MCP-1 macrophage chemoattractant protein-1
  • InsR and IRS are important signaling elements in the insulin signaling pathway: the former is the starting element of insulin signaling, and the IRS is the bridge between the former and the downstream components of the pathway. Numerous studies have shown that oxidative stress can interfere with the phosphorylation of InsR and IRS through multiple pathways, hindering insulin signaling.
  • IKK is an activator of NF- ⁇ B inhibitory subunit I ⁇ B. Under ROS stimulation, IKK acts as a serine/threonine phosphorylation kinase of InsR and IRS, which promotes serine phosphorylation of InsR and IRS, normal tyrosine phosphorylation Inhibition inhibits insulin signaling [32] . Brownlee [33] showed that IKK directly phosphorylates the serine residue at position 307 of IRS, resulting in a decrease in normal tyrosine phosphorylation of IRS, impeding the binding of InsR to IRS, and thus insulin resistance.
  • JNK extracellular regulated protein kinases (ERK) and p38 mitogen-activated protein kinase (p38MAPK) are members of the MAPK family and have serine/threonine protein kinase activity in oxidative stress, cytokines and G - Protein-coupled receptor agonists can be activated by action.
  • ERK extracellular regulated protein kinases
  • p38MAPK mitogen-activated protein kinase
  • Oxidative stress caused by high glucose status in diabetes is one of the key causes of the formation of various chronic complications, and is also an important factor in inducing DNA damage [37] .
  • the extracellular fluid is seen to continue to be high in sugar.
  • the electrons generated by the mitochondrial electron transport chain are significantly increased, resulting in excessive ROS, causing damage to the intracellular environment and biological macromolecules such as lipids, proteins, and DNA.
  • the active oxygen produced by the body in the aerobic metabolic pathway acts as a mutation inducer to oxidize the guanine on the DNA strand to 8-hydroxy-2'-deoxyguanosine (8-OHdG).
  • DNA damage can cause other forms of DNA damage, including DNA strand breaks, DNA site mutations, DNA double-stranded aberrations, and proto-oncogenes and tumor suppressor gene mutations.
  • DNA damage may also aggravate ROS and oxidative stress processes.
  • DNA damage can induce ROS production through the H2AX-reduced coenzyme II oxidase 1 (Nox1)/Rac1 pathway.
  • ROS further causes large Ca 2 + into mitochondria, causing cell necrosis and apoptosis, mitochondrial or directly, causes mitochondrial dysfunction, and thus damage to islet ⁇ cells aggravate pathologic process diabetes [38].
  • ROS In addition to causing insulin resistance, ROS also has an effect on the damage of islet ⁇ cells. Under oxidative stress, the expression of insulin gene transcription factors and insulin binding sites are significantly reduced, thereby affecting the production and secretion of insulin. Other adipocytokines such as TNF- ⁇ also reduce the function of beta cells [15] . The combined action of these cytokines causes more pronounced damage to islet beta cell function. In addition, some inflammatory factors can also act on the key part of insulin receptor substrate 2, which phosphorylates serine/threonine, which leads to accelerated degradation of insulin receptor substrate 2 and promotes apoptosis of islet ⁇ cells.
  • ROS can also act as a signaling molecule to activate some stress-sensitive pathways, regulate the expression of related factors, cause apoptosis or necrosis of ⁇ cells, inhibit insulin secretion, induce insulin resistance, and ultimately cause or aggravate diabetes.
  • Diabetes is usually treated with drugs, and traditional medications include insulin and oral hypoglycemic agents.
  • Sulfonylureas Sulfonylureas are an insulin secretagogue that stimulates islet ⁇ cells to secrete insulin and achieve an effect of improving blood sugar levels.
  • the insulins that are allowed to be marketed in China mainly include glimepiride, glibenclamide, glipizide, gliclazide, gliclazide, etc., but some studies have shown that if these drugs are taken for a long time, It will cause the hypoglycemic effect to fail, and it is prone to complications such as hypoglycemia and increased body mass.
  • thiazolidinedione compounds (TZD): In 1999, FDA approved the use of rosiglitazone and pioglitazone in T2DM. The former may aggravate the risk of heart disease, and was later restricted to use as a second-line treatment and was also banned. In heart failure.
  • ⁇ -Glycosidase inhibitors These insulins inhibit the glycosidase of small intestinal mucosal epithelial cells, thereby alleviating the absorption of carbohydrates and leading to a decrease in postprandial blood glucose levels. Commonly used in these drugs are voglibose, acarbose and miglitol.
  • the drugs for treating diabetes are mainly traditional antidiabetic drugs, including sulfonylureas, glinides, biguanides, thiazolidinediones (TZD), ⁇ -glucosidase inhibitors and insulin.
  • sulfonylureas including sulfonylureas, glinides, biguanides, thiazolidinediones (TZD), ⁇ -glucosidase inhibitors and insulin.
  • TGD thiazolidinediones
  • ⁇ -glucosidase inhibitors ⁇ -glucosidase inhibitors and insulin.
  • There are different levels of adverse reactions in the drug such as causing hypoglycemia, gastrointestinal discomfort, and obesity.
  • the targets associated with the pathogenesis of diabetes are currently found to include glucagon-like peptide-1 (GLP-1) and dipeptide peptidase-4 (DPP-4).
  • glucagon-regulated drugs such as glucagon like peptide-1, GLP-1, GLP-1 receptor agonist and dipeptidyl peptidase -4, DPP-4) inhibitors are thought to be effective in maintaining blood glucose homeostasis, improving beta cell function, delaying the progression of diabetes, and even reversing the course of diabetes.
  • the present invention finds that plasminogen can alleviate the damage of pancreatic tissue of diabetic experimental mice, control inflammation, reduce islet ⁇ cell apoptosis, repair pancreatic tissue, restore pancreatic ⁇ -cell secretion function, and lower blood sugar, which is expected to become comprehensive New drugs in many aspects of the pathogenesis of diabetes.
  • the invention includes the following items:
  • WHAT IS CLAIMED IS 1. A method of reducing glucagon secretion in a diabetic subject comprising administering to the subject an effective amount of plasminogen.
  • any one of items 1 to 5 wherein the plasminogen reduces glucagon secretion in a blood glucose-elevating state of a diabetic subject, and returns blood glucose to a normal or near normal level.
  • a method of promoting insulin secretion in a diabetic subject comprising administering to the subject an effective amount of plasminogen.
  • a method of reducing blood glucose in a diabetic subject comprising administering to the subject an effective amount of plasminogen.
  • the blood glucose is selected from one or more of the group consisting of serum glucose levels, serum fructosamine levels, serum glycated hemoglobin levels.
  • the method of any one of items 18 to 20, wherein the diabetes is T1DM or T2DM.
  • a method of increasing glucose tolerance in a diabetic subject comprising administering to the subject an effective amount of plasminogen.
  • a method of promoting postprandial blood glucose decline in a diabetic subject comprising administering a subject effective Amount of plasminogen.
  • a method of promoting the utilization of glucose by a diabetic subject comprising administering to the subject an effective amount of plasminogen.
  • plasminogen is a protein comprising a plasminogen active fragment and still having plasminogen activity.
  • plasminogen is selected from the group consisting of Glu-plasminogen, Lys-plasminogen, small plasminogen, microplasminogen, and delta-fibrinolysis Proenzymes or their variants that retain plasminogen activity.
  • plasminogen is a natural or synthetic human plasminogen, or a variant or fragment thereof that still retains plasminogen activity.
  • plasminogen is a human plasminogen ortholog from a primate or a rodent or a variant thereof that still retains plasminogen activity Or a fragment.
  • a plasminogen for use in the method of any of items 1-40.
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and plasminogen for use in the method of any of items 1-40.
  • a prophylactic or therapeutic kit comprising: (i) plasminogen for use in the method of any of items 1-40 and (ii) for delivery of said plasminogen to The subject of the subject.
  • kit of clause 43 or 44 further comprising a label or instructions for use, the label or instructions for use instructing administration of the plasminogen to the subject to perform any of items 1-40 method.
  • a plasminogen or a pharmaceutical composition comprising plasminogen comprising the method of any one of clauses 1 to 40, wherein the label indicates administration of the plasminogen or composition
  • the subject is the method of any of items 1-40.
  • kit of claim 43 or the article of item 46 further comprising one or more additional members or containers containing other drugs.
  • kits or article of item 47 wherein the other drug is selected from the group consisting of an anti-diabetic drug, an anti-cardiovascular disease drug, an antithrombotic drug, an antihypertensive drug, an antilipemic drug, an anticoagulant drug, and an anti-infective agent. drug.
  • the invention relates to a method of preventing and treating diabetes comprising administering to a subject an effective amount of plasminogen or plasmin.
  • the invention features a method of reducing blood glucose in a diabetic subject comprising administering to the subject an effective amount of plasminogen.
  • the invention also relates to the use of plasminogen for reducing blood glucose in a diabetic subject.
  • the invention also relates to the use of plasminogen for the manufacture of a medicament for reducing blood glucose in a diabetic subject.
  • the invention relates to plasminogen for reducing blood glucose in a diabetic subject.
  • the blood glucose is selected from one or more of the group consisting of serum glucose levels, serum fructosamine levels, serum glycated hemoglobin levels.
  • the blood Sugar is serum glucose level.
  • the diabetes is T1DM or T2DM.
  • the invention in another aspect, relates to a method of increasing glucose tolerance in a diabetic subject comprising administering to the subject an effective amount of plasminogen.
  • the invention also relates to the use of plasminogen for increasing glucose tolerance in a diabetic subject.
  • the invention also relates to the use of plasminogen for the manufacture of a medicament for increasing glucose tolerance in a diabetic subject.
  • the present invention relates to plasminogen for increasing glucose tolerance in a diabetic subject.
  • the diabetes is T2DM.
  • the invention relates to a method of promoting postprandial blood glucose decline in a diabetic subject comprising administering to the subject an effective amount of plasminogen.
  • the invention also relates to the use of plasminogen for promoting postprandial blood glucose decline in a diabetic subject.
  • the invention further relates to the use of plasminogen for the manufacture of a medicament for promoting postprandial blood glucose decline in a diabetic subject.
  • the present invention also relates to plasminogen for promoting postprandial blood glucose decline in a diabetic subject.
  • the plasminogen is administered from 30 minutes to 1.5 hours prior to the subject's meal. In other embodiments, the plasminogen is administered 30 minutes to 1 hour before the subject's meal.
  • the invention relates to a method of promoting the utilization of glucose by a diabetic subject comprising administering to the subject an effective amount of plasminogen.
  • the invention also relates to the use of plasminogen for promoting the utilization of glucose by a diabetic subject.
  • the invention also relates to the use of plasminogen for the manufacture of a medicament for promoting the utilization of glucose by a diabetic subject.
  • the invention relates to plasminogen for promoting the utilization of glucose by a diabetic subject.
  • the invention relates to a method of promoting insulin secretion in a diabetic subject comprising administering to the subject an effective amount of plasminogen.
  • the plasminogen also promotes expression of insulin in a diabetic subject.
  • the diabetes is T1DM or T2DM.
  • the plasminogen promotes insulin secretion in a diabetic subject after eating. In other embodiments, the plasminogen promotes insulin secretion in a fasted state in a diabetic subject. In some embodiments, the plasminogen promotes insulin secretion stimulated by a diabetic subject in response to elevated blood glucose, returning blood glucose to normal or near normal levels.
  • the plasminogen reduces expression and/or secretion of glucagon in a subject while promoting expression and/or secretion of the insulin, in particular, the plasminogen
  • the plasminogen By reducing the expression and/or secretion of glucagon in the subject while promoting the expression and/or secretion of the insulin, it is achieved that the blood glucose of the subject is returned to normal or near normal levels.
  • the invention relates to a method of reducing glucagon secretion in a diabetic subject, This includes administering to the subject an effective amount of plasminogen.
  • the invention also relates to the use of plasminogen for reducing glucagon secretion in a diabetic subject.
  • the invention also relates to the use of plasminogen for the manufacture of a medicament for reducing glucagon secretion in a diabetic subject.
  • the present invention relates to plasminogen for reducing glucagon secretion in a diabetic subject.
  • the plasminogen also reduces expression of glucagon in a diabetic subject.
  • the diabetes is T1DM or T2DM.
  • the plasminogen reduces glucagon secretion after eating in a diabetic subject. In other embodiments, the plasminogen reduces glucagon secretion in a fasted state of a diabetic subject. In some embodiments, the plasminogen reduces glucagon secretion in a diabetic subject with elevated blood glucose, returning blood glucose to normal or near normal levels. In some embodiments, the plasminogen reduces glucagon secretion in a diabetic subject with elevated blood glucose, returning blood glucose to normal or near normal levels.
  • the plasminogen promotes expression and/or secretion of insulin, while specifically reducing the expression and/or secretion of glucagon in a subject, in particular, the plasminogen
  • the plasminogen By reducing the expression and/or secretion of glucagon in the subject, the insulin expression and/or secretion is promoted, and the blood glucose of the subject is restored to normal or near normal levels.
  • the plasminogen promotes expression of insulin receptor substrate 2 (IRS-2).
  • the invention features a method of promoting repair of islet cell damage in a diabetic subject comprising administering to the subject an effective amount of plasminogen.
  • the invention also relates to the use of plasminogen for promoting repair of islet cell damage in a diabetic subject.
  • the invention also relates to the use of plasminogen for the manufacture of a medicament for promoting repair of islet cell damage in a diabetic subject.
  • the present invention also relates to plasminogen for promoting repair of islet cell damage in a diabetic subject.
  • the plasminogen promotes expression of insulin receptor substrate 2 (IRS-2).
  • the plasminogen promotes expression of the cytokine TNF-[alpha].
  • the plasminogen promotes expression of a multi-directional nuclear transcription factor NF- ⁇ B in a subject.
  • the islet cell damage is one or more selected from the group consisting of: islet beta cell synthesis and insulin secretion functional damage, islet tissue structure damage, islet collagen deposition, islet fibrosis, islet cells Apoptosis and pancreatic islet secretion of glucagon, insulin imbalance, islet secretion of glucagon and insulin levels are not compatible with the subject's blood glucose levels.
  • the plasminogen reduces glucagon secretion, increased insulin secretion, and in particular, normal balance of pancreatic glucagon and insulin secretion.
  • the invention features a method of protecting a islet of a subject, comprising administering a subject An effective amount of plasminogen.
  • the invention also relates to the use of plasminogen for protecting islets of a subject.
  • the invention also relates to the use of plasminogen for the preparation of a medicament for protecting islets of a subject.
  • the invention relates to plasminogen for use in protecting islets of a subject.
  • the plasminogen reduces islet collagen deposition.
  • the plasminogen reduces fibrosis of islets.
  • the plasminogen reduces apoptosis in islet cells.
  • the plasminogen promotes expression of insulin islet receptor substrate 2 (IRS-2). In some embodiments, the plasminogen promotes repair of islet inflammation. In other embodiments, the plasminogen promotes expression of the cytokine TNF-[alpha]. In other embodiments, the plasminogen promotes expression of a multi-directional nuclear transcription factor NF- ⁇ B in a subject.
  • the subject is a diabetic patient, and in particular, the diabetic patient is T1DM or T2DM. In some embodiments, the T1DM subject is a subject with normal PLG activity or impaired PLG activity.
  • the invention features a method of promoting islet inflammation repair in a diabetic subject comprising administering to the subject an effective amount of plasminogen.
  • the invention also relates to the use of plasminogen for promoting islet inflammation repair in a diabetic subject.
  • the invention also relates to the use of plasminogen for the manufacture of a medicament for promoting islet inflammation repair in a diabetic subject.
  • the present invention relates to plasminogen which promotes repair of islet inflammation in a diabetic subject.
  • the plasminogen promotes expression of the cytokine TNF-[alpha].
  • the plasminogen promotes expression of a multi-directional nuclear transcription factor NF- ⁇ B in a subject.
  • the plasminogen reduces islet collagen deposition. In other embodiments, the plasminogen reduces fibrosis of islets. In other embodiments, the plasminogen inhibits islet cell apoptosis.
  • the diabetic patient is T1DM or T2DM, and specifically, the T1DM subject is a subject having normal PLG activity or impaired PLG activity.
  • the invention features a method of promoting expression of a cytokine TNF-[alpha] in a diabetic subject comprising administering to the subject an effective amount of plasminogen.
  • the invention also relates to the use of plasminogen for promoting the expression of the cytokine TNF-[alpha] in a diabetic subject.
  • the invention also relates to the use of plasminogen for the preparation of a medicament for promoting expression of a cytokine TNF-[alpha] in a diabetic subject.
  • the present invention relates to plasminogen for promoting expression of a cytokine TNF- ⁇ in a diabetic subject.
  • the invention in another aspect, relates to a method of promoting expression of a multi-directional nuclear transcription factor NF- ⁇ B in a diabetic subject comprising administering to the subject an effective amount of plasminogen.
  • the invention also relates to the use of plasminogen for promoting expression of a multi-directional nuclear transcription factor NF- ⁇ B in a diabetic subject.
  • the invention also relates to fibrinolysis The use of a zymogen for the preparation of a medicament for promoting expression of a multi-directional nuclear transcription factor NF- ⁇ B in a diabetic subject.
  • the invention features a method of promoting islet insulin receptor substrate 2 (IRS-2) expression comprising administering to a subject an effective amount of plasminogen.
  • the invention also relates to the use of plasminogen for promoting islet insulin receptor substrate 2 (IRS-2) expression.
  • the invention also relates to the use of plasminogen for the manufacture of a medicament for promoting insulin insulin receptor substrate 2 (IRS-2) expression.
  • the invention relates to plasminogen for promoting islet insulin receptor substrate 2 (IRS-2) expression.
  • the invention in another aspect, relates to a method of promoting insulin secretion in a diabetic subject comprising administering to the subject an effective amount of plasminogen to promote expression of insulin receptor substrate 2 (IRS-2).
  • the invention also relates to the use of plasminogen for promoting insulin secretion in a diabetic subject.
  • the invention also relates to the use of plasminogen for the manufacture of a medicament for promoting insulin secretion in a diabetic subject.
  • the invention relates to plasminogen for promoting insulin secretion in a diabetic subject.
  • the invention features a method of promoting an increase in the number of islet beta cells in a diabetic subject comprising administering to the subject an effective amount of plasminogen.
  • the invention also relates to the use of plasminogen for promoting an increase in the number of islet beta cells in a diabetic subject.
  • the invention further relates to the use of plasminogen for the manufacture of a medicament for promoting an increase in the number of islet beta cells in a diabetic subject.
  • the present invention also relates to plasminogen for promoting an increase in the number of islet ⁇ cells in a diabetic subject.
  • the plasminogen promotes insulin receptor substrate 2 (IRS-2) expression.
  • the invention features a method of reducing apoptosis of islet beta cells comprising administering to a subject an effective amount of plasminogen.
  • the invention also relates to the use of plasminogen for reducing apoptosis of islet beta cells.
  • the invention also relates to the use of plasminogen for the preparation of a medicament for reducing apoptosis of islet beta cells.
  • the invention relates to plasminogen for reducing apoptosis of islet beta cells.
  • the plasminogen promotes insulin receptor substrate 2 (IRS-2) expression.
  • the invention features a method of promoting repair of islet beta cell damage comprising administering to a subject an effective amount of plasminogen.
  • the invention also relates to the use of plasminogen for promoting repair of islet beta cell damage.
  • the invention also relates to the use of plasminogen for the manufacture of a medicament for promoting repair of islet beta cell damage.
  • the invention also relates to plasminogen for promoting repair of islet beta cell damage.
  • the plasminogen promotes insulin receptor substrate 2 (IRS-2) expression.
  • the invention features a method of promoting recovery of islet beta cell function comprising administering to a subject an effective amount of plasminogen.
  • the invention also relates to plasminogen for promoting islet ⁇ cells The purpose of functional recovery.
  • the invention also relates to the use of plasminogen for the preparation of a medicament for promoting recovery of islet beta cell function.
  • the present invention also relates to plasminogen for promoting recovery of islet beta cell function.
  • the plasminogen promotes insulin receptor substrate 2 (IRS-2) expression.
  • the plasminogen may be used in combination with one or more other drugs or treatments.
  • the plasminogen may be combined with one or more drugs selected from the group consisting of antidiabetic drugs, anti-cardiovascular disease drugs, antithrombotic drugs, antihypertensive drugs, antilipemic drugs, anticoagulation Drugs, anti-infectives.
  • the plasminogen has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99 with the sequence 2, 6, 8, 10 or 12. % sequence identity and still have plasminogen activity.
  • the amino acid of the plasminogen is shown as sequence 2, 6, 8, 10 or 12.
  • the plasminogen is added, deleted, and/or substituted on the basis of sequence 2, 6, 8, 10, or 12, 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 a protein with plasminogen activity.
  • the plasminogen is a protein comprising a plasminogen active fragment and still having plasminogen activity.
  • the plasminogen is selected from the group consisting of Glu-plasminogen, Lys-plasminogen, small plasminogen, microplasminogen, delta-plasminogen or their retained plasmin A variant of the original activity.
  • the plasminogen is a natural or synthetic human plasminogen, or a variant or fragment thereof that still retains plasminogen activity.
  • the plasminogen is a human plasminogen ortholog from a primate or a rodent or a variant or fragment thereof that still retains plasminogen activity.
  • plasminogen from a primate or rodent is a homologue, such as a plasminogen homolog from gorilla, rhesus, mouse, cow, horse, dog.
  • the amino acid sequence of the plasminogen of the invention is shown as sequence 2, 6, 8, 10 or 12.
  • the subject is a human. In some embodiments, wherein the subject lacks or lacks plasminogen. In particular, the deficiency or deficiency is innate, secondary and/or local.
  • the plasminogen is administered systemically or locally, preferably by: surface, intravenous, intramuscular, subcutaneous, inhalation, intraspinal, local injection, intra-articular injection or passage. rectum.
  • the topical administration is by direct administration to an osteoporotic region, such as by dressings, catheters, and the like.
  • the plasminogen is administered in combination with a suitable polypeptide carrier or stabilizer.
  • the plasminogen is 0.0001-2000 mg/kg, 0.001-800 mg/kg, 0.01-600 mg/kg, 0.1-400 mg/kg, 1-200 mg/kg, 1-100 mg/kg per day, 10-100mg / kg (calculated per kg body weight) or 0.0001-2000mg / cm 2, 0.001-800mg / cm 2, 0.01-600mg / cm 2, 0.1-400mg / cm 2, 1-200mg / cm 2, 1- 100mg / cm 2, 10-100mg / cm 2 ( calculated per square centimeter of body surface area) of the dose administered, preferably repeated at least once, preferably at least daily administration.
  • the above dosages may be further adjusted as appropriate.
  • the invention relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and plasminogen for use in the methods of the invention.
  • the present invention relates to a prophylactic or therapeutic kit comprising: (i) plasminogen for use in the method of the invention and (ii) for delivery of said plasminogen to The subject of the subject, in particular, the member is a syringe or vial.
  • the kit further comprises a label or instructions for use, the label or instructions for use indicating administration of the plasminogen to the subject to perform the methods of the invention.
  • the present invention is also an article comprising: a container containing a label; and (i) a plasminogen or a pharmaceutical composition comprising plasminogen for use in the method of the present invention, wherein The label indicates administration of the plasminogen or composition to the subject to practice the methods of the invention.
  • the kit or article further comprises an additional one or more components or containers containing other drugs.
  • the other drug is selected from the group consisting of an anti-diabetic drug, an anti-cardiovascular disease drug, an antithrombotic drug, an antihypertensive drug, an antilipemic drug, an anticoagulant drug, an anti-infective drug.
  • Diabetes is caused by genetic factors, immune dysfunction, microbial infections and their toxins, free radical toxins, mental factors and other pathogenic factors that cause islet dysfunction, islets
  • a series of metabolic disorders such as sugar, protein, fat, water and electrolytes caused by resistance, etc., are clinically characterized by hyperglycemia.
  • Diabetes complications are damage or dysfunction of other organs or tissues of the body caused by poor glycemic control during diabetes, including damage to the liver, kidneys, heart, retina, nervous system, or dysfunction. According to the World Health Organization, there are more than 100 complications of diabetes, which is the most common complication known.
  • Insulin resistance refers to a decrease in the efficiency of insulin-promoting glucose uptake and utilization for various reasons, and the body's compensatory secretion of excess insulin produces hyperinsulinemia to maintain blood sugar stability.
  • plasma is a very important enzyme found in the blood that is capable of degrading fibrin polymer.
  • Plastinogen (plg) is the zymogen form of plasmin, which is composed of 810 amino acids based on the sequence of the swiss prot, calculated from the natural human plasminogen amino acid sequence (sequence 4) containing the signal peptide.
  • the full-length PLG contains seven domains: a serine protease domain at the C-terminus, a Pan Apple (PAp) domain at the N-terminus, and five Kringle domains (Kringle 1-5).
  • the signal peptide includes the residue Met1-Gly19
  • PAp includes the residue Glu20-Val98
  • Kringle1 includes the residue Cys103-Cys181
  • Kringle2 includes the residue Glu184-Cys262
  • Kringle3 includes the residue Cys275-Cys352
  • Kringle4 Including the residue Cys377-Cys454
  • Kringle5 includes the residue Cys481-Cys560.
  • the serine protease domain includes the residues Val581-Arg804.
  • Glu-plasminogen is a natural full-length plasminogen consisting of 791 amino acids (not containing a 19 amino acid signal peptide), and the cDNA sequence encoding the sequence is shown in SEQ ID NO: 1, and its amino acid sequence is sequence 2. Shown. In vivo, there is also a Lys-plasminogen which is hydrolyzed from amino acids 76-77 of Glu-plasminogen, and as shown in SEQ ID NO: 6, the cDNA sequence encoding the amino acid sequence is as shown in SEQ ID NO: 5 Shown.
  • ⁇ -plasminogen is a fragment of full-length plasminogen deleted from Kringle2-Kringle5 structure, containing only Kringle1 and serine protease domains [39,40] .
  • the amino acid sequence of ⁇ -plasminogen has been reported in the literature (sequence 8) [40] , the cDNA sequence encoding the amino acid sequence is SEQ ID NO: 7.
  • Mini-plasminogen consists of Kringle5 and a serine protease domain, which has been reported to include the residue Val443-Asn791 (the Glu residue of the Glu-plg sequence containing no signal peptide is the starting amino acid) [41] , and its amino acid sequence is as sequence As shown in Figure 10, the cDNA sequence encoding the amino acid sequence is shown in SEQ ID NO: 9. Micro-plasminogen contains only the serine protease domain, and its amino acid sequence has been reported to include the residue Ala543-Asn791 (the Glu residue of the Glu-plg sequence containing no signal peptide is the starting amino acid) [42] , and there is also the patent CN102154253A.
  • sequence includes the residue Lys531-Asn791 (the Glu residue of the Glu-plg sequence not containing the signal peptide is the starting amino acid), and the amino acid sequence of this patent is shown in the sequence 12, and the amino acid sequence is encoded.
  • the cDNA sequence is shown in SEQ ID NO: 11.
  • Plasin of the present invention is used interchangeably with “fibrinolytic enzyme” and “fibrinolytic enzyme”, and has the same meaning; “plasminogen” and “plasminogen”, “fibrinolytic enzyme” "Original” is used interchangeably and has the same meaning.
  • the term "deficiency" of plasminogen means that the content or activity of plasminogen in the subject is lower than that of a normal person, and is low enough to affect the normal physiological function of the subject;
  • the meaning of "deficient" of plasminogen is that the content or activity of plasminogen in the subject is significantly lower than that of normal people, and even the activity or expression is minimal, and only by external supply can maintain normal physiological functions.
  • aging and “premature aging” are used interchangeably to mean the same meaning.
  • plasminogen adopts a closed inactive conformation, but when bound to the surface of a thrombus or cell, it is converted to an open conformational activity mediated by a plasminogen activator (PA).
  • PA plasminogen activator
  • the active PLM can further hydrolyze the fibrin clot into a fibrin degradation product and a D-dimer, thereby dissolving the thrombus.
  • PAp domain of PLG comprises an important determinant that maintains plasminogen in an inactive blocking conformation, while the KR domain is capable of binding to lysine residues present on the receptor and substrate.
  • a variety of enzymes are known that act as PLG activators, including: tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), kallikrein, and coagulation factor XII (Hagman factor). Wait.
  • tissue plasminogen activator tPA
  • uPA urokinase plasminogen activator
  • kallikrein kallikrein
  • coagulation factor XII Hagman factor
  • a "plasminogen active fragment” refers to an active fragment that binds to a target sequence in a substrate and exerts a proteolytic function in a plasminogen protein.
  • the technical solution of the present invention relating to plasminogen covers the technical solution of replacing plasminogen with a plasminogen active fragment.
  • the plasminogen active fragment of the present invention is a protein comprising a serine protease domain of plasminogen, preferably, the present invention
  • the plasminogen active fragment of the present invention comprises the sequence 14, a protein having an amino acid sequence of at least 80%, 90%, 95%, 96%, 97%, 98%, 99% homology to the sequence 14.
  • the plasminogen of the present invention comprises a protein comprising the plasminogen active fragment and still retaining the plasminogen activity.
  • blood plasminogen and its activity assays include: detection of tissue plasminogen activator activity (t-PAA), detection of plasma tissue plasminogen activator antigen (t- PAAg), detection of plasma tissue plasminogen activity (plgA), detection of plasma tissue plasminogen antigen (plgAg), detection of plasma tissue plasminogen activator inhibitor activity, plasma tissue fibrinolysis Detection of zymogen activator inhibitor antigen, plasma plasmin-anti-plasmin complex assay (PAP).
  • t-PAA tissue plasminogen activator activity
  • t- PAAg detection of plasma tissue plasminogen activator antigen
  • plgA plasma tissue plasminogen activity
  • plgAg detection of plasma tissue plasminogen antigen
  • PAP plasma tissue fibrinolysis Detection of zymogen activator inhibitor antigen, plasma plasmin-anti-plasmin complex assay
  • the most commonly used detection method is the chromogenic substrate method: adding streptokinase (SK) and chromogenic substrate to the plasma to be tested, and the PLG in the tested plasma is converted into PLM under the action of SK, and the latter acts on The chromogenic substrate is then measured spectrophotometrically and the increase in absorbance is directly proportional to the plasminogen activity.
  • plasminogen activity in blood can also be measured by immunochemical methods, gel electrophoresis, immunoturbidimetry, and radioimmunoassay.
  • ortholog or ortholog refers to homologs between different species, including both protein homologs and DNA homologs, also known as orthologs, orthologs. It specifically refers to a protein or gene that has evolved from the same ancestral gene in different species.
  • the plasminogen of the present invention includes human natural plasminogen, and also includes plasminogen orthologs or orthologs of plasminogen activity derived from different species.
  • Constant substitution variant refers to a change in one of the given amino acid residues without altering the overall conformation and function of the protein or enzyme, including but not limited to similar properties (eg, acidic, basic, hydrophobic, etc.)
  • the amino acid replaces the amino acid in the amino acid sequence of the parent protein.
  • Amino acids having similar properties are well known. For example, arginine, histidine, and lysine are hydrophilic basic amino acids and are interchangeable.
  • isoleucine is a hydrophobic amino acid that can be replaced by leucine, methionine or valine. Therefore, the similarity of two protein or amino acid sequences of similar function may be different.
  • Constant substitution variants also includes determining polypeptides or enzymes having more than 60% amino acid identity by BLAST or FASTA algorithm. If it is more than 75%, preferably more than 85%, or even more than 90%. Optimal and have the same or substantially similar properties or functions as the native or parent protein or enzyme.
  • Isolated plasminogen refers to a plasminogen protein that is isolated and/or recovered from its natural environment.
  • the plasminogen will purify (1) to a purity greater than 90%, greater than 95%, or greater than 98% by weight, as determined by the Lowry method, eg, over 99% (by weight), (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by using a rotating cup sequence analyzer, or (3) to homogeneity, which is by use Coomassie blue or silver staining was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions.
  • Isolated plasminogen also includes plasminogen prepared from recombinant cells by bioengineering techniques and isolated by at least one purification step.
  • polypeptide peptide
  • protein protein
  • fusion proteins including, but not limited to, fusion proteins having a heterologous amino acid sequence, fusions having heterologous and homologous leader sequences (with or without an N-terminal methionine residue);
  • percent amino acid sequence identity with respect to a reference polypeptide sequence is defined as the introduction of a gap as necessary to achieve maximum percent sequence identity, and without any conservative substitution being considered as part of sequence identity, in the candidate sequence The percentage of amino acid residues that are identical in amino acid residues in the reference polypeptide sequence. Comparisons for the purpose of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art will be able to determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum contrast over the full length of the sequences being compared. However, for the purposes of the present invention, amino acid sequence identity percent values are generated using the sequence comparison computer program ALIGN-2.
  • amino acid sequence identity of a given amino acid sequence A relative to a given amino acid sequence B (or may be expressed as having or comprising relative to, and, or for a given amino acid sequence)
  • a given amino acid sequence A of a certain % amino acid sequence identity of B is calculated as follows:
  • X is the number of amino acid residues scored by the sequence alignment program ALIGN-2 in the A and B alignments of the program, and wherein Y is the total number of amino acid residues in B.
  • % amino acid sequence identity of A with respect to B may not be equal to the % amino acid sequence of B relative to A.
  • All % amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the previous paragraph, unless explicitly stated otherwise.
  • the terms “treating” and “preventing” refer to obtaining a desired pharmacological and/or physiological effect.
  • the effect may be to completely or partially prevent the disease or its symptoms, and/or to partially or completely cure the disease and/or its symptoms, and includes: (a) preventing the disease from occurring in the subject, the subject may have The cause of the disease, but not yet diagnosed as having a disease; (b) inhibiting the disease, ie, retarding its formation; and (c) reducing the disease and/or its symptoms, ie causing the disease and/or its symptoms to subside.
  • the terms "individual”, “subject” and “patient” are used interchangeably herein to refer to a mammal, including but not limited to a mouse (rat, mouse), a non-human primate, a human, a dog, a cat. Hoofed animals (such as horses, cattle, sheep, pigs, goats).
  • “Therapeutically effective amount” or “effective amount” refers to an amount of plasminogen sufficient to effect such prevention and/or treatment of a disease when administered to a mammal or other subject to treat the disease.
  • the “therapeutically effective amount” will vary depending on the plasminogen used, the severity of the disease and/or its symptoms of the subject to be treated, and the age, weight, and the like.
  • Plasminogen can be isolated and purified from nature for further therapeutic use, or it can be synthesized by standard chemical peptide synthesis techniques. When the polypeptide is chemically synthesized, it can be synthesized in a liquid phase or a solid phase.
  • Solid phase polypeptide synthesis SPPS
  • Fmoc and Boc Various forms of SPPS, such as Fmoc and Boc, can be used to synthesize plasminogen.
  • the attached solid phase free N-terminal amine is coupled to a single N-protected amino acid unit. This unit is then deprotected to reveal a new N-terminal amine that can be attached to other amino acids.
  • the peptide remains immobilized on the solid phase and then cut off.
  • the plasminogen of the present invention can be produced using standard recombinant methods.
  • a nucleic acid encoding plasminogen is inserted into an expression vector operably linked to a regulatory sequence in an expression vector.
  • Expression control sequences include, but are not limited to, promoters (eg, naturally associated or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences.
  • Expression regulation can be a eukaryotic promoter system in a vector that is capable of transforming or transfecting eukaryotic host cells (eg, COS or CHO cells). Once the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high level expression of the nucleotide sequence and collection and purification of plasminogen.
  • Suitable expression vectors are typically replicated as an episome in the host organism or as an integral part of the host chromosomal DNA.
  • expression vectors typically contain a selection marker (eg, ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance, or neomycin resistance) to facilitate transformation of the desired DNA sequence with foreign sources. Those cells are tested.
  • a selection marker eg, ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance, or neomycin resistance
  • Escherichia coli is an example of a prokaryotic host cell that can be used to clone a subject antibody-encoding polynucleotide.
  • Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. Genus (Pseudomonas) species.
  • expression vectors can also be generated which will typically contain expression control sequences (e.g., origins of replication) that are compatible with the host cell.
  • promoters such as the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter system, or the promoter system from phage lambda. Promoters typically control expression, optionally in the context of manipulating a gene sequence, and have a ribosome binding site sequence, etc., to initiate and complete transcription and translation.
  • yeast can also be used for expression.
  • Yeast e.g., S. cerevisiae
  • Pichia are examples of suitable yeast host cells in which a suitable vector has expression control sequences (e.g., a promoter), an origin of replication, a termination sequence, and the like, as desired.
  • a typical promoter comprises 3-phosphoglycerate kinase and other saccharolytic enzymes.
  • Inducible yeast is initiated by a promoter specifically comprising an alcohol dehydrogenase, an isocytochrome C, and an enzyme responsible for the utilization of maltose and galactose.
  • mammalian cells e.g., mammalian cells cultured in in vitro cell culture
  • Suitable mammalian host cells include CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, and transformed B cells or hybridomas.
  • Expression vectors for these cells may contain expression control sequences, such as Point, promoter and enhancer (Queen et al, Immnol. Rev.
  • RNA splice sites such as ribosome binding sites, RNA splice sites, polyadenylation sites , and transcription terminator sequences.
  • suitable expression control sequences are promoters derived from the white immunoglobulin gene, SV40, adenovirus, bovine papilloma virus, cytomegalovirus, and the like. See Co et al, J. Immunol. 148: 1149 (1992).
  • the invention may be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity column, column chromatography, high performance liquid chromatography (HPLC), gel electrophoresis, and the like.
  • Plasminogen is substantially pure, such as at least about 80% to 85% pure, at least about 85% to 90% pure, at least about 90% to 95% pure, or 98% to 99% pure. Or more pure, for example, free of contaminants, such as cellular debris, macromolecules other than the subject antibody, and the like.
  • the jelly can be formed by mixing plasminogen of the desired purity with an optional pharmaceutical carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences, 16th Edition, Osol, A. ed. (1980)).
  • the therapeutic formulation is prepared as a dry formulation or as an aqueous solution.
  • Acceptable carriers, excipients, and stabilizers are non-toxic to the recipient at the dosages and concentrations employed, and include buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives such as Octadecyldimethylbenzylammonium chloride; chlorinated hexane diamine; benzalkonium chloride, benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl p-hydroxybenzoic acid Esters such as methyl or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; m-cresol; low molecular weight polypeptide (less than about 10 residues) Protein such as serum albumin, gelatin or immunoglobulin; hydrophilic polymer such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, his
  • the formulations of the invention may also contain more than one active compound as required for the particular condition being treated, preferably those having complementary activities and no side effects to each other.
  • active compound for example, antihypertensive drugs, antiarrhythmic drugs, drugs for treating diabetes, and the like.
  • the plasminogen of the present invention may be encapsulated in microcapsules prepared by, for example, coacervation techniques or interfacial polymerization, for example, may be placed in a glial drug delivery system (eg, liposomes, albumin microspheres, microemulsions, Nanoparticles and nanocapsules are placed in hydroxymethylcellulose or gel-microcapsules and poly-(methyl methacrylate) microcapsules in a macroemulsion.
  • glial drug delivery system eg, liposomes, albumin microspheres, microemulsions, Nanoparticles and nanocapsules are placed in hydroxymethylcellulose or gel-microcapsules and poly-(methyl methacrylate) microcapsules in a macroemulsion.
  • the plasminogen of the invention for in vivo administration must be sterile. This can be easily achieved by filtration through a sterile filter before or after lyophilization and reconstitution.
  • the plasminogen of the present invention can prepare a sustained release preparation.
  • sustained release formulations include solid hydrophobic polymeric semi-permeable matrices having a shape and containing glycoproteins, such as films or microcapsules.
  • sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) (Langer et al, J. Biomed. Mater. Res., 15: 167-277 (1981); Langer, Chem .Tech., 12:98-105 (1982)) or poly(vinyl alcohol), polylactide (U.S.
  • Patent 3,737,919, EP 58,481 copolymer of L-glutamic acid and ethyl-L-glutamic acid ( Sidman, et al, Biopolymers 22: 547 (1983)), non-degradable ethylene-vinyl acetate (Langer, et al, supra), or degradable lactic acid-glycolic acid copolymer such as Lupron DepotTM (Injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly D-(-)-3-hydroxybutyric acid.
  • Lupron DepotTM injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate
  • Polymers such as ethylene-acetic acid Vinyl ester and lactic acid-glycolic acid can release molecules for more than 100 days, while some hydrogels release proteins for a short time.
  • a reasonable strategy for protein stabilization can be designed according to the relevant mechanism. For example, if the mechanism of aggregation is found When thiodisulfide bonds are exchanged to form intermolecular SS bonds, the sulfhydryl residues can be modified, lyophilized from acidic solutions, and humidity controlled. Stabilization is achieved with suitable additives and the development of specific polymer matrix compositions.
  • the invention may be practiced in various ways, for example by intravenous, intraperitoneal, subcutaneous, intracranial, intrathecal, intraarterial (for example via carotid), intramuscular, intranasal, topical or intradermal administration or spinal cord or brain delivery.
  • Administration of the pharmaceutical composition
  • Aerosol formulations such as nasal spray formulations comprise purified aqueous or other solutions of the active agents and preservatives and isotonic agents. Such formulations are adjusted to a pH and isotonic state compatible with the nasal mucosa.
  • the plasminogen pharmaceutical compositions of the invention may be modified or formulated in such a manner as to provide their ability to cross the blood brain barrier.
  • Preparations for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffering media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, or fixed oils.
  • Intravenous vehicles contain liquid and nutritional supplements, electrolyte supplements, and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases, and the like.
  • the plasminogen of the invention is formulated with an agent that promotes across the blood brain barrier.
  • the plasminogen of the invention is fused directly or via a linker to a carrier molecule, peptide or protein that facilitates crossing the blood brain barrier.
  • the plasminogen of the invention is fused to a polypeptide that binds to an endogenous blood brain barrier (BBB) receptor. Linking plasminogen to a polypeptide that binds to the endogenous BBB receptor facilitates passage through the BBB.
  • BBB blood brain barrier
  • Suitable polypeptides that bind to an endogenous BBB receptor include antibodies, such as monoclonal antibodies, or antigen-binding fragments thereof that specifically bind to an endogenous BBB receptor.
  • Suitable endogenous BBB receptors include, but are not limited to, insulin.
  • antibodies are encapsulated in liposomes. See, for example, U.S. Patent Publication No. 2009/0156498.
  • the medical staff will determine the dosage regimen based on various clinical factors. As is well known in the medical arts, the dosage of any patient depends on a variety of factors, including the patient's size, body surface area, age, specific compound to be administered, sex, number and route of administration, overall health, and other medications administered simultaneously. .
  • the pharmaceutical composition of the present invention comprising plasminogen may be, for example, in the range of about 0.0001 to 2000 mg/kg per day, or about 0.001 to 500 mg/kg (e.g., 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 10 mg/kg, 50 mg/kg, etc.) Subject weight.
  • the dose can be 1 mg/kg body weight or 50 mg/kg body weight or in the range of 1-50 mg/kg, or at least 1 mg/kg. Dosages above or below this exemplary range are also contemplated, particularly in view of the above factors. Intermediate doses in the above ranges are also included in the scope of the present invention.
  • the subject can administer such doses daily, every other day, every week, or according to any other schedule determined by empirical analysis.
  • An exemplary dosage schedule includes 1-10 mg/kg for several days. The therapeutic effect and safety of thrombus and thrombosis-related diseases need to be evaluated and periodically evaluated in the drug administration process of the present invention.
  • One embodiment of the invention relates to the determination of therapeutic efficacy and therapeutic safety following treatment of a subject with plasminogen.
  • Commonly used monitoring and evaluation of osteoporosis treatment effects include Interviews (adverse reactions, standardized medications, basic measures, and reassessment of fracture risk factors), new fracture assessment (clinical fractures, height reduction and imaging studies), bone mineral density (BMD) measurements, and bone turnover biochemical markers Bone turnover markers (BTM) detection, and comprehensive reassessment based on these data.
  • BMD is currently the most widely used method for monitoring and evaluating efficacy.
  • BMD can be measured by dual energy X-ray absorptiometry (DXA), quantitative computed tomography (QCT), single photon absorption measurement (SPA), or ultrasonic measurement.
  • DXA dual energy X-ray absorptiometry
  • QCT quantitative computed tomography
  • SPA single photon absorption measurement
  • ultrasonic measurement BMD can be detected once a year after the start of treatment, and the interval can be appropriately extended after the BMD has stabilized, for example, once every 2 years.
  • the bone formation index currently used in serological indicators is procollagen type 1 n-terminal propeptide (PINP), and the bone resorption index is serum type 1 procollagen C-terminal peptide. (serum C-terminal telopeptide, S-CTX). According to the research progress, more reasonable detection indicators can be adjusted in time.
  • PINP procollagen type 1 n-terminal propeptide
  • S-CTX serum C-terminal telopeptide
  • Baseline values should be measured prior to initiation of treatment, 3 months after application of drug-promoting therapy, and 3 to 6 months after application of inhibitory drug therapy.
  • BTM can provide dynamic information of bones, independent of BMD in function and function, and also complements BMD as a monitoring tool. The combination of the two has higher clinical value. In general, if BMD rises or stabilizes after treatment, BTM has an expected change, and no fracture occurs during treatment, the treatment response is considered to be good.
  • the present invention relates to the use of plasminogen and variants thereof for the treatment of a subject during and after treatment, the determination of the safety of the treatment regimen, including but not limited to serum for the drug in the subject Half-life, treatment half-life, half-toxicity (TD50), and median lethal dose (LD50) were counted, or various adverse events such as sensitization reactions occurred during or after treatment were observed.
  • TD50 half-life
  • LD50 median lethal dose
  • the article preferably includes a container, label or package insert.
  • Suitable containers are bottles, vials, syringes, and the like.
  • the container can be made of various materials such as glass or plastic.
  • the container contains a composition that is effective to treat a disease or condition of the invention and has a sterile access port (eg, the container can be an intravenous solution or vial containing a stopper that can be penetrated by a hypodermic needle) of).
  • At least one active agent in the composition is plasminogen.
  • the label on or attached to the container indicates that the composition is used to treat the aging or aging related conditions of the present invention.
  • the article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as phosphate buffered saline, Ringer's solution And a glucose solution. It may further comprise other materials required from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes.
  • the article comprises a package insert with instructions for use, including, for example, a user instructing the composition to administer the plasminogen composition and other drugs to treat the accompanying disease.
  • FIG. 1 Immunohistochemical observation of islet glucagon after 35 days of plasminogen administration in 24-25-week-old diabetic mice.
  • A is a normal control group
  • B is a vehicle PBS control group
  • C is a plasminogen group.
  • the results showed that glucagon was expressed in the ⁇ -cell region surrounding the islet in normal control mice.
  • the glucagon-positive cells (arrow markers) in the control group of PBS were significantly increased, and the positive cells infiltrated into the central region of the islets; the glucagon-positive cells in the plasminogen group were scattered.
  • the distribution was distributed around the islets, and the islet morphology was closer to normal mice than the PBS group. It indicated that plasminogen can significantly inhibit the proliferation of islet ⁇ cells and the secretion of glucagon, and correct the disorder of islet ⁇ cell distribution, thus promoting the repair of islet injury.
  • FIG. 1 Immunohistochemical observation of islet glucagon after 35 days of plasminogen administration in 26-week-old diabetic mice.
  • A is the normal control group
  • B is the vehicle control group
  • C is the plasminogen group
  • D is the quantitative analysis result. The results showed that glucagon was expressed in the ⁇ -cell region surrounding the islet in normal control mice.
  • the glucagon-positive cells (arrow markers) in the control group of PBS were significantly increased, the positive cells infiltrated into the central region of the islets, and the mean optical density quantitative analysis results were statistically different (* P ⁇ 0.05); the glucagon-positive cells in the plasminogen group were scattered around the islets, and the plasminogen group was more similar to the normal mice than the PBS group. It indicated that plasminogen can significantly inhibit the proliferation of islet ⁇ cells and the secretion of glucagon, and correct the disorder of islet ⁇ cell distribution, thus promoting the repair of islet injury.
  • FIG. 3 shows the results of islet glucagon immunohistochemistry in a T1DM model of mice with normal PLG activity 28 days after administration of plasminogen.
  • A is a blank control group
  • B is a vehicle control group
  • C is a plasminogen group
  • D is a quantitative analysis result.
  • the results showed that the positive expression of glucagon in the vehicle control group was significantly higher than that in the lysin group, and the statistical difference of the mean optical density quantitative analysis was significant (* indicates P ⁇ 0.05). This indicates that plasminogen can significantly reduce the secretion of glucagon from islet ⁇ cells in diabetic mice and promote the repair of islet injury.
  • Figure 4 Results of blood glucose test after 10 and 31 days of administration of plasminogen in 24-25-week-old diabetic mice. The results showed that the blood glucose in the plasminogen group was significantly lower than that in the vehicle control group, and the statistical difference Significant (* indicates P ⁇ 0.05, ** indicates P ⁇ 0.01). In addition, with the prolongation of the administration time, the blood glucose of the control vehicle PBS control group increased, while the blood glucose to the plasminogen group gradually decreased. This indicates that plasminogen has a hypoglycemic effect.
  • Figure 5 shows the effect of plasminogen on serum fructosamine concentration in diabetic rats.
  • the test results showed that the concentration of serum fructosamine was significantly decreased after administration of plasminogen, and the statistical difference was extremely significant compared with that before administration (** means P ⁇ 0.01). This indicates that plasminogen can significantly reduce blood sugar in diabetic mice.
  • Figure 7 Results of plasma glycated hemoglobin detection after administration of plasminogen for 35 days in 26-week-old diabetic mice. The results showed that the OD value of glycosylated hemoglobin in the plasminogen group was significantly lower than that in the vehicle control group, and the statistical difference was extremely significant (** means P ⁇ 0.01). This indicates that plasminogen has a role in reducing blood sugar in diabetic mice.
  • FIG. 8 IPGTT assay results after 10 days of administration of plasminogen in 26-week-old diabetic mice. The results showed that after intraperitoneal injection of glucose, the blood glucose level of the plasminogen group was lower than that of the vehicle PBS control group, and the glucose tolerance curve of the plasminogen group was closer to normal mice than the vehicle PBS control group. group. This indicates that plasminogen can significantly improve the glucose tolerance of diabetic mice.
  • Fig. 9 Results of blood glucose test after fasting in 10 days after administration of plasminogen in normal mice with PLG activity in T1DM model. The results showed that the blood glucose of the control vehicle PBS control group was significantly higher than that of the plasminogen group, and the statistical difference was extremely significant (*** indicates P ⁇ 0.001). This indicates that plasminogen can significantly reduce blood glucose levels in T1DM models in mice with normal PLG activity.
  • FIG. 10 Results of IPGTT assay after 28 days of administration of plasminogen in T1DM model normal PLG-active mice. The results showed that the blood glucose concentration of the mice in the vehicle control group was significantly higher than that of the plasminogen group, and the glucose tolerance curve of the plasminogen group was closer to that of the normal mice. This indicates that plasminogen can increase the glucose tolerance of normal mice with PLG activity in the T1DM model.
  • Figure 11 shows the results of blood glucose test after 20 days of administration of plasminogen in T1DM model mice.
  • FIG. 12 Serum insulin test results after 35 days of administration of plasminogen in 26-week-old diabetic mice. The results showed that the serum insulin level in the plasminogen group was significantly higher than that in the vehicle control group, and the statistical difference was significant (* indicates P ⁇ 0.05). This indicates that plasminogen can effectively promote insulin secretion.
  • FIG. 13 HE staining of pancreas and islet area ratio after 31 days of administration of plasminogen in 24-25-week-old diabetic mice.
  • a and B are the control group for the vehicle PBS
  • C and D are for the plasminogen group
  • E is the quantitative analysis of the islet area.
  • the results showed that most of the islets in the vehicle control group were atrophied, and the atrophic islet cells were replaced by acinar (marked by sputum).
  • the acinar hyperplasia at the edge of the islets caused the boundary between the islets and the acinus to be unclear; Most of the islets in the zymogen group had larger area than the control group, and there was no acinar hyperplasia in the islets.
  • Figure 14 shows the results of islet Sirius red staining after administration of plasminogen for 23 days in 24-25-week-old diabetic mice.
  • A is the control vehicle PBS control group
  • B is the plasminogen group
  • C is the quantitative analysis result.
  • the results showed that the islet collagen deposition (arrow mark) in the plasminogen group was significantly less than that in the vehicle PBS control group, and the statistical difference was significant (* indicates P ⁇ 0.05). This indicates that plasminogen can improve the fibrosis of islets in diabetic animals.
  • FIG. 15 Immunohistochemical staining of islet Caspase-3 after administration of plasminogen for 23 days in 24-25-week-old diabetic mice.
  • A is the vehicle PBS control group and B is the plasminogen group.
  • the results showed that the expression of Caspase-3 in the plasminogen group (arrow mark) was significantly lower than that in the vehicle PBS control group. This indicates that plasminogen can reduce the apoptosis of islet cells and protect the pancreatic tissue of diabetic mice.
  • Figure 16 Results of immunohistochemical staining of islet insulin 35 days after administration of plasminogen in 18-week-old diabetic mice.
  • A is the control vehicle PBS control group
  • B is the plasminogen group
  • C is the quantitative analysis result.
  • Figure 17 shows the results of insulin immunohistochemical staining of islets after administration of plasminogen for 25 days in 24-25-week-old diabetic mice.
  • A is the control vehicle PBS control group
  • B is the plasminogen group
  • C is the quantitative analysis result.
  • the results showed that the expression of insulin in the plasminogen group (arrow mark) was significantly higher than that in the vehicle control group, and the statistical difference was significant (* indicates P ⁇ 0.05). It indicates that plasminogen can promote islet function repair and promote insulin production and secretion.
  • FIG. 18 Results of insulin immunohistochemical staining of islets after 35 days of administration of plasminogen in 26-week-old diabetic mice.
  • A is the control vehicle PBS control group
  • B is the plasminogen group
  • C is the quantitative analysis result.
  • the results showed that the expression of insulin in the plasminogen group (arrow mark) was significantly higher than that in the vehicle control group, and the statistical difference was extremely significant (** indicates P ⁇ 0.01). It indicates that plasminogen can effectively promote islet function repair and promote insulin production and secretion.
  • FIG. 19 Immunohistochemical staining of NF- ⁇ B in pancreatic tissue after administration of plasminogen for 23 days in 24-25-week-old diabetic mice.
  • A is the normal control group
  • B is the vehicle control group
  • C is the plasminogen group
  • D is the quantitative analysis result.
  • the results showed that the expression of NF- ⁇ B in the plasminogen group (arrow mark) was significantly higher than that in the vehicle-treated PBS control group, and the statistical difference was significant (* indicates P ⁇ 0.05). This indicates that plasminogen can promote the expression of multi-directional nuclear transcription factor NF- ⁇ B, thereby promoting the repair of islet inflammation in 24-25-week-old diabetic mice.
  • FIG. 20 Immunohistochemical observation of islet glucagon after administration of plasminogen for 18 days in 18-week-old diabetic mice.
  • A is the normal control group
  • B is the vehicle control group
  • C is the plasminogen group
  • D is the quantitative analysis result.
  • the results showed that glucagon was expressed in the ⁇ -cell region surrounding the islet in normal control mice.
  • glucagon-positive cells arrow markers
  • glucagon-positive cells were significantly increased in the vehicle PBS control group, glucagon-positive cells infiltrated into the central region of the islets, and statistical differences in mean optical density quantitative analysis results.
  • FIG. 21 Immunohistochemical results of islet IRS-2 after administration of plasminogen for 18 days in 18-week-old diabetic mice.
  • A is the normal control group
  • B is the vehicle control group
  • C is the plasminogen group
  • D is the quantitative analysis result.
  • the results showed that the expression of IRS-2 in the PBS control group was significantly less than that in the plasminogen group, and the statistical difference was extremely significant (** indicates P ⁇ 0.01); the plasminogen group was given.
  • the expression level of IRS-2 was closer to that of normal control mice than to the vehicle PBS group. This indicates that plasminogen can effectively increase the expression of IRS-2 in islet cells, improve insulin signaling, and reduce islet ⁇ cell damage in diabetic mice.
  • FIG. 22 Immunohistochemical observation of islet IRS-2 after 31 days of administration of plasminogen in 24-25-week-old diabetic mice.
  • A is the normal control group
  • B is the vehicle control group
  • C is the plasminogen group
  • D is the quantitative analysis result.
  • the results showed that the islet IRS-2 positive in the control vehicle PBS control group.
  • Sexual expression was significantly less than that given to the plasminogen group, and the statistical difference was significant (* indicates P ⁇ 0.05); the expression level of IRS-2 in the plasminogen group was closer to the normal control group than in the vehicle PBS group. mouse. This indicates that plasminogen can effectively increase the expression of IRS-2 in islet cells, improve insulin signaling, and reduce islet ⁇ cell damage in diabetic mice.
  • FIG. 23 Immunohistochemical results of islet IRS-2 after 35 days of administration of plasminogen in 26-week-old diabetic mice.
  • A is the normal control group
  • B is the vehicle control group
  • C is the plasminogen group
  • D is the quantitative analysis result.
  • the results showed that the positive expression of IRS-2 in the PBS control group (arrow mark) was significantly less than that in the plasminogen group; the expression level of IRS-2 in the plasminogen group was closer to the normal control than the vehicle PBS group. Group of mice. This indicates that plasminogen can effectively increase the expression of IRS-2 in islet cells, improve insulin signaling, and reduce islet ⁇ cell damage in diabetic mice.
  • Figure 24 shows the results of immunohistochemical observation of islet IRS-2 in T1DM mice with normal PLG activity 28 days after administration of plasminogen.
  • A is a normal control group
  • B is a vehicle PBS control group
  • C is a plasminogen group.
  • the results showed that the positive expression of IRS-2 in the PBS control group (arrow mark) was significantly less than that in the plasminogen group; the expression level of IRS-2 in the plasminogen group was closer to the normal control than the vehicle PBS group. Group of mice. This indicates that plasminogen can effectively increase the expression of IRS-2 in islet cells, improve insulin signaling, and reduce islet ⁇ -cell damage in normal T1DM mice with PLG activity.
  • FIG. 25 Results of islet neutrophil immunohistochemistry after administration of plasminogen for 26 days in 26-week-old diabetic mice.
  • A is a normal control group
  • B is a vehicle PBS control group
  • C is a plasminogen group.
  • the results showed that the positive expression cells (arrow markers) in the plasminogen group were less than the vehicle control group, and the plasminogen group was closer to the normal control group than the vehicle PBS group. This indicates that plasminogen can reduce the infiltration of neutrophils.
  • FIG. 26 Immunohistochemical observation of islet neutrophils 28 days after administration of plasminogen in mice with impaired PLG activity in the T1DM model.
  • A is a blank control group
  • B is a vehicle PBS control group
  • C is a plasminogen group.
  • the results showed that the positive expression cells (arrow markers) in the plasminogen group were less than the vehicle control group, and the plasminogen group was closer to the blank control group than the vehicle PBS group. This indicates that plasminogen plasminogen can reduce islet neutrophil infiltration in T1DM model in mice with impaired PLG activity.
  • Figure 27 Results of islet neutrophil immunohistochemistry 28 days after administration of plasminogen in P1 active normal mice.
  • A is a blank control group
  • B is a vehicle PBS control group
  • C is a plasminogen group.
  • the results showed that the positive expression cells (arrow markers) were given to the plasminogen group.
  • the vehicle was administered to the PBS control group, and the plasminogen group was closer to the blank control group than the vehicle PBS group. This indicates that plasminogen can promote islet neutrophil infiltration in T1DM model in mice with normal PLG activity.
  • FIG. 28 Immunohistochemical observation of islet insulin 28 days after administration of plasminogen in mice with impaired PLG activity in the T1DM model.
  • A is a blank control group
  • B is a vehicle PBS control group
  • C is a plasminogen group.
  • the results of immunohistochemistry showed that the positive expression of insulin in the plasminogen group (arrow mark) was significantly higher than that in the vehicle control group, and the plasminogen group was closer to the blank control group than the vehicle PBS group. This indicates that plasminogen can promote the synthesis and secretion of insulin in mice with impaired PLG activity in the T1DM model.
  • FIG. 29 Immunohistochemical observation of islet insulin 28 days after administration of plasminogen in P1 active normal mice.
  • A is a blank control group
  • B is a vehicle PBS control group
  • C is a plasminogen group.
  • the results of immunohistochemistry showed that the positive expression of insulin in the plasminogen group (arrow mark) was significantly higher than that in the vehicle control group, and the plasminogen group was closer to the blank control group than the vehicle PBS group. This indicates that plasminogen promotes the synthesis and expression of insulin in mice with normal PLG activity in the T1DM model.
  • FIG. 30 Immunohistochemical observation of islet NF- ⁇ B 28 days after administration of plasminogen in PIL-activated mice.
  • A is a blank control group
  • B is a vehicle PBS control group
  • C is a plasminogen group.
  • the results showed that the expression of NF- ⁇ B in the plasminogen group (arrow mark) was significantly higher than that in the vehicle-treated PBS control group. This indicates that plasminogen can promote the expression of inflammatory repair factor NF- ⁇ B, thereby promoting the repair of islet inflammation.
  • FIG. 31 Immunohistochemical observation of islet NF- ⁇ B after administration of plasminogen for 18 days in 18-week-old diabetic mice.
  • A is the vehicle PBS control group and B is the plasminogen group.
  • the results of the experiment showed that the expression of NF- ⁇ B (arrow mark) in the plasminogen group was significantly higher than that in the vehicle control group. This indicates that plasminogen can promote the expression of multi-directional nuclear transcription factor NF- ⁇ B, thereby promoting the repair of islet inflammation in relatively young (18 weeks old) diabetic mice.
  • FIG. 32 Immunohistochemical observation of islet NF- ⁇ B after 35 days of administration of plasminogen in 26-week-old diabetic mice.
  • A is a normal control group
  • B is a vehicle PBS control group
  • C is a plasminogen group.
  • the experimental results of the present invention showed that the expression of NF- ⁇ B (arrow mark) in the plasminogen group was significantly higher than that in the vehicle PBS control group. This indicates that plasminogen can promote the expression of multi-directional nuclear transcription factor NF- ⁇ B, thereby promoting the repair of islet inflammation in relatively old (26 weeks old) diabetic mice.
  • FIG. 33 Immunohistochemical observation of islet TNF- ⁇ after 31 days of administration of plasminogen in 24-25-week-old diabetic mice.
  • A is a normal control group
  • B is a vehicle PBS control group
  • C is a plasminogen group.
  • the results showed that the positive expression of TNF- ⁇ in the plasminogen group (arrow mark) was significantly higher than that in the vehicle control group, and the plasminogen group was closer to the normal control group than the vehicle PBS group. This indicates that plasminogen can promote the expression of TNF- ⁇ , thereby promoting the repair of islet injury in mice with 24-25 weeks old diabetes.
  • FIG. 34 Immunohistochemical observation of islet TNF- ⁇ after 31 days of administration of plasminogen in 26-week-old diabetic mice.
  • A is a normal control group
  • B is a vehicle PBS control group
  • C is a plasminogen group.
  • the results showed that the positive expression of TNF- ⁇ in the plasminogen group (arrow mark) was significantly higher than that in the vehicle control group, and the plasminogen group was closer to the normal control group than the vehicle PBS group. This indicates that plasminogen can promote the expression of TNF- ⁇ , thereby promoting the repair of islet injury in 26-week-old diabetic mice.
  • Figure 35 shows the results of immunohistochemical observation of islet TNF- ⁇ 28 days after administration of plasminogen in a T1DM model of PLG-inactivated mice.
  • A is the vehicle PBS control group and B is the plasminogen group.
  • the results of the study showed that the positive expression of TNF- ⁇ in the plasminogen group (arrow mark) was significantly higher than that in the vehicle control group. This indicates that plasminogen can promote the expression of TNF- ⁇ , thereby promoting the repair of islet injury in the T1DM model of mice with impaired PLG activity.
  • Figure 36 shows the results of islet IgM immunohistochemistry after 28 days of administration of plasminogen in mice with impaired PLG activity.
  • A is a blank control group
  • B is a vehicle PBS control group
  • C is a plasminogen group.
  • the results of this experiment showed that the positive expression of IgM in the plasminogen group (arrow mark) was significantly lower than that in the vehicle control group, and the plasminogen group was closer to the normal control group than the vehicle PBS group. This indicates that plasminogen can reduce the expression of IgM, thereby reducing islet injury in T1DM models in mice with impaired PLG activity.
  • FIG. 37 Results of islet TUNEL staining after 31 days of plasminogen administration in 24-25-week-old diabetic mice.
  • A is a normal control group
  • B is a vehicle PBS control group
  • C is a plasminogen group.
  • the results of this experiment showed that the number of positive cells (arrow mark) given to the plasminogen group was significantly less than that of the vehicle PBS control group.
  • TUNEL positive staining was extremely low in the normal control group.
  • the apoptosis rate in the normal control group was about 8%
  • the apoptosis rate in the PBS group was about 93%
  • the apoptosis rate in the plasminogen group was about 16%. This indicates that the plasminogen group can significantly reduce the apoptosis of islet cells in diabetic mice.
  • Example 1 Plasminogen reduces proliferation of islet alpha cells in diabetic mice 24-25 weeks of age, restores normal distribution of islet alpha cells and reduces glucagon secretion
  • mice There were 11 male db/db mice and 5 male db/m mice at 24-25 weeks of age. The day of the experiment was recorded as day 0 and weighed. The db/db mice were weighed and randomly divided into two groups. Five plasminogen groups were given to the vehicle PBS control group, and db/m mice were used as the normal control group. Start with plasminogen or PBS on day 1. The plasminogen group was injected with human plasminogen 2 mg/0.2 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group or no liquid was injected for 31 days. Mice were sacrificed on day 32 and pancreas were fixed in 4% paraformaldehyde.
  • the fixed pancreatic tissue was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • the thickness of the tissue section was 3 ⁇ m, and the sections were dewaxed and rehydrated and washed once with water.
  • the PAP pen looped out of the tissue, incubated with 3% hydrogen peroxide for 15 minutes, and washed twice with 0.01 M PBS for 5 minutes each time.
  • 5% normal sheep serum (Vector laboratories, Inc., USA) was blocked for 30 minutes; after the time, the sheep serum was discarded, and rabbit anti-mouse glucagon antibody (Abeam) was added dropwise at 4 ° C overnight, 0.01 Wash twice with M PBS for 5 minutes each time.
  • Goat anti-rabbit IgG (HRP) antibody (Abeam) secondary antibody was incubated for 1 hour at room temperature and washed twice with 0.01 M PBS for 5 minutes each time. The color was developed according to the DAB kit (Vector Laboratories, Inc., USA), washed three times with water, and counterstained with hematoxylin for 30 seconds, and rinsed with running water for 5 minutes. The gradient alcohol was dehydrated, the xylene was transparent and neutral gum was mounted, and the sections were observed under a 200-fold optical microscope.
  • Islet ⁇ cells synthesize and secrete glucagon, which is mainly distributed in the peripheral region of islets.
  • Example 2 Plasminogen inhibits proliferation of islet alpha cells in 26-week-old diabetic mice, restores normal distribution of islet alpha cells and reduces glucagon secretion
  • mice 9 male db/db male mice and 3 db/m male mice at 26 weeks of age were recorded on day 0 of the experiment and weighed.
  • the db/db mice were weighed and randomly divided into two groups to give fibrinolysis.
  • Four zymogen groups were given to the vehicle PBS control group, and db/m mice were used as the normal control group.
  • the plasminogen group was injected with human plasminogen 2 mg/0.2 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group for 35 days. Mice were sacrificed on day 36 and pancreas were fixed in 4% paraformaldehyde.
  • the fixed pancreatic tissue was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • the thickness of the tissue section was 3 ⁇ m, and the sections were dewaxed and rehydrated and washed once with water.
  • the PAP pen looped out of the tissue, incubated with 3% hydrogen peroxide for 15 minutes, and washed twice with 0.01 M PBS for 5 minutes each time.
  • 5% normal sheep serum (Vector laboratories, Inc., USA) was blocked for 30 minutes; after the time, the sheep serum was discarded, and rabbit anti-mouse glucagon antibody (Abeam) was added dropwise at 4 ° C overnight, 0.01 Wash twice with M PBS for 5 minutes each time.
  • Goat anti-rabbit IgG (HRP) antibody (Abeam) secondary antibody was incubated for 1 hour at room temperature and washed twice with 0.01 M PBS for 5 minutes each time. The color was developed according to the DAB kit (Vector Laboratories, Inc., USA), washed three times with water, and counterstained with hematoxylin for 30 seconds, and rinsed with running water for 5 minutes. The gradient alcohol was dehydrated, the xylene was transparent and neutral gum was mounted, and the sections were observed under a 200-fold optical microscope.
  • Islet ⁇ cells synthesize and secrete glucagon, which is mainly distributed in the peripheral region of islets.
  • plasminogen can significantly inhibit the proliferation of pancreatic islet ⁇ cells and the secretion of glucagon in 26-week-old diabetic mice, and correct the disorder of islet ⁇ cell distribution, suggesting that plasminogen can promote the repair of islet injury.
  • mice were sacrificed on day 29 and pancreas were fixed in 4% paraformaldehyde.
  • the fixed pancreatic tissue was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • the thickness of the tissue section was 3 ⁇ m, and the sections were dewaxed and rehydrated and washed once with water.
  • the PAP pen looped out of the tissue, incubated with 3% hydrogen peroxide for 15 minutes, and washed twice with 0.01 M PBS for 5 minutes each time.
  • Islet ⁇ cells synthesize and secrete glucagon, which is mainly distributed in the peripheral region of islets.
  • mice Eight male db/db mice aged 24-25 weeks were randomly divided into two groups, 5 in the plasminogen group and 3 in the vehicle control group. On the day of the experiment, the day 0 was recorded and weighed. On the first day, plasminogen or PBS was given, and the plasminogen group was injected with human plasminogen 2 mg/0.2 ml/day/day. The vehicle PBS control group was injected with the same volume of PBS in the tail vein for 31 consecutive days. After fasting for 16 hours on days 10 and 31, blood glucose testing was performed using blood glucose test paper (Roche, Mannheim, Germany).
  • mice Five male db/db mice, 24-25 weeks old, took 50 ⁇ l of blood from each mouse eye venous plexus one day before administration to detect serum fructosamine concentration, and recorded it as day 0, starting on the first day. The zymogen was administered continuously for 31 days. On the 32nd day, the eyeballs were removed and blood was taken, and the concentration of serum fructosamine was measured. Fructosamine concentration was measured using a fructosamine detection kit (Nanjing, A037-2).
  • the fructosamine concentration reflects the average level of blood glucose within 1 to 3 weeks.
  • the results showed that the concentration of serum fructosamine was significantly reduced after administration of plasminogen, and the statistical difference was extremely significant compared with that before administration (Fig. 5). This indicates that plasminogen can effectively reduce blood sugar in diabetic animals.
  • Example 6 plasminogen reduces serum fructosamine levels in 26-week-old diabetic mice
  • mice at 26 weeks of age were recorded as day 0 on the day of the experiment and weighed. They were randomly divided into two groups according to body weight, 4 in the plasminogen group and 5 in the vehicle PBS control group.
  • the plasminogen group was injected with human plasminogen 2 mg/0.2 mL/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group.
  • plasminogen or PBS was administered continuously for 35 days. Mice were sacrificed on day 36 and serum fructosamine concentrations were measured. Fructosamine concentration was measured using a fructosamine detection kit (Nanjing, A037-2).
  • mice at 26 weeks of age were randomly divided into two groups according to body weight, and 4 rats were given plasminogen group and 5 rats in vehicle control group.
  • plasminogen or PBS was administered to the plasminogen group for injection of human plasminogen 2 mg/0.2 ml/day/day, and the same volume of PBS was injected into the tail of the vehicle PBS control group.
  • the mice were fasted for 16 hours, and on the 36th day, the eyeballs were taken for blood to detect the concentration of plasma glycated hemoglobin.
  • Glycated hemoglobin content usually reflects the patient's blood glucose control for nearly 8 to 12 weeks.
  • the results showed that the concentration of glycated hemoglobin in the plasminogen group was significantly lower than that in the vehicle PBS control group, and the statistical difference was significant (Fig. 7). This indicates that plasminogen can effectively reduce blood sugar levels in diabetic animals.
  • mice There were 9 male db/db male mice and 3 db/m mice at 26 weeks of age.
  • db/db mice were weighed and randomly divided into two groups according to body weight, 4 in the plasminogen group and 5 in the vehicle control group, and db/m mice as the normal control group.
  • plasminogen or PBS was administered to the plasminogen group for injection of human plasminogen 2 mg/0.2 ml/day/day, and the same volume of PBS was injected into the tail of the vehicle PBS control group. Dosing for 10 days.
  • mice On day 11 after the mice were fasted for 16 hours, each mouse was intraperitoneally injected with 5% glucose solution at 5 g/kg body weight, and blood glucose test paper (Roche, Mannheim, Germany) at 0, 30, 60, 90, 120, 180 minutes. Check blood sugar concentration.
  • IPGTT intraperitoneal glucose tolerance test
  • mice in the two groups were given a single intraperitoneal injection of 200 mg/kg streptozotocin (STZ) (sigma S0130) to induce T1DM [43] .
  • STZ streptozotocin
  • the drug was administered and recorded as the first day of administration.
  • the plasminogen group was injected with human plasmin 1 mg/0.1 ml/day/day into the tail vein, and the same volume of the vehicle was injected into the tail of the PBS control group.
  • PBS was administered continuously for 10 days. After the mice were fasted for 6 hours on day 11, blood glucose was measured with blood glucose test paper (Roche, Mannheim, Germany).
  • mice were fasted for 6 hours, 5% glucose solution was intraperitoneally injected at 5 g/kg body weight, and blood glucose concentration was measured with blood glucose test paper (Roche, Mannheim, Germany) at 0, 15, 30, 60, and 90 minutes after the injection.
  • blood glucose test paper (Roche, Mannheim, Germany) at 0, 15, 30, 60, and 90 minutes after the injection.
  • IPGTT intraperitoneal glucose tolerance test
  • mice Eight male C57 male mice, 9-10 weeks old, were randomly divided into two groups, the vehicle PBS control group and the plasminogen group, 4 in each group.
  • the vehicle PBS control group and the plasminogen group mice were fasted for 4 hours and then intraperitoneally injected with 200 mg/kg streptozotocin (STZ) (sigma S0130) to induce T1DM [43].
  • STZ streptozotocin
  • the plasminogen group was injected with human plasmin 1 mg/0.1 ml/day/day into the tail vein, and the same volume of the PBS control group was injected into the tail vein.
  • PBS streptozotocin
  • mice at 26 weeks of age were recorded as day 0 on the day of the experiment, weighed and randomly divided into two groups according to body weight, 4 to plasminogen group and 5 to vehicle PBS control group.
  • plasminogen or PBS was administered to the plasminogen group for injection of human plasminogen 2 mg/0.2 ml/day/day, and the same volume of PBS was injected into the tail of the vehicle PBS control group.
  • the test results showed that the serum insulin level in the plasminogen group was significantly higher than that in the vehicle control group, and the statistical difference was significant (Fig. 12). This indicates that plasminogen can significantly increase insulin secretion in diabetic mice.
  • mice Seven male db/db mice aged 24-25 weeks were recorded as day 0 on the day of the experiment and weighed. They were randomly divided into two groups according to body weight, 4 rats in the plasminogen group and 3 rats in the vehicle control group. . On the first day, plasminogen or PBS was administered to the plasminogen group, and human plasminogen was injected into the plasminogen group at a dose of 2 mg/0.2 ml/day/day. The same volume of PBS was injected into the tail of the vehicle PBS control group. Continuous administration for 31 days. Mice were sacrificed on day 32 and pancreas were fixed in 4% paraformaldehyde.
  • the fixed pancreatic tissue was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • the thickness of the tissue section was 3 ⁇ m, and the sections were dewaxed and rehydrated. It was stained with hematoxylin and eosin (HE staining), differentiated with 1% hydrochloric acid alcohol, returned to blue with ammonia, and dehydrated with an alcohol gradient.
  • the sections were observed under a 200 and 400x optical microscope.
  • mice Sixteen male db/db mice aged 24-25 weeks were recorded on the 0th day of the experiment and weighed. They were randomly divided into two groups according to body weight, 10 in the plasminogen group and 6 in the vehicle PBS control group. . On the first day, plasminogen or PBS was administered to the plasminogen group for injection of human plasminogen 2 mg/0.2 ml/day/day, and the same volume of PBS was injected into the tail of the vehicle PBS control group. Dosing for 31 days. Mice were sacrificed on day 32 and pancreas were fixed in 4% paraformaldehyde. The fixed pancreatic tissue was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • the thickness of the tissue section was 3 ⁇ m.
  • the sections were dewaxed to water, washed once with water, stained with 0.1% Sirius red for 60 minutes, rinsed with running water, stained with hematoxylin for 1 minute, rinsed with running water, 1% hydrochloric acid and ammonia were rehydrated to blue, rinsed with water. After drying, the sheets were mounted and sections were observed under a 200x optical microscope.
  • Sirius red staining can make collagen staining for a long time. As a special staining method for pathological sections, Sirius red staining can specifically display collagen tissue.
  • mice Six male db/db mice aged 24-25 weeks were recorded on day 0 and weighed on the day of the experiment. They were randomly divided into two groups according to body weight, 4 for plasminogen group and 2 for vehicle control PBS. . On the first day, plasminogen or PBS was administered to the plasminogen group for injection of human plasminogen 2 mg/0.2 ml/day/day, and the same volume of PBS was injected into the tail of the vehicle PBS control group. Dosing for 31 days. Mice were sacrificed on day 32 and pancreas were fixed in 4% paraformaldehyde. The fixed pancreatic tissue is removed by alcohol gradient Water and xylene were transparent and then paraffin embedded.
  • the thickness of the tissue section was 3 ⁇ m, and the sections were dewaxed and rehydrated and washed once with water. Incubate for 15 minutes with 3% hydrogen peroxide and wash twice with water for 5 minutes each time. 5% of normal sheep serum (Vector laboratories, Inc., USA) was blocked for 1 hour; after that, the serum of the sheep was discarded and the tissue was circled with a PAP pen. Rabbit anti-mouse Caspase-3 (Abeam) was incubated overnight at 4 ° C and washed twice with PBS for 5 minutes each time. Goat anti-rabbit IgG (HRP) antibody (Abeam) secondary antibody was incubated for 1 hour at room temperature and washed twice with PBS for 5 minutes each time.
  • HRP Goat anti-rabbit IgG
  • the color was developed according to the DAB kit (Vector Laboratories, Inc., USA), washed three times with water, and counterstained with hematoxylin for 30 seconds, and rinsed with running water for 5 minutes. The gradient was dehydrated and sealed and the sections were observed under a 200x optical microscope.
  • Caspase-3 is the most important terminal cleavage enzyme in the process of apoptosis, and the more its expression, the more cells are in apoptotic state [44] .
  • the experimental results of the present invention showed that the expression of Caspase-3 (arrow mark) to the plasminogen group (Fig. 15B) was significantly lower than that of the vehicle PBS control group (Fig. 15A). This indicates that plasminogen can reduce the apoptosis of islet cells.
  • mice at 18 weeks of age were recorded as day 0 on the day of the experiment and weighed. They were randomly divided into two groups according to body weight. The plasminogen group and the vehicle control group were given PBS control group, 4 in each group. . On the first day, plasminogen or PBS was administered to the plasminogen group for injection of human plasminogen 2 mg/0.2 ml/day/day, and the same volume of PBS was injected into the tail of the vehicle PBS control group. Dosing for 31 days. Mice were sacrificed on day 36 and pancreas were fixed in 4% paraformaldehyde. The fixed pancreatic tissue was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • the thickness of the tissue section was 3 ⁇ m, and the sections were dewaxed and rehydrated and washed once with water. Incubate for 15 minutes with 3% hydrogen peroxide and wash twice with water for 5 minutes each time. 5% of normal sheep serum (Vector laboratories, Inc., USA) was blocked for 1 hour; after that, the serum of the sheep was discarded and the tissue was circled with a PAP pen. Rabbit anti-mouse insulin antibody (Abeam) was incubated overnight at 4 ° C and washed twice with PBS for 5 minutes each time. Goat anti-rabbit IgG (HRP) antibody (Abeam) secondary antibody was incubated for 1 hour at room temperature and washed twice with PBS for 5 minutes each time.
  • HRP Goat anti-rabbit IgG
  • the color was developed according to the DAB kit (Vector Laboratories, Inc., USA), washed three times with water, and counterstained with hematoxylin for 30 seconds, and rinsed with running water for 5 minutes. The gradient was dehydrated and sealed, and the sections were observed under a microscope at 200 times.
  • mice Eight male db/db mice aged 24-25 weeks were recorded as day 0 on the day of the experiment and weighed. They were randomly divided into two groups according to body weight, 5 rats in the plasminogen group and 3 rats in the vehicle control group. . On the first day, plasminogen or PBS was administered to the plasminogen group for injection of human plasminogen 2 mg/0.2 ml/day/day, and the same volume of PBS was injected into the tail of the vehicle PBS control group. Dosing for 31 days. Mice were sacrificed on day 32 and pancreas were fixed in 4% paraformaldehyde. The fixed pancreatic tissue was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • the thickness of the tissue section was 3 ⁇ m, and the sections were dewaxed and rehydrated and washed once with water. Incubate for 15 minutes with 3% hydrogen peroxide and wash twice with water for 5 minutes each time. 5% of normal sheep serum (Vector laboratories, Inc., USA) was blocked for 1 hour; after that, the serum of the sheep was discarded and the tissue was circled with a PAP pen. Rabbit anti-mouse insulin antibody (Abeam) was incubated overnight at 4 ° C and washed twice with PBS for 5 minutes each time. Goat anti-rabbit IgG (HRP) antibody (Abeam) secondary antibody was incubated for 1 hour at room temperature and washed twice with PBS for 5 minutes each time.
  • HRP Goat anti-rabbit IgG
  • the color was developed according to the DAB kit (Vector Laboratories, Inc., USA), washed three times with water, and counterstained with hematoxylin for 30 seconds, and rinsed with running water for 5 minutes. The gradient was dehydrated and sealed, and the sections were observed under a microscope at 200 times.
  • mice at 26 weeks of age were recorded on day 0 and weighed on the day of the experiment. They were randomly divided into two groups according to body weight, and 4 rats in the plasminogen group were given vehicle PBS control group.
  • plasminogen or PBS was administered to the plasminogen group for injection of human plasminogen 2 mg/0.2 ml/day/day, and the same volume of PBS was injected into the tail of the vehicle PBS control group. Administered for 35 days. After the mice were fasted for 16 hours on day 35, the mice were sacrificed on the 36th day and the pancreas was fixed in 4% paraformaldehyde.
  • the fixed pancreatic tissue was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • the thickness of the tissue section was 3 ⁇ m, and the sections were dewaxed and rehydrated and washed once with water. Incubate for 15 minutes with 3% hydrogen peroxide and wash twice with water for 5 minutes each time. 5% of normal sheep serum (Vector laboratories, Inc., USA) was blocked for 1 hour; after that, the serum of the sheep was discarded and the tissue was circled with a PAP pen. Rabbit anti-mouse insulin antibody (Abeam) was incubated overnight at 4 ° C and washed twice with PBS for 5 minutes each time.
  • Abeam rabbit anti-mouse insulin antibody
  • Goat anti-rabbit IgG (HRP) antibody (Abeam) secondary antibody was incubated for 1 hour at room temperature and washed twice with PBS for 5 minutes each time. The color was developed according to the DAB kit (Vector Laboratories, Inc., USA), washed three times with water, and counterstained with hematoxylin for 30 seconds, and rinsed with running water for 5 minutes. The gradient was dehydrated and sealed, and the sections were observed under a microscope at 200 times.
  • Example 19 Plasminogen Promotes Expression of Islet Multi-directional Nuclear Factor NF- ⁇ B in 24-25 Weeks Old Diabetic Mice
  • mice of 24-25 weeks old db/db were recorded on the 0th day of the experiment and weighed. They were randomly divided into two groups according to body weight, 4 for plasminogen group and 6 for vehicle control PBS. Another db/m was taken as the normal control group, and the normal control group was not treated.
  • plasminogen or PBS was administered to the plasminogen group for injection of human plasminogen 2 mg/0.2 ml/day/day, and the same volume of PBS was injected into the tail of the vehicle PBS control group. Dosing for 31 days. Mice were sacrificed on day 32 and pancreas were fixed in 4% paraformaldehyde.
  • the fixed pancreatic tissue was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • the thickness of the tissue section was 3 ⁇ m, and the sections were dewaxed and rehydrated and washed once with water. Incubate for 15 minutes with 3% hydrogen peroxide and wash twice with water for 5 minutes each time. 5% of normal sheep serum (Vector laboratories, Inc., USA) was blocked for 1 hour; after that, the serum of the sheep was discarded and the tissue was circled with a PAP pen. Rabbit anti-mouse NF- ⁇ B (Abeam) was incubated overnight at 4° C. and washed twice with PBS for 5 minutes each time.
  • Goat anti-rabbit IgG (HRP) antibody (Abeam) secondary antibody was incubated for 1 hour at room temperature and washed twice with PBS for 5 minutes each time. The color was developed according to the DAB kit (Vector Laboratories, Inc., USA), washed three times with water, and counterstained with hematoxylin for 30 seconds, and rinsed with running water for 5 minutes. The gradient was dehydrated and sealed, and the sections were observed under a microscope at 200 times.
  • NF- ⁇ B is a member of the transcription factor protein family and plays an important role in the process of inflammatory repair [45] .
  • Example 20 Plasminogen reduces proliferation of islet alpha cells in 18-week-old diabetic mice, restores normal distribution of islet alpha cells and reduces glucagon secretion
  • db/db mice Eighteen db/db male mice and three db/m male mice at 18 weeks of age were recorded as day 0 on the day of the experiment and weighed. db/db mice were randomly divided into two groups according to body weight, giving plasmin. The original group and the control vehicle PBS control group, 4 in each group, db / m mice as a normal control group. Start with plasminogen or PBS on day 1. The plasminogen group was injected with human plasminogen 2 mg/0.2 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group for 35 days. On the 36th day The mice were sacrificed and the pancreas was fixed in 4% paraformaldehyde.
  • the fixed pancreatic tissue was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • the thickness of the tissue section was 3 ⁇ m, and the sections were dewaxed and rehydrated and washed once with water.
  • the PAP pen looped out of the tissue, incubated with 3% hydrogen peroxide for 15 minutes, and washed twice with 0.01 M PBS for 5 minutes each time.
  • 5% normal sheep serum (Vector laboratories, Inc., USA) was blocked for 30 minutes; after the time, the sheep serum was discarded, and rabbit anti-mouse glucagon antibody (Abeam) was added dropwise at 4 ° C overnight, 0.01 Wash twice with M PBS for 5 minutes each time.
  • Goat anti-rabbit IgG (HRP) antibody (Abeam) secondary antibody was incubated for 1 hour at room temperature and washed twice with 0.01 M PBS for 5 minutes each time. The color was developed according to the DAB kit (Vector Laboratories, Inc., USA), washed three times with water, and counterstained with hematoxylin for 30 seconds, and rinsed with running water for 5 minutes. The gradient alcohol was dehydrated, the xylene was transparent and neutral gum was mounted, and the sections were observed under a 200-fold optical microscope.
  • Islet ⁇ cells synthesize and secrete glucagon, which is mainly distributed in the peripheral region of islets.
  • plasminogen can significantly inhibit the proliferation of pancreatic islet ⁇ cells and the secretion of glucagon in 18-week-old diabetic mice, and correct the disorder of islet ⁇ cell distribution, suggesting that plasminogen can promote the repair of islet injury.
  • Example 21 Plasminogen promotes expression of insulin receptor substrate 2 (IRS-2) in 18-week-old diabetic mice
  • db/db mice Seventeen db/db male mice and three db/m male mice at 18 weeks of age were recorded as day 0 on the day of the experiment and weighed. db/db mice were randomly divided into two groups according to body weight, giving plasmin. Three of the original groups were given to the vehicle PBS control group, and db/m mice were used as the normal control group. Start with plasminogen or PBS on day 1. The plasminogen group was injected with human plasminogen 2 mg/0.2 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group for 35 days. Mice were sacrificed on day 36 and pancreas were fixed in 4% paraformaldehyde.
  • the fixed pancreatic tissue was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • the thickness of the tissue section was 3 ⁇ m, and the sections were dewaxed and rehydrated and washed once with water.
  • the PAP pen looped out of the tissue, incubated with 3% hydrogen peroxide for 15 minutes, and washed twice with 0.01 M PBS for 5 minutes each time.
  • 5% normal sheep serum (Vector laboratories, Inc., USA) was blocked for 30 minutes; after the time, the sheep serum was discarded, and rabbit anti-mouse IRS-2 antibody (Abeam) was added dropwise and incubated at 4 ° C overnight, 0.01 M. Wash twice with PBS for 5 minutes each time.
  • Goat anti-rabbit IgG (HRP) antibody (Abcam) The secondary antibody was incubated for 1 hour at room temperature and washed twice with 0.01 M PBS for 5 minutes each time. The color was developed according to the DAB kit (Vector Laboratories, Inc., USA), washed three times with water, and counterstained with hematoxylin for 30 seconds, and rinsed with running water for 5 minutes. The gradient alcohol was dehydrated, the xylene was transparent and neutral gum was mounted, and the sections were observed under a 200-fold optical microscope.
  • Insulin Receptor Substrate-2 is a substrate that acts on the activated insulin receptor tyrosine kinase, is an important molecule in the insulin signal transduction pathway, and is an islet The survival of beta cells is very important. IRS-2 has a protective effect on the expression of islet ⁇ cells and is essential for the maintenance of functional islet ⁇ cells [46,47] .
  • mice There were 11 male db/db mice and 5 male db/m mice at 24-25 weeks of age. The day of the experiment was recorded as day 0 and weighed. The db/db mice were randomly divided into two groups according to their body weight. Five lysozyme groups were given to the vehicle PBS control group, and db/m mice were used as the normal control group. Start with plasminogen or PBS on day 1. The plasminogen group was injected with human plasminogen 2 mg/0.2 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group or no liquid was injected for 31 days.
  • mice were sacrificed on day 32 and pancreas were fixed in 4% paraformaldehyde.
  • the fixed pancreatic tissue was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • the thickness of the tissue section was 3 ⁇ m, and the sections were dewaxed and rehydrated and washed once with water.
  • the PAP pen looped out of the tissue, incubated with 3% hydrogen peroxide for 15 minutes, and washed twice with 0.01 M PBS for 5 minutes each time.
  • IRS-2 immunohistochemistry showed that the expression of IRS-2 in the PBS control mice (Fig. 22B) was significantly less than that in the plasminogen group (Fig. 22C), and the statistical difference was significant (Fig. 22). 22D), and the plasminogen group was closer to the normal control group (22A) than the vehicle PBS group. Description Plasminogen can effectively increase the expression of IRS-2 in islet cells of 24-25 weeks old diabetic mice.
  • mice 9 male db/db male mice and 3 db/m male mice at 26 weeks of age, recorded as day 0 on the day of the experiment and weighed, db/db mice were randomly divided into two groups according to body weight, giving plasmin Four of the original groups were given to the vehicle PBS control group, and db/m mice were used as the normal control group. Start with plasminogen or PBS on day 1. The plasminogen group was injected with human plasminogen 2 mg/0.2 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group for 35 days. Mice were sacrificed on day 36 and pancreas were fixed in 4% paraformaldehyde.
  • the fixed pancreatic tissue was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • the thickness of the tissue section was 3 ⁇ m, and the sections were dewaxed and rehydrated and washed once with water.
  • the PAP pen looped out of the tissue, incubated with 3% hydrogen peroxide for 15 minutes, and washed twice with 0.01 M PBS for 5 minutes each time.
  • 5% normal sheep serum (Vector laboratories, Inc., USA) was blocked for 30 minutes; after the time, the sheep serum was discarded, and rabbit anti-mouse IRS-2 antibody (Abeam) was added dropwise and incubated at 4 ° C overnight, 0.01 M. Wash twice with PBS for 5 minutes each time.
  • Goat anti-rabbit IgG (HRP) antibody (Abeam) secondary antibody was incubated for 1 hour at room temperature and washed twice with 0.01 M PBS for 5 minutes each time. The color was developed according to the DAB kit (Vector Laboratories, Inc., USA), washed three times with water, and counterstained with hematoxylin for 30 seconds, and rinsed with running water for 5 minutes. The gradient alcohol was dehydrated, the xylene was transparent and neutral gum was mounted, and the sections were observed under a 200-fold optical microscope.
  • IRS-2 immunohistochemistry showed that the islet IRS-2 positive expression (arrow mark) was significantly less in the vehicle PBS control group (Fig. 23B) than in the plasminogen group (Fig. 23C); the plasminogen group was given. IRS-2 expression levels were close to normal control mice (Fig. 23A). This indicates that plasminogen can effectively increase the expression of IRS-2 in islet cells of 26-week-old diabetic mice.
  • mice were sacrificed on day 29 and pancreas were fixed in 4% paraformaldehyde.
  • the fixed pancreatic tissue was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • the thickness of the tissue section was 3 ⁇ m, and the sections were dewaxed and rehydrated and washed once with water.
  • the PAP pen looped out of the tissue, incubated with 3% hydrogen peroxide for 15 minutes, and washed twice with 0.01 M PBS for 5 minutes each time.
  • IRS-2 immunohistochemistry showed that the islet IRS-2 positive expression (arrow mark) was significantly less in the vehicle PBS control group (Fig. 24B) than in the plasminogen group (Fig. 24C), and plasminogen was given.
  • the group was closer to the blank control group (24A) than the vehicle PBS group. This indicates that plasminogen can effectively increase the expression of IRS-2 in islet cells of normal PDL-active mice aged 9-10 weeks.
  • db/db mice There were 9 male db/db mice and 3 db/m mice at 24-26 weeks of age.
  • the db/db mice were randomly divided into two groups, 4 in the plasminogen group and 5 in the vehicle PBS control group. Db/m mice served as normal controls.
  • the group On the day of the start of the experiment, the group was weighed on the 0th day, and the day after the start of the experiment, plasminogen or PBS was given and recorded as the first day.
  • the plasminogen group was injected with human plasminogen 2 mg/0.2 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group for 35 days.
  • mice were sacrificed on day 36 and pancreas were fixed in 4% paraformaldehyde.
  • the fixed pancreatic tissue was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • the thickness of the tissue section was 3 ⁇ m, and the sections were dewaxed and rehydrated and washed once with water.
  • the PAP pen looped out of the tissue, incubated with 3% hydrogen peroxide for 15 minutes, and washed twice with 0.01 M PBS for 5 minutes each time.
  • Centroblasts are important members of the non-specific cellular immune system, and when inflammation occurs, they are attracted to the site of inflammation by chemotactic substances.
  • mice Ten male rats with impaired PLG activity at 9-10 weeks old were randomly divided into three groups, 3 in the blank control group, 3 in the PBS control group, and 4 in the plasminogen group.
  • the vehicle PBS control group and the plasminogen group mice were fasted for 4 hours, and a single intraperitoneal injection of 200 mg/kg STZ (sigma S0130) induced type I diabetes [43] , and the blank control group was not treated.
  • the drug was administered 12 days after the injection and was designated as the first day of administration.
  • the plasminogen group was injected with human plasmin 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group. , continuous administration for 28 days.
  • mice were sacrificed on day 29 and pancreas were fixed in 4% paraformaldehyde.
  • the fixed pancreatic tissue was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • the thickness of the tissue section was 3 ⁇ m, and the sections were dewaxed and rehydrated and washed once with water.
  • the PAP pen looped out of the tissue, incubated with 3% hydrogen peroxide for 15 minutes, and washed twice with 0.01 M PBS for 5 minutes each time.
  • 11 male rats with normal PLG activity at 9-10 weeks old were randomly divided into three groups, 3 in the blank control group, 4 in the vehicle PBS control group, and 4 in the plasminogen group.
  • the vehicle PBS control group and the plasminogen group mice were fasted for 4 hours, and a single intraperitoneal injection of 200 mg/kg STZ (sigmaS0130) induced type I diabetes [43] , and the blank control group was not treated.
  • the drug was administered 12 days after the injection and was designated as the first day of administration.
  • the plasminogen group was injected with human plasmin 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group. , continuous administration for 28 days.
  • mice were sacrificed on day 29 and pancreas were fixed in 4% paraformaldehyde.
  • the fixed pancreatic tissue was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • the thickness of the tissue section was 3 ⁇ m, and the sections were dewaxed and rehydrated and washed once with water.
  • the PAP pen looped out of the tissue, incubated with 3% hydrogen peroxide for 15 minutes, and washed twice with 0.01 M PBS for 5 minutes each time.
  • Example 28 Lysozyme promotes synthesis and secretion of insulin in mice with impaired PLG activity in the T1DM model
  • mice Ten male rats with impaired PLG activity at 9-10 weeks were randomly divided into three groups, 3 in the blank control group, 3 in the PBS control group, and 4 in the plasminogen group.
  • the vehicle PBS control group and the plasminogen group mice were fasted for 4 hours, and a single intraperitoneal injection of 200 mg/kg STZ (sigma S0130) induced type I diabetes [43] , and the blank control group was not treated.
  • the drug was administered 12 days after the injection and was designated as the first day of administration.
  • the plasminogen group was injected with human plasmin 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group. , continuous administration for 28 days.
  • mice were sacrificed on day 29 and pancreas were fixed in 4% paraformaldehyde.
  • the fixed pancreatic tissue was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • the thickness of the tissue section was 3 ⁇ m, and the sections were dewaxed and rehydrated and washed once with water.
  • the PAP pen looped out of the tissue, incubated with 3% hydrogen peroxide for 15 minutes, and washed twice with 0.01 M PBS for 5 minutes each time.
  • Example 29 Plasminogen promotes synthesis and expression of insulin in mice with normal PLG activity in T1DM model
  • 11 male rats with normal PLG activity at 9-10 weeks old were randomly divided into three groups, 3 in the blank control group, 4 in the vehicle PBS control group, and 4 in the plasminogen group.
  • the vehicle PBS control group and the plasminogen group mice were fasted for 4 hours, and a single intraperitoneal injection of 200 mg/kg STZ (sigma S0130) induced type I diabetes [43] , and the blank control group was not treated.
  • the drug was administered 12 days after the injection and was designated as the first day of administration.
  • the plasminogen group was injected with human plasmin 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group. , continuous administration for 28 days.
  • mice were sacrificed on day 29 and pancreas were fixed in 4% paraformaldehyde.
  • the fixed pancreatic tissue was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • the thickness of the tissue section was 3 ⁇ m, and the sections were dewaxed and rehydrated and washed once with water.
  • the PAP pen looped out of the tissue, incubated with 3% hydrogen peroxide for 15 minutes, and washed twice with 0.01 M PBS for 5 minutes each time.
  • Example 30 Plasminogen Promotes Expression of Islet Multi-directional Nuclear Factor NF- ⁇ B in T1DM Model of Impaired PLG Activity in Mice
  • mice Ten male rats with impaired PLG activity at 9-10 weeks were randomly divided into three groups, 3 in the blank control group, 3 in the PBS control group, and 4 in the plasminogen group.
  • the vehicle PBS control group and the plasminogen group mice were fasted for 4 hours, and a single intraperitoneal injection of 200 mg/kg STZ (sigma S0130) induced type I diabetes [43] , and the blank control group was not treated.
  • the drug was administered 12 days after the injection and was designated as the first day of administration.
  • the plasminogen group was injected with human plasmin 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group. , continuous administration for 28 days.
  • mice were sacrificed on day 29 and pancreas were fixed in 4% paraformaldehyde.
  • the fixed pancreatic tissue was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • the thickness of the tissue section was 3 ⁇ m, and the sections were dewaxed and rehydrated and washed once with water.
  • the PAP pen looped out of the tissue, incubated with 3% hydrogen peroxide for 15 minutes, and washed twice with 0.01 M PBS for 5 minutes each time.
  • NF- ⁇ B As a multi-directional nuclear transcription factor, NF- ⁇ B is involved in the regulation of various genes such as cell proliferation, apoptosis, inflammation and immunity after activation [24] .
  • Example 31 Plasminogen promotes expression of islet multi-directional nuclear transcription factor NF- ⁇ B in 18-week-old diabetic mice
  • mice at 18 weeks of age were recorded as day 0 on the day of the experiment and weighed. They were randomly divided into two groups according to body weight, 3 in the plasminogen group and 4 in the vehicle PBS control group.
  • plasminogen or PBS was administered to the plasminogen group for injection of human plasminogen 2 mg/0.2 ml/day/day, and the same volume of PBS was injected into the tail of the vehicle PBS control group.
  • Mice were sacrificed on day 36 and pancreas were fixed in 4% paraformaldehyde. The fixed pancreatic tissue was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • the thickness of the tissue section was 3 ⁇ m, and the sections were dewaxed and rehydrated and washed once with water.
  • the PAP pen looped out of the tissue, incubated with 3% hydrogen peroxide for 15 minutes, and washed twice with 0.01 M PBS for 5 minutes each time.
  • 5% normal sheep serum (Vector Laboratories, Inc., USA) was blocked for 30 minutes; after the time was over, the serum of the sheep was discarded, rabbit anti-mouse NF- ⁇ B antibody (Cell Signal) was added and incubated overnight at 4 ° C, and washed twice with 0.01 M PBS for 5 times each time. minute.
  • Goat anti-rabbit IgG (HRP) antibody (Abeam) secondary antibody was incubated for 1 hour at room temperature and washed twice with 0.01 M PBS for 5 minutes each time. The color was developed according to the DAB kit (Vector Laboratories, Inc., USA), washed three times with water, and counterstained with hematoxylin for 30 seconds, and rinsed with running water for 5 minutes. The gradient alcohol was dehydrated, the xylene was transparent and neutral gum was mounted, and the sections were observed under a 200-fold optical microscope.
  • Example 32 Plasminogen inhibits the expression of multi-directional nuclear transcription factor NF- ⁇ B in 26-week-old diabetic mice
  • mice 9 male db/db male mice and 3 db/m male mice at 26 weeks of age, recorded as day 0 on the day of the experiment and weighed, db/db mice were randomly divided into two groups according to body weight, giving plasmin Four of the original groups were given to the vehicle PBS control group, and db/m mice were used as the normal control group.
  • plasminogen or PBS was given and recorded as the first day.
  • the plasminogen group was injected with human plasminogen 2 mg/0.2 ml/day/day into the tail vein of the plasminogen group.
  • the same volume of PBS was administered continuously for 35 days. Mice were sacrificed on day 36 and pancreas were fixed in 4% paraformaldehyde.
  • the fixed pancreatic tissue was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • the thickness of the tissue section was 3 ⁇ m, and the sections were dewaxed and rehydrated and washed once with water.
  • the PAP pen looped out of the tissue, incubated with 3% hydrogen peroxide for 15 minutes, and washed twice with 0.01 M PBS for 5 minutes each time.
  • 5% normal sheep serum (Vector laboratories, Inc., USA) was blocked for 30 minutes; after the time was over, the serum of the sheep was discarded, and rabbit anti-mouse NF- ⁇ B antibody (Cell Signal) was added and incubated at 4 ° C overnight, 0.01 Wash twice with M PBS for 5 minutes each time.
  • Goat anti-rabbit IgG (HRP) antibody (Abeam) secondary antibody was incubated for 1 hour at room temperature and washed twice with 0.01 M PBS for 5 minutes each time. The color was developed according to the DAB kit (Vector Laboratories, Inc., USA), washed three times with water, and counterstained with hematoxylin for 30 seconds, and rinsed with running water for 5 minutes. The gradient alcohol was dehydrated, the xylene was transparent and neutral gum was mounted, and the sections were observed under a 200-fold optical microscope.
  • db/db mice There were 11 male db/db mice and 5 male db/m mice at 24-25 weeks of age. The day of the experiment was recorded as day 0 and weighed. The db/db mice were randomly divided into two groups according to their body weight. Five lysozyme groups were given to the vehicle PBS control group, and db/m mice were used as the normal control group. On the first day, plasminogen or PBS was administered to the plasminogen group. The human plasminogen was injected into the plasminogen group at a dose of 2 mg/0.2 ml/day/day. The vehicle was injected into the same volume of PBS or the same volume of PBS. Any liquid was injected and administered continuously for 31 days.
  • mice were sacrificed on day 32 and pancreas were fixed in 4% paraformaldehyde.
  • the fixed pancreatic tissue was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • the thickness of the tissue section was 3 ⁇ m, and the sections were dewaxed and rehydrated and washed once with water.
  • the PAP pen looped out of the tissue, incubated with 3% hydrogen peroxide for 15 minutes, and washed twice with 0.01 M PBS for 5 minutes each time.
  • Tumor Necrosis Factor- ⁇ ( TNF - ⁇ ) is mainly produced by activated monocytes/macrophages and is an important pro-inflammatory factor [48] .
  • Example 34 Plasminogen inhibits the expression of TNF- ⁇ in islets of 26-week-old diabetic mice
  • mice 9 male db/db male mice and 3 db/m male mice at 26 weeks of age, recorded as day 0 on the day of the experiment and weighed, db/db mice were randomly divided into two groups according to body weight, giving plasmin Four of the original groups were given to the vehicle PBS control group, and db/m mice were used as the normal control group. Start with plasminogen or PBS on day 1. The plasminogen group was injected with human plasminogen 2 mg/0.2 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group or no liquid was injected for 35 days. Mice were sacrificed on day 36 and pancreas were fixed in 4% paraformaldehyde.
  • the fixed pancreatic tissue was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • the thickness of the tissue section was 3 ⁇ m, and the sections were dewaxed and rehydrated and washed once with water.
  • the PAP pen looped out of the tissue, incubated with 3% hydrogen peroxide for 15 minutes, and washed twice with 0.01 M PBS for 5 minutes each time.
  • 5% normal sheep serum (Vector Laboratories, Inc., USA) was blocked for 30 minutes; after the time was over, the serum of the sheep was discarded, rabbit anti-mouse TNF- ⁇ antibody (Abeam) was added dropwise and incubated overnight at 4 ° C, and washed twice with 0.01 M PBS for 5 minutes each time. .
  • Goat anti-rabbit IgG (HRP) antibody (Abeam) secondary antibody was incubated for 1 hour at room temperature and washed twice with 0.01 M PBS for 5 minutes each time. The color was developed according to the DAB kit (Vector Laboratories, Inc., USA), washed three times with water, and counterstained with hematoxylin for 30 seconds, and rinsed with running water for 5 minutes. The gradient alcohol was dehydrated, the xylene was transparent and neutral gum was mounted, and the sections were observed under a 200-fold optical microscope.
  • mice Seven male rats with impaired PLG activity at 9-10 weeks of age were randomly divided into two groups, three in the PBS control group and four in the plasminogen group. Two groups of mice were fasted for 4 hours and a single intraperitoneal injection of 200 mg/kg STZ (sigma S0130) induced type 1 diabetes [43] . The drug was administered 12 days after the injection and was designated as the first day of administration.
  • the plasminogen group was injected with human plasmin 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group. , continuous administration for 28 days. Mice were sacrificed on day 29 and pancreas were fixed in 4% paraformaldehyde.
  • the fixed pancreatic tissue was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • the thickness of the tissue section was 3 ⁇ m, and the sections were dewaxed and rehydrated and washed once with water.
  • the PAP pen looped out of the tissue, incubated with 3% hydrogen peroxide for 15 minutes, and washed twice with 0.01 M PBS for 5 minutes each time.
  • 5% normal sheep serum (Vector laboratories, Inc., USA) was blocked for 30 minutes; after the time, the sheep serum was discarded, and the rabbit anti-mouse antibody TNF- ⁇ (Abeam) was added dropwise at 4 ° C overnight, 0.01 M Wash twice with PBS for 5 minutes each time.
  • Goat anti-rabbit IgG (HRP) antibody (Abeam) secondary antibody was incubated for 1 hour at room temperature and washed twice with 0.01 M PBS for 5 minutes each time. The color was developed according to the DAB kit (Vector Laboratories, Inc., USA), washed three times with water, and counterstained with hematoxylin for 30 seconds, and rinsed with running water for 5 minutes. The gradient alcohol was dehydrated, the xylene was transparent and neutral gum was mounted, and the sections were observed under a 200-fold optical microscope.
  • Example 36 Plasminogen attenuates PLG activity in injured mice in islet injury in a T1DM model
  • mice Ten male rats with impaired PLG activity at 9-10 weeks were randomly divided into three groups, 3 in the blank control group, 3 in the PBS control group, and 4 in the plasminogen group.
  • the vehicle PBS control group and the plasminogen group mice were fasted for 4 hours, and a single intraperitoneal injection of 200 mg/kg STZ (sigma S0130) induced type I diabetes [43] , and the blank control group was not treated.
  • the drug was administered 12 days after the injection and was designated as the first day of administration.
  • the plasminogen group was injected with human plasmin 1 mg/0.1 ml/day/day into the tail vein, and the same volume of PBS was injected into the tail of the vehicle PBS control group. , continuous administration for 28 days.
  • mice were sacrificed on day 29 and pancreas were fixed in 4% paraformaldehyde.
  • the fixed pancreatic tissue was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • the thickness of the tissue section was 3 ⁇ m, and the sections were dewaxed and rehydrated and washed once with water.
  • the PAP pen looped out of the tissue, incubated with 3% hydrogen peroxide for 15 minutes, and washed twice with 0.01 M PBS for 5 minutes each time.
  • IgM antibodies play an important role in the clearance of apoptotic and necrotic cells.
  • the level of local IgM antibodies in tissue and organ damage is positively correlated with the degree of injury [49,50] . Therefore, detecting the level of local IgM antibodies in tissues and organs can reflect the damage of the tissues and organs.
  • db/db mice There were 11 male db/db mice and 5 male db/m mice at 24-25 weeks of age. The day of the experiment was recorded as day 0 and weighed. The db/db mice were randomly divided into two groups according to their body weight. Five lysozyme groups were given to the vehicle PBS control group, and db/m mice were used as the normal control group. On the first day, plasminogen or PBS was administered to the plasminogen group. The human plasminogen was injected into the plasminogen group at a dose of 2 mg/0.2 ml/day/day. The vehicle was injected into the same volume of PBS or the same volume of PBS. Any liquid was injected and administered continuously for 31 days.
  • mice were sacrificed on day 32 and pancreas were fixed in 4% paraformaldehyde.
  • the fixed pancreatic tissue was dehydrated by alcohol gradient and transparent to xylene for paraffin embedding.
  • the thickness of the tissue section was 3 ⁇ m, and the sections were dewaxed and rehydrated and washed once with water.
  • PAP pen circle out tissue, drop protease K working solution
  • the tissues were covered, incubated for 7 min at room temperature, and washed 3 times with 0.01 M PBS for 3 minutes each time.
  • TUNEL kit (Roche) reagent 1 and reagent 2 were mixed with liquid (5:45), incubated at 37 ° C for 40 min, and washed with 0.01 M PBS for 3 minutes.
  • Add tunel kit reagent 3 incubate at 37 °C for 30 min, wash with 0.01 M PBS for 3 times, DAB kit (Vector laboratories, Inc., USA), color wash, wash with water for 3 times, hematoxylin counterstaining for 30 seconds, rinse with water for 5 minutes. .
  • the gradient alcohol was dehydrated, the xylene was transparent and neutral gum was mounted, and the sections were observed under a 200-fold optical microscope.
  • TUNEL staining can be used to detect the breakage of nuclear DNA in tissue cells during the late stage of apoptosis.
  • the results of this experiment showed that the number of positive cells (arrow mark) given to the plasminogen group (Fig. 37C) was significantly less than that of the vehicle PBS control group (Fig. 37B). TUNEL positive staining was extremely low in the normal control group (Fig. 37A). The apoptotic rate was about 8% in the normal control group, about 93% in the vehicle PBS group, and about 16% in the plasminogen group. This indicates that the plasminogen group can significantly reduce the apoptosis of islet cells in diabetic mice.
  • mice Six male C57 male mice, 9-10 weeks old, were randomly divided into two groups, three for each of the vehicle PBS control group and the plasminogen group. After two hours of fasting, the mice in the two groups were given a single intraperitoneal injection of 200 mg/kg streptozotocin (STZ) (sigma S0130) to induce T1DM [43] . After 12 days of STZ injection, the drug was administered and designated as the first day of administration.
  • STZ streptozotocin
  • the plasminogen group was injected with human plasmin 1 mg/0.1 ml/day/day into the tail vein, and the same volume of the PBS control group was injected into the tail vein. PBS.
  • PI 3-k inase is a dual specif icity enzyme: autoregulation by an intrinsic p rotein-serin e k inase activity [J] . Em bo J, 1994, 13 (3) : 522-331.
  • TNF tumor necrosis factor

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Abstract

一种使胰高血糖素、胰岛素恢复正常平衡的方法，包括给药受试者有效量的纤溶酶原，同时涉及用于使胰高血糖素、胰岛素恢复正常平衡的药物。

Description

一种使胰高血糖素、胰岛素恢复正常平衡的方法 技术领域
本发明涉及一种使胰高血糖素、胰岛素恢复正常平衡的方法，包括给药受试者有效量的纤溶酶原，同时本发明涉及用于使胰高血糖素、胰岛素恢复正常平衡的药物。
背景技术
糖尿病(diabetes mellitus，DM)是一种常见的具有遗传倾向的葡萄糖代谢异常和内分泌障碍性疾病，是由绝对性或相对性胰岛素分泌不足所引起。2015年，全世界有4.15亿糖尿病患者，预计到2040年，糖尿病患病人数将达到6.42亿[1]。糖尿病是严重危害人类健康的重大疾病之一。
糖尿病的主要表现为糖代谢异常以及脂肪、蛋白质等物质的代谢紊乱，而长期的高血糖状态会导致严重的糖尿病并发症，包括微血管并发症、糖尿病肾病、糖尿病心肌病、糖尿病神经系统病变、糖尿病皮肤病变和糖尿病合并感染等。其中糖尿病肾病以及糖尿病神经系统病变对患者生活质量影响巨大，危害严重。
临床上常见的糖尿病可以分为四种类型：1型糖尿病(type 1 diabetes，T1DM)、2型糖尿病(type 2 diabetes，T2DM)、妊娠期糖尿病、特殊类型糖尿病。其中，以T1DM和T2DM患者最为多见，妊娠期糖尿病和特殊类型糖尿病患者相对较少。
T1DM被认为与遗传因素、环境因素(如病毒感染、致糖尿病化学物质、饮食因素)和自身免疫因素相关。研究表明，T1DM相关的基因位点至少有17个，定位在不同的染色体。环境因素方面，对T1DM发病有影响的环境因素包括病毒感染、致糖尿病化学物质及饮食因素，其中病毒因素最为重要。目前已经发现腮腺炎、风疹病毒、巨细胞病毒等与T1DM发病有关。其机制在于病毒可以直接破坏胰岛β细胞，并在病毒损伤胰岛β细胞后激发自身免疫反应进一步损伤胰岛β细胞。致糖尿病的化学物质如四氧嘧啶、链脲佐菌素(STZ)、喷他脒等作用于胰岛β细胞，导致胰岛β细胞的破坏。自身免疫因素包括体液免疫和细胞免疫。体液免疫表现为患者血液循环中存在多种抗胰岛β细胞的自身抗体。细胞免疫主要表现为在胰岛炎症浸润细胞和胰岛β细胞表面可以观察到HLA-DA抗原的异常表达和IL- 2受体与胰岛细胞表面HLA-1类抗原的过度表达，而外周血的CD4+/CD8+比例，以及IL-1、TNF-α、INF-γ水平升高。这些因素导致的病理变化集中于胰岛β细胞破坏，使得体内胰岛素水平绝对降低，引起T1DM，因此T1DM被考虑是一种自身免疫性疾病。
T2DM是一种多基因遗传性疾病，一般认为它的发生是多源性的，其中环境因素和遗传因素共同作用导致胰岛素抵抗，表现为相同水平浓度的胰岛素因为机体的抵抗作用而无法起到正常水平的作用。而机体为了达到正常血糖水平，将会过量分泌胰岛素以缓解胰岛素使用的“低效”状态，长此以往对胰岛β细胞的要求越来越高，最终导致胰岛β细胞“过度工作”而自身损伤，进展为胰岛素绝对缺乏。
DM的发病机制
DM发病机制复杂，主要与家族遗传倾向、种族异质性、胰岛素受体缺陷、胰岛素受体底物损伤、蛋白酪氨酸磷酸酶相关基因上调、过度免疫炎性反应、脂毒性、氧化应激、及线粒体损伤等相关[2-3]。
1.游离脂肪酸
游离脂肪酸水平升高既是胰岛素抵抗的发病原因之一，也是胰岛素抵抗状态的重要特征之一。在遗传因素或环境因素的作用下，血液中的游离脂肪酸水平升高，当超过脂肪组织的存储能力就会导致胰岛素抵抗的发生。研究显示，长期的高脂饮食将会导致胰岛β细胞发生功能异常，这是因为高脂饮食除了引发外周胰岛素抵抗还会使腹腔脂肪含量升高和胰岛素抑制脂肪分解能力降低，从而促使游离脂肪酸含量升高，继而抑制胰岛素受体及其底物IRS-1、1RS-2的酪氨酸位点磷酸化，抑制P13K的活性，导致胰岛素信号转导通路受阻形成胰岛素抵抗。
2.炎性反应
1)炎症与胰岛素抵抗
T2DM是一种轻度非特异性炎性疾病。近年来的研究显示，炎症导致胰岛素抵抗的主要机制是炎性因子与胰岛素受体底物的信号转导出现交叉，一方面非特异性炎症产生的炎性因子对IRS/PI3K信号通路出现了阻碍作用，而另一方面炎性因子激活的一系列激酶会诱导IRS的丝、苏氨酸位点的磷酸化从而对正常的酪氨酸磷酸化产生阻碍，最终使得胰岛素信号转导能力下降诱发胰岛素抵抗[2-3]。
在靶细胞中，胰岛素与其受体结合能激活受体，之后细胞内的信号转导通路产生一系列细胞内转导分子与酶促级联反应完成信号在胞内的逐级传递并放大，信号最后传至靶器官而产生一系列的生物学效应。信号传导通路主要有两条，一个是IRS-1-PI3K-PKB/AKT途径，另一个是丝裂原活化蛋白激酶(Shc/Raf/MAPK)途径。在第一条通路中，首先是在外源性胰岛素和/或葡萄糖刺激下发生胰岛素与其受体结合，从而激活了受体的内源性酪氨酸激酶。激活的酪氨酸激酶在实现自身的磷酸化的同时诱导了胰岛素受体底物IRS的酪氨酸位点磷酸化。活化的IRS迁移至细胞膜上，通过磷酸酪氨酸结合域(PTB)将磷酸酪氨酸锚定在IRS酪氨酸激酶上，酪氨酸磷酸化的IRS通过其SH2结构域招募到PI3K的调节亚单位P85。P85与磷酸肌醇的3磷酸分子结合，将磷脂酰肌醇一磷酸(PIP)转化为磷脂酰肌醇二磷酸(PIP2)及磷脂酰肌醇三磷酸(PIP3)，它们是胰岛素和其他生长因子的第二信使，是下游信号分子磷酸肌醇依赖的蛋白激酶-1(PDK1)和(或)蛋白激酶c(PKC)的某一亚型的锚定位点。PDK1可以激活蛋白激酶B(PKB，也称为Akt)和某一非典型PKC亚型。激活的PKB一边通过丝/苏氨酸磷酸化让糖原合成激酶-3(GSK3)失活，另一方面激活哺乳动物的雷帕霉素靶点(mTOR)蛋白激酶，从而诱导其下游70ku-S6激酶(p70S6K)磷酸化激活。mTOR蛋白激酶可作为“ATP感受器”，激活p70s6K而不需要通过Ca2+/cAMP，实现控制蛋白的合成、加强基因的转录、促使胰岛β细胞肥大及其他生物效应。PKB可以直接诱导某些转录因子丝/苏氨酸磷酸化促进细胞有丝分裂的发生[4-5]。在第二条通路中，Ras的激活可通过两条通路实现。1)活化的胰岛素受体激活IRS-2蛋白，而IRS-2蛋白可将信号传递给适配蛋白生长因子受体结合蛋白2(Grb2)，再与信号蛋白GDP/GTP交换因子(mSOS)相互作用进而能活化失活的Ras-GDP转变成的Ras-GT从而实现激活Ras。胰岛素受体直接作用使信号蛋白Shc的酪氨酸磷酸化，然后Shc与Grb2结合经mSOS途径激活Ras。激活的Ras-GTP招募Raf丝氨酸激酶，依次使MAPK激酶、MAPK磷酸化。激活的MAPK可激活其他蛋白激酶参与诱导基因转录、调控细胞凋亡等过程[6]。
目前已证实IRS-1的丝氨酸残基可被多种炎症激酶磷酸化，如c-Jun氨基末端激酶(JNK)、IκB激酶β(IκKβ)和蛋白激酶C(PKC)-θ。放射免疫分析法显示丝氨酸307位点是JNK磷酸化IRS-1的主要位点，它的突变会使 JNK诱导的IRS-1磷酸化和TNF对胰岛素引起的IRS-1酪氨酸磷酸化的抑制作用消失。JNK通过磷酸化IRS-1的丝氨酸307，减少了胰岛素受体底物酪氨酸磷酸化，抑制胰岛素信号的转导[7]。Hiorsumi等发现饮食型肥胖鼠和ob/ob鼠的肝脏、肌肉、脂肪组织中JNK活性显著升高。基因敲除(JNK1-/-)能够使饮食诱型肥胖鼠胰岛素抵抗现象减弱，缓解ob/ob鼠的肥胖、高血糖和高胰岛素血症。肥胖鼠肝脏组织IRS-1丝氨酸307位点的磷酸化水平比瘦鼠高，可是在基因敲除(JNK1-/-)的肥胖鼠中并未见升高，可见IRS-1的丝氨酸307位点是JNK在体内作用的靶点[，8]。研究显示TNFα刺激诱导肝细胞胰岛素抵抗的模型中，JNK抑制剂可以完全阻断丝氨酸307的磷酸化。IκKβ可通过至少两个途径影响胰岛素信号传导，可以是直接诱导IRS-1的Ser307位点磷酸化，也可以通过IκB的磷酸化，进而活化NF-κB，通过刺激多种炎症因子的表达间接引发胰岛素抵抗。
炎症反应是感染、组织损伤和应激反应后人体免疫系统对抗这些损伤的防御性反应，同时也是糖尿病、心血管疾病和肿瘤的病因或发病机制。
早在1993年，Hotmamisligil等[9]通过动物实验证明，胰岛素抵抗的肥胖大鼠其脂肪组织中促炎性细胞因子、TNF-α水平高。从此，众多研究者开始探讨炎症与肥胖、胰岛素抵抗之间的关系，并探究其分子发病机制。2006年Hotmamisligil[10]第一次提出代谢性炎症(metabolic inflammation)这一新的医学定义，强调这种低度、慢性的全身炎症主要是由多余的营养物质和代谢物质导致的。代谢性炎症可能存在与典型炎症相类似的分子与信号的传导通路，与既往我们所认识的典型炎症不同的是，代谢性炎症并不存在红、肿、热、痛和功能障碍的症状。正常情况下，机体内环境处于稳态水平，炎症和代谢各自及相互之间均保持一种动态平衡状态。当机体发生代谢紊乱时，打破了机体这种平衡状态，引起免疫系统的失衡，激发炎症信号传导通路，促使机体释放一系列炎症因子，某些炎症因子甚至放大自身炎症反应，形成炎症瀑布效应，进一步使机体发生胰岛素抵抗，从而导致代谢综合征的发生。
研究证明TNF-α与代谢综合征有密切关系。TNF又叫恶液质素，主要由活化的巨噬细胞、自然杀伤(NK)细胞及T淋巴细胞产生，把由巨噬细胞产生分泌的TNF称为TNF-α，把由T淋巴细胞产生分泌的淋巴毒素叫做TNF-β。TNF-α的生物学活性占TNF总活性的70％～95％，因此目前经常 涉及的TNF大多指的是TNF-α。经过多年的研究探讨，目前已明确TNF-α与胰岛素抵抗、自身免疫性疾病、肿瘤、慢性乙型肝炎等多种疾病有关。在胰岛素抵抗的发生发展过程中TNF-α起到至关重要的作用。Swaroop等[11]通过检测50例T2DM患者的血清TNF-α水平，得出T2DM患者TNF-α水平升高，并且与BMI、空腹胰岛素水平以及稳态模型胰岛素抵抗指数(HOMA-IR)显著相关，提示TNF-α在T2DM发病机制中起重要作用。还有研究指出，TNF-α可以使胰岛素受体的磷酸化受到抑制，当胰岛素受体的磷酸化受到抑制时可以减少葡萄糖转运蛋白的基因表达，从而使脂蛋白脂酶的活性降低，最终可以造成脂肪的分解[12]。
2)炎症与胰岛β细胞的凋亡
慢性低度炎症反应与胰岛β细胞功能障碍密切相关。β细胞数量减少导致的胰岛β细胞功能障碍是T2DM发病的另一重要原因，而β细胞的凋亡又是β细胞数量减少最重要的原因。由于遗传或饮食的原因，T2DM患者易发生胰岛素抵抗，患者血糖升高，高血糖状态又能促进IL-6产生，IL-6不仅可以减少GLUT4表达，降低脂肪细胞对葡萄糖的转运，阻碍糖原合成，降低胰岛素的敏感性；同时也可以促进胰岛细胞分泌IL-6，造成恶性循环。高血糖诱导IL-1β大量产生，通过激活NF-κB、MAPK、Fas、NO等通路导致胰岛细胞凋亡，多种炎症通路相互交叉促进，加剧了胰岛细胞的凋亡，最终导致胰岛功能的衰竭[13]。此外，IL-1β还可以介导白细胞间的相互作用，并与其他细胞因子如IFN-γ、TNF-α等相互影响制约，在β细胞损伤过程中起着重要的作用。T2DM的血脂异常会引起激素类物质如瘦素和IL-6水平增加。瘦素可增加IL-1β的释放来诱导β细胞凋亡，还可负相调控胰岛素的分泌[14]。ROS除导致胰岛素抵抗外，对于胰岛β细胞的损伤也有作用，氧化应激状态下，胰岛素基因转录因子的表达以及胰岛素结合位点明显减少，从而影响胰岛素的产生及分泌。其他脂肪细胞因子如TNF-α和瘦也能降低β细胞的功能[15]。这些细胞因子的联合作用，对胰岛β细胞功能造成更明显的损伤。此外，部分炎症因子还可作用于胰岛素受体底物2的关键部位，使其丝氨酸/苏氨酸磷酸化，导致胰岛素受体底物2的降解加快，促进胰岛β细胞的凋亡。
3.氧化应激
研究表明，氧化应激是引起T2DM的发生及发展的重要因素。氧化应 激是指活性氧(reactive oxygen species，ROS)和活性氮(reactive nitrogen species，RNS)的产生与机体内抗氧化防御系统的清除之间失衡，导致ROS和RNS产生过多，造成机体组织细胞及蛋白和核酸等生物大分子损伤[13]。高血糖是产生氧化应激的主要原因，其通过线粒体电子传递链[14]、葡萄糖自氧化和多元醇通路等途径[15]增加机体内的ROS与RNS含量，其中线粒体电子传递链是产生ROS的主要途径。线粒体电子传递链主要涉及酶复合物I～IV、细胞色素c和辅酶Q，在酶复合物I和III中会持续产生少量的超氧产物，包括超氧阴离子，过氧化氢和羟基自由基，而超氧化物歧化酶、过氧化氢酶和谷胱甘肽过氧化物酶会将超氧产物催化转化成氧气和水。但在肥胖或高血糖条件下，超氧产物会大幅增加，当超氧产物的产生速率超过其移除速率时即会产生氧化应激。
多项研究[16-18]表明，ROS可直接损伤β细胞，特别是破坏细胞线粒体结构，促进β细胞凋亡；ROS还可通过影响胰岛素信号转导通路间接抑制β细胞功能，如激活核转录因子κB(nuclear transcription factorκB，NF-κB)信号通路，引起β细胞炎症反应；抑制胰十二指肠同源盒因子1(pancreatic and duodenal homeobox 1，PDX-1)的核质易位，抑制线粒体能量代谢，减少胰岛素合成与分泌等。氧化应激通过NF-κB通路引起β细胞损伤NF-κB为p50和RelA两个亚基组成的二聚体，在静息细胞中，与抑制蛋白IκB结合，以无活性的三聚体形式存在于胞浆中，主要参与细胞对应激、细胞因子、自由基及细菌病毒等刺激的应答及瞬时调控基因表达等[19]。研究表明，高血糖诱导生成的ROS会通过扰乱细胞内信号转导激活NF-κB，诱导β细胞损伤[20]。Mariappan等[21]用吡咯烷二硫代氨基甲酸(PDTC)抑制肥胖db/db小鼠体内NF-κB表达，发现氧化应激对小鼠β细胞线粒体的损伤程度明显减轻；Hofmann等[22]利用抗氧化药物α-硫辛酸对糖尿病患者进行治疗，发现患者体内NF-κB活性显著降低，患者病情也有改善；Eldor等[23]利用转基因技术特异性地抑制小鼠NF-κB的表达，明显降低了STZ诱导后小鼠的糖尿病发病率。
NF-κB作为一种多向核转录因子，激活后参与细胞增殖、细胞凋亡及炎症和免疫等多种基因的调节[24]。在糖尿病机体中，NF-κB通过调控细胞因子和趋化因子的基因表达，如IL-1(interleukin-1)和MCP-1(monocyte/macrophage chemoattractant protein-1)因子等，引起胰岛白细胞 增多，导致β细胞损伤[25]。另外NF-κB调控的许多基因产物如肿瘤坏死因子α(tumor necrosis factorα，TNF-α)等又会进一步激活NF-κB，加重β细胞损伤[26]。
Mahadev等[27]研究显示，ROS对胰岛素信号传导有调控作用，且这种作用有多面性。在胰岛素刺激下，机体会通过Nox(NADPH oxidase)依赖机制快速产生微量的ROS，后者作为第二信使，主要通过氧化作用抑制PTP1B的活性促进胰岛素级联反应[28]，而用DPI(diphenyleneiodonium)抑制Nox后，胰岛素刺激的胰岛素受体(insulin receptor，InsR)与胰岛素受体底物(insulin receptor substrate，IRS)磷酸化下降48％[29]。Loh等[30]的研究显示生理性ROS可促进机体对胰岛素的敏感性。虽然在生理状态下，由胰岛素刺激产生的微量ROS会促进胰岛素的作用，但是长期高血糖会使机体通过线粒体途径产生大量ROS[31]，引起胰岛素抵抗。
InsR和IRS是胰岛素信号传导通路中重要的信号元件：前者是胰岛素信号传导的起始元件，而IRS是前者与通路下游元件的连接桥梁。大量研究表明，氧化应激可通过多个途径干扰InsR和IRS的磷酸化反应，阻碍胰岛素信号传导。IKK是NF-κB的抑制亚基IκB的激活剂，在ROS刺激下IKK可作为InsR和IRS的丝氨酸/苏氨酸磷酸化激酶，促使InsR和IRS发生丝氨酸磷酸化，正常的酪氨酸磷酸化受抑制，阻碍胰岛素信号传导[32]。Brownlee[33]研究显示，IKK可直接磷酸化IRS 307位的丝氨酸残基，导致IRS正常的酪氨酸磷酸化减弱，阻碍InsR与IRS的结合，从而引起胰岛素抵抗。
除IKK外，MAPK家族中的多个成员对InsR和IRS也有影响。JNK、细胞外调节蛋白激酶(extracellularregulated protein kinases，ERK)和p38丝裂原活化蛋白激酶(p38MAPK)是MAPK家族成员，具有丝氨酸/苏氨酸蛋白激酶活性，在受到氧化应激、细胞因子和G-蛋白偶联受体激动剂等作用下可被激活。多项研究表明，JNK、ERK和p38MAPK的激活会加重InsR和IRS的丝氨酸/苏氨酸磷酸化程度，使InsR与IRS之间的蛋白结合能力及IRS活化下游含有SH-2结构域的信号分子的能力降低[34-36]。
糖尿病高糖状态所致的氧化应激是多种慢性并发症形成的关键原因之一，也是诱发DNA损伤的重要因素[37]。糖尿病发生时，细胞外液可见持续高糖。在该状态下，线粒体电子传递链产生的电子明显增多，产生过多 ROS，造成细胞内环境和脂质、蛋白质和DNA等生物大分子损伤。机体在有氧代谢途径中产生的活性氧作为一种突变诱导剂，可将DNA链上的鸟嘌呤氧化为8-羟基鸟嘌呤(8-hydroxy-2′-deoxyguanosine，8-OHdG)。在DNA复制过程中，8-OHdG易与腺嘌呤错配，导致G：C到T：A颠换突变，形成DNA损伤。此外，ROS还会引起其他形式的DNA损伤，包括DNA链断裂、DNA位点突变、DNA双链畸变和原癌基因与肿瘤抑制基因突变等。同时，DNA损伤也可能加剧ROS及氧化应激过程，如DNA损伤可通过H2AX-还原型辅酶II氧化酶1(Nox1)/Rac1通路诱导ROS产生。ROS进一步促使大量Ca2 +进入线粒体，引起细胞坏死和凋亡，或直接损伤线粒体，引起线粒体功能障碍，进而损伤胰岛β细胞，加剧糖尿病的病理过程[38]。
ROS除导致胰岛素抵抗外，对于胰岛β细胞的损伤也有作用，氧化应激状态下，胰岛素基因转录因子的表达以及胰岛素结合位点明显减少，从而影响胰岛素的产生及分泌。其他脂肪细胞因子如TNF-α也能降低β细胞的功能[15]。这些细胞因子的联合作用，对胰岛β细胞功能造成更明显的损伤。此外，部分炎症因子还可作用于胰岛素受体底物2的关键部位，使其丝氨酸/苏氨酸磷酸化，导致胰岛素受体底物2的降解加快，促进胰岛β细胞的凋亡。
从以上可见，氧化应激在糖尿病发生和发展过程中的作用十分复杂。ROS除直接损伤胰岛β细胞外，还可作为信号分子激活一些应激敏感通路，调节相关因子的表达，引起β细胞凋亡或坏死，抑制胰岛素分泌，诱发胰岛素抵抗，最终引发或加重糖尿病。
DM的治疗
糖尿病通常采用药物治疗，传统的药物治疗包括胰岛素类药物和口服类降糖药物。
胰岛素早期主要是从猪牛等动物的胰脏中提取而来，在人体应用后会发生明显的过敏反应。20世纪90年越来越成熟，使得胰岛素类似物逐渐应用起来，这种胰岛素能明显改变传统类胰岛素的药代动力学，有着低血糖发生率低、起效快、作用持久等优势。目前，随着胰岛素制剂探索的不断深入，一些口服胰岛素制剂已步入试验阶段，但因为技术上的困难，至今尚无有效的口服制剂应用在临床。
传统口服类降糖药物较多，常见的有如下几种：(1)双胍类如二甲双 胍。二甲双胍有良好的心血管保护作用，降糖效果也不错，目前已有多个国家将其作为一线药物治疗T2DM。(2)磺脲类：磺脲类属于一种胰岛素促泌剂，刺激胰岛β细胞，使其分泌出胰岛素，达到改善血糖水平的效果。目前，我国允许上市的该类胰岛素主要有格列美脲、格列本脲、格列吡嗪、格列齐特、格列喹酮等，不过从一些研究中显示若长期服用该类药物可能会造成降糖效果失败，极易发生低血糖与体质量增加等并发症。(3)噻唑烷二酮(thiazolidinedionecompounds，TZD)类：1999年FDA将罗格列酮与吡格列酮批准用在T2DM中，前者可能加重心脏病风险，为此之后被限制作为二线治疗药物使用，同时禁用于心衰病症。2013年6月FDA对罗格列酮进行重新审核，指出该药物可继续用于临床，甚至放松或完全解除此药及其复方制剂的应用。(4)α-糖苷酶抑制剂：这类胰岛素会抑制小肠黏膜上皮细胞的糖苷酶，进而缓解碳水化合物的吸收作用，导致餐后血糖水平降低。此类药物常用的有伏格列波糖、阿卡波糖及米格列醇等。
现阶段治疗糖尿病的药物主要为传统抗糖尿病药物，包括磺酰脲类、格列奈类、双胍类、噻唑烷二酮类(thiazolidinediones，TZD)、α-葡萄糖苷酶抑制剂及胰岛素等，这些药物均存在不同程度的不良反应，如引发低血糖、胃肠道不适、肥胖等。随着对糖尿病基础理论研究的深入，为了避免传统降糖药物的副作用、对胰岛β细胞带来保护作用，人们也在积极地寻找新的糖尿病治疗靶点。目前发现与糖尿病发病机制相关的靶点主要包括胰高血糖素样肽-1(glucagon-like peptide-1，GLP-1)、二肽基肽酶-4(dipeptide peptidase-4，DPP-4)、钠-葡萄糖共转运蛋白-2(sodium-glucose cotransporter-2，SGLT-2)、糖原合酶激酶-3(glycogen synthase kinase-3，GSK-3)、蛋白酪氨酸磷酸酶(protein tyrosine phosphates，PTP)、葡萄糖激酶(glucokinase，GK)等。其中基于调整胰高血糖素的药物如胰高血糖素样肽-1(glucagon like peptide-1，GLP-1)类似物、GLP-1受体激动剂及二肽基肽酶-4(dipeptidyl peptidase-4，DPP-4)抑制剂被认为可有效维持血糖稳态、改善β细胞功能、延缓糖尿病发展，甚至逆转糖尿病病程。
对于糖尿病目前尚没有一种有效的药物或手段能完全治愈，目前的药物治疗集中在通过控制血糖在一定的范围降低并延缓并发症的发生。随着 对糖尿病发病机制更深入、全面的了解，对于糖尿病的治疗药物研究，也从对传统机制的药物研究过渡到对具有新靶点和新作用机制的药物研究，其中有些已上市，如GLP-1受体激动剂、DPP-4抑制剂及SGLT-2抑制剂等，还有一些药物处在临床或临床前研究阶段，如GPR119受体激动剂、11β-HSD1抑制剂、PTP1B抑制剂及GK激动剂等，其疗效和安全性还有待进一步临床验证。尽管近年来新靶点抗糖尿病药物的出现为DM治疗提供了更多的选择，但由于糖尿病的发病机制复杂，涉及的激素、酶和受体众多，新药物研究领域还存在诸如单靶点药物作用范围较窄、降糖作用较弱、作用于全身系统引起不良反应等问题均有待于进一步研究。因此，人们需要寻找可作用于糖尿病发病机制诸多方面的、更加有效的治疗药物。
本发明发现纤溶酶原能够减轻糖尿病实验小鼠胰腺组织的损伤、控制炎症、减少胰岛β细胞凋亡、修复胰腺组织、恢复胰岛β细胞的分泌功能、降低血糖，是一种有望成为全面针对糖尿病发病机制诸多方面的全新药物。
发明简述
本发明包括下述各项：
1.一种降低糖尿病受试者胰高血糖素分泌的方法，包括给药受试者有效量的纤溶酶原。
2.项1的方法，其中所述纤溶酶原还降低糖尿病受试者胰高血糖素的表达。
3.项1或2的方法，其中所述糖尿病为T1DM或T2DM。
4.项1-3任一项的方法，其中所述纤溶酶原降低糖尿病受试者进食后的胰高血糖素分泌。
5.项1-4任一项的方法，其中所述纤溶酶原降低糖尿病受试者禁食状态下的胰高血糖素分泌。
6.项1-5任一项的方法，其中所述纤溶酶原在糖尿病受试者血糖升高状态下降低胰高血糖素的分泌，使血糖回复到正常或接近正常水平。
7.项1-6任一项的方法，其中所述纤溶酶原在降低受试者胰高血糖素的表达和/或分泌的同时，促进所述胰岛素表达和/或分泌。
8.项7的方法，其中所述纤溶酶原通过降低受试者胰高血糖素的表达和/或分泌的同时，促进所述胰岛素表达和/或分泌，实现使受试者血糖回复到正常或接近正常水平。
9.项1-8任一项的方法，其中所述纤溶酶原促进胰岛素受体底物2(IRS-2)的表达。
10.一种促进糖尿病受试者胰岛素分泌的方法，包括给药受试者有效量的纤溶酶原。
11.项10的方法，其中所述纤溶酶原还促进糖尿病受试者胰岛素的表达。
12.项10或11的方法，其中所述糖尿病为T1DM或T2DM。
13.项10-12任一项的方法，其中所述纤溶酶原促进糖尿病受试者进食后的胰岛素分泌。
14.项10-12任一项的方法，其中所述纤溶酶原促进糖尿病受试者禁食状态下的胰岛素分泌。
15.项10-14任一项的方法，其中所述纤溶酶原促进糖尿病受试者应答血糖升高刺激的胰岛素分泌，使血糖回复到正常或接近正常水平。
16.项10-15任一项的方法，其中所述纤溶酶原在促进所述胰岛素表达和/或分泌的同时，降低受试者胰高血糖素的表达和/或分泌。
17.项16的方法，其中所述纤溶酶原通过促进所述胰岛素表达和/或分泌的同时，降低受试者胰高血糖素的表达和/或分泌，实现使受试者血糖回复到正常或接近正常水平。
18.一种降低糖尿病受试者血糖的方法，包括给药受试者有效量的纤溶酶原。
19.项18的方法，其中所述血糖选自如下的一项或多项：血清葡萄糖水平、血清果糖胺水平、血清糖化血红蛋白水平。
20.项19的方法，其中所述血糖为血清葡萄糖水平。
21.项18-20任一项的方法，其中所述糖尿病为T1DM或T2DM。
22.一种提高糖尿病受试者糖耐量的方法，包括给药受试者有效量的纤溶酶原。
23.项22的方法，其中所述糖尿病为T2DM。
24.一种促进糖尿病受试者餐后血糖下降的方法，包括给药受试者有效 量的纤溶酶原。
25.项24的方法，其中所述纤溶酶原在受试者餐前30分钟至1.5小时给予。
26.项25的方法，其中所述纤溶酶原在受试者餐前30分钟至1小时给予。
27.一种促进糖尿病受试者对葡萄糖的利用的方法，包括给药受试者有效量的纤溶酶原。
28.项1-27任一项的方法，其中所述纤溶酶原可与一种或多种其它药物或治疗方法联用。
29.项28的方法，其中所述纤溶酶原可与一种或多种选自如下的药物联用：抗糖尿病药物、抗心脑血管疾病药物、抗血栓药物、抗高血压药物，抗血脂药物、抗凝药物、抗感染药物。
30.项1-29任一项的方法，其中所述纤溶酶原与序列2、6、8、10或12具有至少75％、80％、85％、90％、95％、96％、97％、98％或99％的序列同一性，并且仍然具有纤溶酶原活性。
31.项1-30任一项的方法，所述纤溶酶原是在序列2、6、8、10或12的基础上，添加、删除和/或取代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个氨基酸，并且仍然具有纤溶酶原活性的蛋白质。
32.项1-31任一项的方法，所述纤溶酶原是包含纤溶酶原活性片段、并且仍然具有纤溶酶原活性的蛋白质。
33.项1-32任一项的方法，所述纤溶酶原选自Glu-纤溶酶原、Lys-纤溶酶原、小纤溶酶原、微纤溶酶原、delta-纤溶酶原或它们的保留纤溶酶原活性的变体。
34.项1-33任一项的方法，所述纤溶酶原为天然或合成的人纤溶酶原、或其仍然保留纤溶酶原活性的变体或片段。
35.项1-33任一项的方法，所述纤溶酶原为来自灵长类动物或啮齿类动物的人纤溶酶原直向同系物或其仍然保留纤溶酶原活性的变体或片段。
36.项1-35任一项的方法，所述纤溶酶原的氨基酸如序列2、6、8、10或12所示。
37.项1-36任一项的方法，其中所述纤溶酶原是人天然纤溶酶原。
38.项1-37任一项的方法，其中所述受试者是人。
39.项1-38任一项的方法，其中所述受试者缺乏或缺失纤溶酶原。
40.项1-39任一项的方法，所述缺乏或缺失是先天的、继发的和/或局部的。
41.一种用于项1-40任一项的方法的纤溶酶原。
42.一种药物组合物，其包含药学上可接受的载剂和用于项1-40中任一项所述方法的纤溶酶原。
43.一种预防性或治疗性试剂盒，其包含：(i)用于项1-40中任一项所述方法的纤溶酶原和(ii)用于递送所述纤溶酶原至所述受试者的构件(means)。
44.根据项43所述的试剂盒，其中所述构件为注射器或小瓶。
45.项43或44的试剂盒，其还包含标签或使用说明书，该标签或使用说明书指示将所述纤溶酶原投予所述受试者以实施项1-40中任一项所述方法。
46.一种制品，其包含：
含有标签的容器；和
包含(i)用于项1-40中任一项所述方法的纤溶酶原或包含纤溶酶原的药物组合物，其中所述标签指示将所述纤溶酶原或组合物投予所述受试者以实施项1-40中任一项所述方法。
47.项43-45中任一项的试剂盒或项46的制品，还包含另外的一个或多个构件或容器，该构件或容器中含有其他药物。
48.项47的试剂盒或制品，其中所述其他药物选自下组：抗糖尿病药物、抗心脑血管疾病药物、抗血栓药物、抗高血压药物，抗血脂药物、抗凝药物、抗感染药物。
一方面，本发明涉及一种预防和治疗糖尿病的方法，包括给药受试者有效量的纤维蛋白溶酶原或纤溶酶。
另一方面，本发明涉及一种降低糖尿病受试者血糖的方法，包括给药受试者有效量的纤溶酶原。本发明还涉及纤溶酶原用于降低糖尿病受试者血糖的用途。本发明还涉及纤溶酶原用于制备降低糖尿病受试者血糖的药物的用途。此外，本发明还涉及用于降低糖尿病受试者血糖的纤溶酶原。在一些实施方案中，所述血糖选自如下的一项或多项：血清葡萄糖水平、血清果糖胺水平、血清糖化血红蛋白水平。在另一些实施方案中，所述血 糖为血清葡萄糖水平。在上述实施方案中，所述糖尿病为T1DM或T2DM。
另一方面，本发明涉及一种提高糖尿病受试者糖耐量的方法，包括给药受试者有效量的纤溶酶原。本发明还涉及纤溶酶原用于提高糖尿病受试者糖耐量的用途。本发明还涉及纤溶酶原用于制备提高糖尿病受试者糖耐量的药物的用途。此外，本发明还涉及用于提高糖尿病受试者糖耐量的纤溶酶原。在一些实施方案中，所述糖尿病为T2DM。
一方面，本发明涉及一种促进糖尿病受试者餐后血糖下降的方法，包括给药受试者有效量的纤溶酶原。本发明还涉及纤溶酶原用于促进糖尿病受试者餐后血糖下降的用途。本发明还涉及纤溶酶原用于制备促进糖尿病受试者餐后血糖下降的药物的用途。此外，本发明还涉及用于促进糖尿病受试者餐后血糖下降的纤溶酶原。在一些实施方案中，所述纤溶酶原在受试者餐前30分钟至1.5小时给予。在另一些实施方案中，所述纤溶酶原在受试者餐前30分钟至1小时给予。
一方面，本发明涉及一种促进糖尿病受试者对葡萄糖的利用的方法，包括给药受试者有效量的纤溶酶原。本发明还涉及纤溶酶原用于促进糖尿病受试者对葡萄糖的利用的用途。本发明还涉及纤溶酶原用于制备促进糖尿病受试者对葡萄糖的利用的药物的用途。此外，本发明还涉及用于促进糖尿病受试者对葡萄糖的利用的纤溶酶原。另一方面，本发明涉及一种促进糖尿病受试者胰岛素分泌的方法，包括给药受试者有效量的纤溶酶原。在一些实施方案中，所述纤溶酶原还促进糖尿病受试者胰岛素的表达。在上述实施方案中，所述糖尿病为T1DM或T2DM。在一些实施方案中，所述纤溶酶原促进糖尿病受试者进食后的胰岛素分泌。在另一些实施方案中，所述纤溶酶原促进糖尿病受试者禁食状态下的胰岛素分泌。在一些实施方案中，所述纤溶酶原促进糖尿病受试者应答血糖升高刺激的胰岛素分泌，使血糖回复到正常或接近正常水平。在另一些实施方案中，所述纤溶酶原在促进所述胰岛素表达和/或分泌的同时，降低受试者胰高血糖素的表达和/或分泌，具体地，所述纤溶酶原通过促进所述胰岛素表达和/或分泌的同时，降低受试者胰高血糖素的表达和/或分泌，实现使受试者血糖回复到正常或接近正常水平。
一方面，本发明涉及一种降低糖尿病受试者胰高血糖素分泌的方法， 包括给药受试者有效量的纤溶酶原。本发明还涉及纤溶酶原用于降低糖尿病受试者胰高血糖素分泌的用途。本发明还涉及纤溶酶原用于制备降低糖尿病受试者胰高血糖素分泌的药物的用途。此外，本发明还涉及用于降低糖尿病受试者胰高血糖素分泌的纤溶酶原。在一些实施方案中，所述纤溶酶原还降低糖尿病受试者胰高血糖素的表达。在上述实施方案中，所述糖尿病为T1DM或T2DM。在一些实施方案中，所述纤溶酶原降低糖尿病受试者进食后的胰高血糖素分泌。在另一些实施方案中，所述纤溶酶原降低糖尿病受试者禁食状态下的胰高血糖素分泌。在一些实施方案中，所述纤溶酶原在糖尿病受试者血糖升高状态下降低胰高血糖素的分泌，使血糖回复到正常或接近正常水平。在一些实施方案中，所述纤溶酶原在糖尿病受试者血糖升高状态下降低胰高血糖素的分泌，使血糖回复到正常或接近正常水平。在另一些实施方案中，所述纤溶酶原在降低受试者胰高血糖素的表达和/或分泌的同时，促进所述胰岛素表达和/或分泌，具体地，所述纤溶酶原通过降低受试者胰高血糖素的表达和/或分泌的同时，促进所述胰岛素表达和/或分泌，实现使受试者血糖回复到正常或接近正常水平。在上述实施方案中，所述纤溶酶原促进胰岛素受体底物2(IRS-2)的表达。
一方面，本发明涉及一种促进糖尿病受试者胰岛细胞损伤修复的方法，包括给药受试者有效量的纤溶酶原。本发明还涉及纤溶酶原用于促进糖尿病受试者胰岛细胞损伤修复的用途。本发明还涉及纤溶酶原用于制备促进糖尿病受试者胰岛细胞损伤修复的药物的用途。此外，本发明还涉及用于促进糖尿病受试者胰岛细胞损伤修复的纤溶酶原。在一些实施方案中，所述纤溶酶原促进胰岛素受体底物2(IRS-2)的表达。在另一些实施方案中，所述纤溶酶原促进细胞因子TNF-α的表达。在另一些实施方案中，所述纤溶酶原促进受试者多向核转录因子NF-κB的表达。在一些实施方案中，所述胰岛细胞损伤为选自下述的一种或多种：胰岛β细胞合成和分泌胰岛素的功能损伤、胰岛组织结构损伤、胰岛胶原沉积、胰岛的纤维化、胰岛细胞凋亡和胰岛分泌胰高血糖素、胰岛素的平衡紊乱、胰岛分泌胰高血糖素和胰岛素的水平不能与受试者血糖水平相适应。在一些实施方案中，所述纤溶酶原使所述糖尿病受试者胰高血糖素分泌减少，胰岛素分泌增加，具体地，所述胰岛胰高血糖素和胰岛素分泌的正常平衡得到修复。
另一方面，本发明涉及一种保护受试者胰岛的方法，包括给药受试者 有效量的纤溶酶原。本发明还涉及纤溶酶原用于保护受试者胰岛的用途。本发明还涉及纤溶酶原用于制备保护受试者胰岛的药物的用途。此外，本发明还涉及用于保护受试者胰岛的纤溶酶原。在一些实施方案中，所述纤溶酶原减少胰岛胶原沉积。在另一些实施方案中，所述纤溶酶原减轻胰岛的纤维化。在另一些实施方案中，所述纤溶酶原减轻胰岛细胞凋亡。在另一些实施方案中，所述纤溶酶原促进胰岛胰岛素受体底物2(IRS-2)的表达。在一些实施方案中，所述纤溶酶原促进胰岛炎症的修复。在另一些实施方案中，所述纤溶酶原促进细胞因子TNF-α的表达。在另一些实施方案中，所述纤溶酶原促进受试者多向核转录因子NF-κB的表达。在上述实施方案中，所述受试者为糖尿病患者，具体地，所述糖尿病患者为T1DM或T2DM。在一些实施方案中，所述T1DM受试者为PLG活性正常或PLG活性受损受试者。
另一方面，本发明涉及一种促进糖尿病受试者胰岛炎症修复的方法，包括给药受试者有效量的纤溶酶原。本发明还涉及纤溶酶原用于促进糖尿病受试者胰岛炎症修复的用途。本发明还涉及纤溶酶原用于制备促进糖尿病受试者胰岛炎症修复的药物的用途。此外，本发明还涉及促进糖尿病受试者胰岛炎症修复的纤溶酶原。在一些实施方案中，所述纤溶酶原促进细胞因子TNF-α的表达。在另一些实施方案中，所述纤溶酶原促进受试者多向核转录因子NF-κB的表达。在另一些实施方案中，所述纤溶酶原减少胰岛胶原沉积。在另一些实施方案中，所述纤溶酶原减轻胰岛的纤维化。在另一些实施方案中，所述纤溶酶原抑制胰岛细胞凋亡。在上述实施方案中，所述糖尿病患者为T1DM或T2DM，具体地，所述T1DM受试者为PLG活性正常或PLG活性受损受试者。
一方面，本发明涉及一种促进糖尿病受试者细胞因子TNF-α表达的方法，包括给药受试者有效量的纤溶酶原。本发明还涉及纤溶酶原用于促进糖尿病受试者细胞因子TNF-α表达的用途。本发明还涉及纤溶酶原用于制备促进糖尿病受试者细胞因子TNF-α表达的药物的用途。此外，本发明还涉及用于促进糖尿病受试者细胞因子TNF-α表达的纤溶酶原。
另一方面，本发明涉及促进糖尿病受试者多向核转录因子NF-κB表达的方法，包括给药受试者有效量的纤溶酶原。本发明还涉及纤溶酶原用于促进糖尿病受试者多向核转录因子NF-κB表达的用途。本发明还涉及纤溶 酶原用于制备促进糖尿病受试者多向核转录因子NF-κB表达的药物的用途。
另一方面，本发明涉及一种促进胰岛胰岛素受体底物2(IRS-2)表达的方法，包括给药受试者有效量的纤溶酶原。本发明还涉及纤溶酶原用于促进胰岛胰岛素受体底物2(IRS-2)表达的用途。本发明还涉及纤溶酶原用于制备促进胰岛胰岛素受体底物2(IRS-2)表达的药物的用途。此外，本发明还涉及用于促进胰岛胰岛素受体底物2(IRS-2)表达的纤溶酶原。
另一方面，本发明涉及一种促进糖尿病受试者胰岛素分泌的方法，包括给药受试者有效量的纤溶酶原促进胰岛素受体底物2(IRS-2)的表达。本发明还涉及纤溶酶原用于促进糖尿病受试者胰岛素分泌的用途。本发明还涉及纤溶酶原用于制备促进糖尿病受试者胰岛素分泌的药物的用途。此外，本发明还涉及用于促进糖尿病受试者胰岛素分泌的纤溶酶原。
另一方面，本发明涉及一种促进糖尿病受试者胰岛β细胞数量增加的方法，包括给药受试者有效量的纤溶酶原。本发明还涉及纤溶酶原用于促进糖尿病受试者胰岛β细胞数量增加的用途。本发明还涉及纤溶酶原用于制备促进糖尿病受试者胰岛β细胞数量增加的药物的用途。此外，本发明还涉及用于促进糖尿病受试者胰岛β细胞数量增加的纤溶酶原。在一些实施方案中，所述纤溶酶原促进胰岛素受体底物2(IRS-2)表达。
另一方面，本发明涉及一种减少胰岛β细胞凋亡的方法，包括给药受试者有效量的纤溶酶原。本发明还涉及纤溶酶原用于减少胰岛β细胞凋亡的用途。本发明还涉及纤溶酶原用于制备减少胰岛β细胞凋亡的药物的用途。此外，本发明还涉及用于减少胰岛β细胞凋亡的纤溶酶原。在一些实施方案中，所述纤溶酶原促进胰岛素受体底物2(IRS-2)表达。
另一方面，本发明涉及一种促进胰岛β细胞损伤修复的方法，包括给药受试者有效量的纤溶酶原。本发明还涉及纤溶酶原用于促进胰岛β细胞损伤修复的用途。本发明还涉及纤溶酶原用于制备促进胰岛β细胞损伤修复的药物的用途。本发明还涉及用于促进胰岛β细胞损伤修复的纤溶酶原。在一些实施方案中，所述纤溶酶原促进胰岛素受体底物2(IRS-2)表达。
另一方面，本发明涉及一种促进胰岛β细胞功能恢复的方法，包括给药受试者有效量的纤溶酶原。本发明还涉及纤溶酶原用于促进胰岛β细胞 功能恢复的用途。本发明还涉及纤溶酶原用于制备促进胰岛β细胞功能恢复的药物的用途。此外，本发明还涉及用于促进胰岛β细胞功能恢复的纤溶酶原。在一些实施方案中，所述纤溶酶原促进胰岛素受体底物2(IRS-2)表达。
在上述实施方案中，所述纤溶酶原可与一种或多种其它药物或治疗方法联用。具体地，所述纤溶酶原可与一种或多种选自如下的药物联用：抗糖尿病药物、抗心脑血管疾病药物、抗血栓药物、抗高血压药物，抗血脂药物、抗凝药物、抗感染药物。
在上述实施方案中，所述纤溶酶原与序列2、6、8、10或12具有至少75％、80％、85％、90％、95％、96％、97％、98％或99％的序列同一性，并且仍然具有纤溶酶原活性。
在上述实施方案中，所述纤溶酶原的氨基酸如序列2、6、8、10或12所示。在一些实施方案中，所述纤溶酶原是在序列2、6、8、10或12的基础上，添加、删除和/或取代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个氨基酸，并且仍然具有纤溶酶原活性的蛋白质。
在上述实施方案中，所述纤溶酶原是包含纤溶酶原活性片段、并且仍然具有纤溶酶原活性的蛋白质。具体地，所述纤溶酶原选自Glu-纤溶酶原、Lys-纤溶酶原、小纤溶酶原、微纤溶酶原、delta-纤溶酶原或它们的保留纤溶酶原活性的变体。
在上述实施方案中，纤溶酶原为天然或合成的人纤溶酶原、或其仍然保留纤溶酶原活性的变体或片段。在一些实施方案中，所述纤溶酶原为来自灵长类动物或啮齿类动物的人纤溶酶原直向同系物或其仍然保留纤溶酶原活性的变体或片段。例如，来自灵长类动物或啮齿类动物的纤维蛋白溶酶原直向同系物，例如来自大猩猩，恒河猴、鼠、牛、马，狗的纤维蛋白溶酶原直向同系物。最优选，本发明的纤维蛋白溶酶原的氨基酸序列如序列2、6、8、10或12所示。
在上述实施方案中，所述受试者是人。在一些实施方案中，其中所述受试者缺乏或缺失纤溶酶原。具体地，所述缺乏或缺失是先天的、继发的和/或局部的。
在一个实施方案中，所述纤维蛋白溶酶原通过全身或局部给药，优选通过以下途径施用：表面、静脉内、肌内、皮下、吸入、椎管内、局部注射、关节内注射或通过直肠。在一个实施方案中，所述局部给药为直接向骨质疏松区域给药，例如通过敷料，导管等方式来进行。
在一个实施方案中，所述纤溶酶原与适当的多肽载体或稳定剂组合施用。在一个实施方案中，所述纤溶酶原以每天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(以每公斤体重计算)或0.0001-2000mg/cm2、0.001-800mg/cm2、0.01-600mg/cm2、0.1-400mg/cm2、1-200mg/cm2、1-100mg/cm2、10-100mg/cm2(以每平方厘米体表面积计算)的剂量施用，优选至少重复一次，优选至少每天施用。在局部施用的情况下，上述剂量还可以根据情况进一步调整。一方面，本发明涉及一种药物组合物，其包含药学上可接受的载剂和用于本发明所述方法的纤溶酶原。
另一方面，本发明涉及一种预防性或治疗性试剂盒，其包含：(i)用于本发明所述方法的纤溶酶原和(ii)用于递送所述纤溶酶原至所述受试者的构件(means)，具体地，所述构件为注射器或小瓶。在一些实施方案中，所述试剂盒还包含标签或使用说明书，该标签或使用说明书指示将所述纤溶酶原投予所述受试者以实施本发明所述的方法。
另一方面，本发明还涉及一种制品，其包含：含有标签的容器；和(i)用于本发明所述方法的纤溶酶原或包含纤溶酶原的药物组合物，其中所述标签指示将所述纤溶酶原或组合物投予所述受试者以实施本发明所述方法。
在上述实施方案中，所述试剂盒或制品还包含另外的一个或多个构件或容器，该构件或容器中含有其他药物。在一些实施方案中，所述其他药物选自下组：抗糖尿病药物、抗心脑血管疾病药物、抗血栓药物、抗高血压药物，抗血脂药物、抗凝药物、抗感染药物。
发明详述
“糖尿病”是由遗传因素、免疫功能紊乱、微生物感染及其毒素、自由基毒素、精神因素等等各种致病因子作用于机体导致胰岛功能减退、胰岛 素抵抗等而引发的糖、蛋白质、脂肪、水和电解质等一系列代谢紊乱综合征，临床上以高血糖为主要特点。
“糖尿病并发症”是由糖尿病过程中血糖控制不良导致的身体其他器官或组织的损害或功能障碍，其中包括肝脏、肾脏、心脏、视网膜、神经系统的损害或功能障碍等。据世界卫生组织统计，糖尿病并发症高达100多种，是目前已知并发症最多的一种疾病。
“胰岛素抵抗”是指各种原因使胰岛素促进葡萄糖摄取和利用的效率下降，机体代偿性的分泌过多胰岛素产生高胰岛素血症，以维持血糖的稳定。
“纤溶酶”是存在于血液中的一种非常重要的酶，其能够降解纤维蛋白多聚体。
“纤溶酶原(plasminogen，plg)”是纤溶酶的酶原形式，根据swiss prot中的序列，按含有信号肽的天然人源plasminogen氨基酸序列(序列4)计算由810个氨基酸组成，分子量约为90kD，主要在肝脏中合成并能够在血液中循环的糖蛋白，编码该氨基酸序列的cDNA序列如序列3所示。全长的PLG包含七个结构域：位于C末端的丝氨酸蛋白酶结构域、N末端的Pan Apple(PAp)结构域以及5个Kringle结构域(Kringle1-5)。参照swiss prot中的序列，其信号肽包括残基Met1-Gly19，PAp包括残基Glu20-Val98，Kringle1包括残基Cys103-Cys181，Kringle2包括残基Glu184-Cys262，Kringle3包括残基Cys275-Cys352，Kringle4包括残基Cys377-Cys454，Kringle5包括残基Cys481-Cys560。根据NCBI数据，丝氨酸蛋白酶域包括残基Val581-Arg804。
Glu-纤溶酶原是天然全长的纤溶酶原，由791个氨基酸组成(不含有19个氨基酸的信号肽)，编码该序列的cDNA序列如序列1所示，其氨基酸序列如序列2所示。在体内，还存在一种是从Glu-纤溶酶原的第76-77位氨基酸处水解从而形成的Lys-纤溶酶原，如序列6所示，编码该氨基酸序列的cDNA序列如序列5所示。δ-plasminogen(δ-plasminogen)是全长纤溶酶原缺失了Kringle2-Kringle5结构的片段，仅含有Kringle1和丝氨酸蛋白酶域[39，40]，有文献报道了δ-plasminogen的氨基酸序列(序列8)[40]，编码该氨基酸序列的cDNA序列如序列7。Mini-plasminogen由Kringle5和丝氨酸蛋白酶域组成，有文献报道其包括残基Val443-Asn791(以不含有信号肽 的Glu-plg序列的Glu残基为起始氨基酸)[41]，其氨基酸序列如序列10所示，编码该氨基酸序列的cDNA序列如序列9所示。而Micro-plasminogen仅含有丝氨酸蛋白酶结构域，有文献报道其氨基酸序列包括残基Ala543-Asn791(以不含有信号肽的Glu-plg序列的Glu残基为起始氨基酸)[42]，也有专利CN102154253A报道其序列包括残基Lys531-Asn791(以不含有信号肽的Glu-plg序列的Glu残基为起始氨基酸)，本专利序列参考专利CN102154253A，其氨基酸序列如序列12所示，编码该氨基酸序列的cDNA序列如序列11所示。
本发明的“纤溶酶”与“纤维蛋白溶酶”、“纤维蛋白溶解酶”可互换使用，含义相同；“纤溶酶原”与“纤维蛋白溶酶原”、“纤维蛋白溶解酶原”可互换使用，含义相同。
在本申请中，所述纤溶酶原“缺乏”的含义为受试者体内纤溶酶原的含量或活性比正常人低，低至足以影响所述受试者的正常生理功能；所述纤溶酶原“缺失”的含义为受试者体内纤溶酶原的含量或活性显著低于正常人，甚至活性或表达极微，只有通过外源提供才能维持正常生理功能。
本领域技术人员可以理解，本发明纤溶酶原的所有技术方案适用于纤溶酶，因此，本发明描述的技术方案涵盖了纤溶酶原和纤溶酶。
在本发明的实施方案中，“衰老”和“早衰”可以互换使用，表示同样的含义。
在循环过程中，纤溶酶原采用封闭的非活性构象，但当结合至血栓或细胞表面时，在PLG激活剂(plasminogen activator，PA)的介导下，其转变为呈开放性构象的活性PLM。具有活性的PLM可进一步将纤维蛋白凝块水解为纤维蛋白降解产物和D-二聚体，进而溶解血栓。其中PLG的PAp结构域包含维持纤溶酶原处于非活性封闭构象的重要决定簇，而KR结构域则能够与存在于受体和底物上的赖氨酸残基结合。已知多种能够作为PLG激活剂的酶，包括：组织纤溶酶原激活剂(tPA)、尿激酶纤溶酶原激活剂(uPA)、激肽释放酶和凝血因子XII(哈格曼因子)等。
“纤溶酶原活性片段”是指在纤溶酶原蛋白中，能够与底物中的靶序列结合并发挥蛋白水解功能的活性片段。本发明涉及纤溶酶原的技术方案涵盖了用纤溶酶原活性片段代替纤溶酶原的技术方案。本发明所述的纤溶酶原活性片段为包含纤溶酶原的丝氨酸蛋白酶结构域的蛋白质，优选，本发 明所述的纤溶酶原活性片段包含序列14、与序列14具有至少80％、90％、95％、96％、97％、98％、99％同源性的氨基酸序列的蛋白质。因此，本发明所述的纤溶酶原包括含有该纤溶酶原活性片段、并且仍然保持该纤溶酶原活性的蛋白。
目前，对于血液中纤维蛋白溶酶原及其活性测定方法包括：对组织纤维蛋白溶酶原激活剂活性的检测(t-PAA)、血浆组织纤维蛋白溶酶原激活剂抗原的检测(t-PAAg)、对血浆组织纤溶酶原活性的检测(plgA)、血浆组织纤溶酶原抗原的检测(plgAg)、血浆组织纤维蛋白溶酶原激活剂抑制物活性的检测、血浆组织纤维蛋白溶酶原激活剂抑制物抗原的检测、血浆纤维蛋白溶酶-抗纤维蛋白溶酶复合物检测(PAP)。其中最常用的检测方法为发色底物法：向受检血浆中加链激酶(SK)和发色底物，受检血浆中的PLG在SK的作用下，转变成PLM，后者作用于发色底物，随后用分光光度计测定，吸光度增加与纤维蛋白溶酶原活性成正比。此外也可采用免疫化学法、凝胶电泳、免疫比浊法、放射免疫扩散法等对血液中的纤维蛋白溶酶原活性进行测定。
“直系同源物或直系同系物(ortholog)”指不同物种之间的同源物，既包括蛋白同源物也包括DNA同源物，也称为直向同源物、垂直同源物。其具体指不同物种中由同一祖先基因进化而来的蛋白或基因。本发明的纤溶酶原包括人的天然纤溶酶原，还包括来源于不同物种的、具有纤溶酶原活性的纤溶酶原直系同源物或直系同系物。
“保守取代变体”是指其中一个给定的氨基酸残基改变但不改变蛋白质或酶的整体构象和功能，这包括但不限于以相似特性(如酸性，碱性，疏水性，等)的氨基酸取代亲本蛋白质中氨基酸序列中的氨基酸。具有类似性质的氨基酸是众所周知的。例如，精氨酸、组氨酸和赖氨酸是亲水性的碱性氨基酸并可以互换。同样，异亮氨酸是疏水氨基酸，则可被亮氨酸，蛋氨酸或缬氨酸替换。因此，相似功能的两个蛋白或氨基酸序列的相似性可能会不同。例如，基于MEGALIGN算法的70％至99％的相似度(同一性)。“保守取代变体”还包括通过BLAST或FASTA算法确定具有60％以上的氨基酸同一性的多肽或酶，若能达75％以上更好，最好能达85％以上，甚至达90％以上为最佳，并且与天然或亲本蛋白质或酶相比具有相同或基本相似的性质或功能。
“分离的”纤溶酶原是指从其天然环境分离和/或回收的纤溶酶原蛋白。在一些实施方案中，所述纤溶酶原会纯化(1)至大于90％、大于95％、或大于98％的纯度(按重量计)，如通过Lowry法所确定的，例如超过99％(按重量计)，(2)至足以通过使用旋转杯序列分析仪获得N端或内部氨基酸序列的至少15个残基的程度，或(3)至同质性，该同质性是通过使用考马斯蓝或银染在还原性或非还原性条件下的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)确定的。分离的纤溶酶原也包括通过生物工程技术从重组细胞制备，并通过至少一个纯化步骤分离的纤溶酶原。
术语“多肽”、“肽”和“蛋白质”在本文中可互换使用，指任何长度的氨基酸的聚合形式，其可以包括遗传编码的和非遗传编码的氨基酸，化学或生物化学修饰的或衍生化的氨基酸，和具有经修饰的肽主链的多肽。该术语包括融合蛋白，包括但不限于具有异源氨基酸序列的融合蛋白，具有异源和同源前导序列(具有或没有N端甲硫氨酸残基)的融合物；等等。
关于参照多肽序列的“氨基酸序列同一性百分数(％)”定义为在必要时引入缺口以实现最大百分比序列同一性后，且不将任何保守替代视为序列同一性的一部分时，候选序列中与参照多肽序列中的氨基酸残基相同的氨基酸残基的百分率。为测定百分比氨基酸序列同一性目的的对比可以以本领域技术范围内的多种方式实现，例如使用公众可得到的计算机软件，诸如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员能决定用于比对序列的适宜参数，包括对所比较序列全长实现最大对比需要的任何算法。然而，为了本发明的目的，氨基酸序列同一性百分数值是使用序列比较计算机程序ALIGN-2产生的。
在采用ALIGN-2来比较氨基酸序列的情况中，给定氨基酸序列A相对于给定氨基酸序列B的％氨基酸序列同一性(或者可表述为具有或包含相对于、与、或针对给定氨基酸序列B的某一％氨基酸序列同一性的给定氨基酸序列A)如下计算：
分数X/Y乘100
其中X是由序列比对程序ALIGN-2在该程序的A和B比对中评分为相同匹配的氨基酸残基的数目，且其中Y是B中的氨基酸残基的总数。应当领会，在氨基酸序列A的长度与氨基酸序列B的长度不相等的情况下，A相对于B的％氨基酸序列同一性会不等于B相对于A的％氨基酸序列同 一性。除非另有明确说明，本文中使用的所有％氨基酸序列同一性值都是依照上一段所述，使用ALIGN-2计算机程序获得的。
如本文中使用的，术语“治疗”和“预防”指获得期望的药理和/或生理效果。所述效果可以是完全或部分预防疾病或其症状，和/或部分或完全治愈疾病和/或其症状，并且包括：(a)预防疾病在受试者体内发生，所述受试者可以具有疾病的素因，但是尚未诊断为具有疾病；(b)抑制疾病，即阻滞其形成；和(c)减轻疾病和/或其症状，即引起疾病和/或其症状消退。
术语“个体”、“受试者”和“患者”在本文中可互换使用，指哺乳动物，包括但不限于鼠(大鼠，小鼠)、非人灵长类、人、犬、猫、有蹄动物(例如马、牛、绵羊、猪、山羊)等。
“治疗有效量”或“有效量”指在对哺乳动物或其它受试者施用以治疗疾病时足以实现对疾病的所述预防和/或治疗的纤溶酶原的量。“治疗有效量”会根据所使用的纤溶酶原、要治疗的受试者的疾病和/或其症状的严重程度以及年龄、体重等而变化。
2.本发明纤溶酶原的制备
纤溶酶原可以从自然界分离并纯化用于进一步的治疗用途，也可以通过标准的化学肽合成技术来合成。当通过化学合成多肽时，可以经液相或固相进行合成。固相多肽合成(SPPS)(其中将序列的C末端氨基酸附接于不溶性支持物，接着序贯添加序列中剩余的氨基酸)是适合纤溶酶原化学合成的方法。各种形式的SPPS，诸如Fmoc和Boc可用于合成纤溶酶原。用于固相合成的技术描述于Barany和Solid-Phase Peptide Synthesis；第3-284页于The Peptides：Analysis，Synthesis，Biology.第2卷：Special Methods in Peptide Synthesis，Part A.，Merrifield，等J.Am.Chem.Soc.，85：2149-2156(1963)；Stewart等，Solid Phase Peptide Synthesis，2nd ed.Pierce Chem.Co.，Rockford，Ill.(1984)；和Ganesan A.2006Mini Rev.Med Chem.6：3-10和Camarero JA等2005Protein Pept Lett.12：723-8中。简言之，用其上构建有肽链的功能性单元处理小的不溶性多孔珠。在偶联/去保护的重复循环后，将附接的固相游离N末端胺与单个受N保护的氨基酸单元偶联。然后，将此单元去保护，露出可以与别的氨基酸附接的新的N末端胺。肽保持固定在固相上，之后将其切掉。
可以使用标准重组方法来生产本发明的纤溶酶原。例如，将编码纤溶酶原的核酸插入表达载体中，使其与表达载体中的调控序列可操作连接。表达调控序列包括但不限于启动子(例如天然关联的或异源的启动子)、信号序列、增强子元件、和转录终止序列。表达调控可以是载体中的真核启动子系统，所述载体能够转化或转染真核宿主细胞(例如COS或CHO细胞)。一旦将载体掺入合适的宿主中，在适合于核苷酸序列的高水平表达及纤溶酶原的收集和纯化的条件下维持宿主。
合适的表达载体通常在宿主生物体中作为附加体或作为宿主染色体DNA的整合部分复制。通常，表达载体含有选择标志物(例如氨苄青霉素抗性、潮霉素抗性、四环素抗性、卡那霉素抗性或新霉素抗性)以有助于对外源用期望的DNA序列转化的那些细胞进行检测。
大肠杆菌(Escherichia coli)是可以用于克隆主题抗体编码多核苷酸的原核宿主细胞的例子。适合于使用的其它微生物宿主包括杆菌，诸如枯草芽孢杆菌(Bacillus subtilis)和其他肠杆菌科(enterobacteriaceae)，诸如沙门氏菌属(Salmonella)、沙雷氏菌属(Serratia)、和各种假单胞菌属(Pseudomonas)物种。在这些原核宿主中，也可以生成表达载体，其通常会含有与宿主细胞相容的表达控制序列(例如复制起点)。另外，会存在许多公知的启动子，诸如乳糖启动子系统，色氨酸(trp)启动子系统，beta-内酰胺酶启动子系统，或来自噬菌体λ的启动子系统。启动子通常会控制表达，任选在操纵基因序列的情况中，并且具有核糖体结合位点序列等，以启动并完成转录和翻译。
其他微生物，诸如酵母也可用于表达。酵母(例如酿酒酵母(S.cerevisiae))和毕赤酵母(Pichia)是合适的酵母宿主细胞的例子，其中合适的载体根据需要具有表达控制序列(例如启动子)、复制起点、终止序列等。典型的启动子包含3-磷酸甘油酸激酶和其它糖分解酶。诱导型酵母启动于特别包括来自醇脱氢酶、异细胞色素C、和负责麦芽糖和半乳糖利用的酶的启动子。
在微生物外，哺乳动物细胞(例如在体外细胞培养物中培养的哺乳动物细胞)也可以用于表达本发明的纤溶酶原。参见Winnacker，From Genes to Clones，VCH Publishers，N.Y.，N.Y.(1987)。合适的哺乳动物宿主细胞包括CHO细胞系、各种Cos细胞系、HeLa细胞、骨髓瘤细胞系、和经转化的B细胞或杂交瘤。用于这些细胞的表达载体可以包含表达控制序列，如复制起 点，启动子和增强子(Queen等，Immnol.Rev.89：49(1986))，以及必需的加工信息位点，诸如核糖体结合位点，RNA剪接位点，多聚腺苷酸化位点，和转录终止子序列。合适的表达控制序列的例子是白免疫球蛋白基因、SV40、腺病毒、牛乳头瘤病毒、巨细胞病毒等衍生的启动子。参见Co等，J.Immunol.148：1149(1992)。
一旦合成(化学或重组方式)，可以依照本领域的标准规程，包括硫酸铵沉淀，亲和柱，柱层析，高效液相层析(HPLC)，凝胶电泳等来纯化本发明所述的纤溶酶原。该纤溶酶原是基本上纯的，例如至少约80％至85％纯的，至少约85％至90％纯的，至少约90％至95％纯的，或98％至99％纯的或更纯的，例如不含污染物，所述污染物如细胞碎片，除主题抗体以外的大分子，等等。
3.药物配制剂
可以通过将具有所需纯度的纤溶酶原与可选的药用载体，赋形剂，或稳定剂(Remington′s Pharmaceutical Sciences，16版，Osol，A.ed.(1980))混合形成冻干制剂或水溶液制备治疗配制剂。可接受的载体、赋形剂、稳定剂在所用剂量及浓度下对受者无毒性，并包括缓冲剂例如磷酸盐，柠檬酸盐及其它有机酸；抗氧化剂包括抗坏血酸和蛋氨酸；防腐剂(例如十八烷基二甲基苄基氯化铵；氯化己烷双胺；氯化苄烷铵(benzalkonium chloride)，苯索氯铵；酚、丁醇或苯甲醇；烷基对羟基苯甲酸酯如甲基或丙基对羟基苯甲酸酯；邻苯二酚；间苯二酚；环己醇；3-戊醇；间甲酚)；低分子量多肽(少于约10个残基)；蛋白质如血清白蛋白，明胶或免疫球蛋白；亲水聚合物如聚乙烯吡咯烷酮；氨基酸如甘氨酸，谷氨酰胺、天冬酰胺、组氨酸、精氨酸或赖氨酸；单糖，二糖及其它碳水化合物包括葡萄糖、甘露糖、或糊精；螯合剂如EDTA；糖类如蔗糖、甘露醇、岩藻糖或山梨醇；成盐反离子如钠；金属复合物(例如锌-蛋白复合物)；和/或非离子表面活性剂，例如TWEENTM，PLURONICSTM或聚乙二醇(PEG)。优选冻干的抗-VEGF抗体配制剂在WO 97/04801中描述，其包含在本文中作为参考。
本发明的配制剂也可含有需治疗的具体病症所需的一种以上的活性化合物，优选活性互补并且相互之间没有副作用的那些。例如，抗高血压的药物，抗心律失常的药物，治疗糖尿病的药物等。
本发明的纤溶酶原可包裹在通过诸如凝聚技术或界面聚合而制备的微胶囊中，例如，可置入在胶质药物传送系统(例如，脂质体，白蛋白微球，微乳剂，纳米颗粒和纳米胶囊)中或置入粗滴乳状液中的羟甲基纤维素或凝胶-微胶囊和聚-(甲基丙烯酸甲酯)微胶囊中。这些技术公开于Remington′s Pharmaceutical Sciences 16th edition，Osol，A.Ed.(1980)。
用于体内给药的本发明的纤溶酶原必需是无菌的。这可以通过在冷冻干燥和重新配制之前或之后通过除菌滤膜过滤而轻易实现。
本发明的纤溶酶原可制备缓释制剂。缓释制剂的适当实例包括具有一定形状且含有糖蛋白的固体疏水聚合物半通透基质，例如膜或微胶囊。缓释基质实例包括聚酯、水凝胶(如聚(2-羟基乙基-异丁烯酸酯)(Langer等，J.Biomed.Mater.Res.，15：167-277(1981)；Langer，Chem.Tech.，12：98-105(1982))或聚(乙烯醇)，聚交酯(美国专利3773919，EP 58,481)，L-谷氨酸与乙基-L-谷氨酸的共聚物(Sidman，等，Biopolymers 22：547(1983))，不可降解的乙烯-乙烯乙酸酯(ethylene-vinyl acetate)(Langer，等，出处同上)，或可降解的乳酸-羟基乙酸共聚物如Lupron DepotTM(由乳酸-羟基乙酸共聚物和亮氨酰脯氨酸(leuprolide)乙酸酯组成的可注射的微球体)，以及聚D-(-)-3-羟丁酸。聚合物如乙烯-乙酸乙烯酯和乳酸-羟基乙酸能持续释放分子100天以上，而一些水凝胶释放蛋白的时间却较短。可以根据相关机理来设计使蛋白稳定的合理策略。例如，如果发现凝聚的机理是通过硫代二硫键互换而形成分子间S-S键，则可通过修饰巯基残基、从酸性溶液中冻干、控制湿度、采用合适的添加剂、和开发特定的聚合物基质组合物来实现稳定。
4.给药和剂量
可以通过不同方式，例如通过静脉内，腹膜内，皮下，颅内，鞘内，动脉内(例如经由颈动脉)，肌内，鼻内，表面或皮内施用或脊髓或脑投递来实现本发明药物组合物的施用。气溶胶制剂如鼻喷雾制剂包含活性剂的纯化的水性或其它溶液及防腐剂和等渗剂。将此类制剂调节至与鼻粘膜相容的pH和等渗状态。
在一些情况中，可以以下方式修饰或配制本发明的纤溶酶原药物组合物，从而提供其穿过血脑屏障的能力。
用于胃肠外施用的制备物包括无菌水性或非水性溶液、悬浮液和乳剂。非水性溶剂的例子是丙二醇、聚乙二醇、植物油如橄榄油，和可注射有机酯，如油酸乙酯。水性载体包括水、醇性/水性溶液、乳剂或悬浮液，包括盐水和缓冲介质。胃肠外媒介物包含氯化钠溶液、林格氏右旋糖、右旋糖和氯化钠、或固定油。静脉内媒介物包含液体和营养补充物、电解质补充物，等等。也可以存在防腐剂和其他添加剂，诸如例如，抗微生物剂、抗氧化剂、螯合剂、和惰性气体，等等。
在一些实施方案中，本发明的纤溶酶原与促进穿过血脑屏障的药剂配制在一起。在一些情况中，本发明的纤溶酶原直接或经接头与促进穿过血脑屏障的载体分子、肽或蛋白质融合。在一些实施方案中，本发明的纤溶酶原与结合内源血脑屏障(BBB)受体的多肽融合。连接纤溶酶原与结合内源BBB受体的多肽，促进穿过BBB。结合内源BBB受体的合适的多肽包括抗体，例如单克隆抗体，或其抗原结合片段，其特异性结合内源BBB受体。合适的内源BBB受体包括但不限于胰岛素受在一些情况中，抗体是囊封于脂质体中的。参见例如美国专利公开文本No.2009/0156498。
医务人员会基于各种临床因素确定剂量方案。如医学领域中公知的，任一患者的剂量取决于多种因素，包括患者的体型、体表面积、年龄、要施用的具体化合物、性别、施用次数和路径、总体健康、和同时施用的其它药物。本发明包含纤溶酶原的药物组合物的剂量范围可以例如为例如每天约0.0001至2000mg/kg，或约0.001至500mg/kg(例如0.02mg/kg，0.25mg/kg，0.5mg/kg，0.75mg/kg，10mg/kg，50mg/kg等等)受试者体重。例如，剂量可以是1mg/kg体重或50mg/kg体重或在1-50mg/kg的范围，或至少1mg/kg。高于或低于此例示性范围的剂量也涵盖在内，特别是考虑到上述的因素。上述范围中的中间剂量也包含在本发明的范围内。受试者可以每天、隔天、每周或根据通过经验分析确定的任何其它日程表施用此类剂量。例示性的剂量日程表包括连续几天1-10mg/kg。在本发明的药物施用过程中需要实时评估、定期评估血栓和血栓相关疾病的治疗效果和安全性。
5.治疗效力和治疗安全性
本发明的一个实施方案涉及使用纤溶酶原治疗受试者后，对治疗效力和治疗安全性的判断。常用的骨质疏松症治疗效果监测与评估内容包括随 访(不良反应、规范服药、基础措施以及骨折风险因子再评估等)，新发骨折评估(临床骨折、身高降低和影像学检查)、骨密度(bone mineral density，BMD)测量和骨转换生化标志物(bone turnover markers，BTM)检测，以及基于这些数据的综合再评估等。其中BMD是目前应用最广泛的疗效监测和评估方法。例如，可以通过双能X线骨密度仪(dual energy X-ray absorptiometry，DXA)、定量CT(quantitative computed tomography，QCT)、单光子吸收测定法(SPA)、或超声波测定法来测量BMD。治疗开始后可每年检测1次BMD，在BMD达到稳定后可以适当延长间隔，例如2年监测1次。针对BTM，目前在血清学指标中较多使用的骨形成指标是血清1型原胶原N端前肽(procollagen type 1 n-terminal propeptide，PINP)，骨吸收指标是血清1型原胶原C末端肽(serum C-terminal telopeptide，S-CTX)。可根据研究进展，适时调整更合理的检测指标。应在开始治疗前检测基线值，应用促形成药物治疗后3个月、应用抑制吸收药物治疗后3～6个月时进行检测。BTM能够提供骨骼的动态信息，在作用和功能上独立于BMD，同时也与BMD成为互为补充的监测手段，二者结合起来具有更高的临床价值。一般地，如果治疗后BMD上升或稳定，BTM有预期变化，同时治疗期间无骨折发生，可认为治疗反应良好。此外，本发明还涉及使用纤溶酶原及其变体对受试者进行治疗过程中和治疗后，所述该治疗方案安全性的判断，包括但不限于对药物在受试者体内的血清半衰期、治疗半衰期、半数中毒量(TD50)、半数致死量(LD50)进行统计，或对在治疗过程中或治疗后发生的各种不良事件如致敏反应进行观察。
6.制品或药盒
本发明的一个实施方案涉及一种制品或药盒，其包含本发明纤溶酶原。所述制品优选包括一个容器，标签或包装插页。适当的容器有瓶子，小瓶，注射器等。容器可由各种材料如玻璃或塑料制成。所述容器含有组合物，所述组合物可有效治疗本发明的疾病或病症并具有无菌入口(例如所述容器可为静脉内溶液包或小瓶，其含有可被皮下注射针穿透的塞子的)。所述组合物中至少一种活性剂为纤溶酶原。所述容器上或所附的标签说明所述组合物用于治疗本发明所述衰老或衰老相关病症。所述制品可进一步包含含有可药用缓冲液的第二容器，诸如磷酸盐缓冲的盐水，林格氏溶液 以及葡萄糖溶液。其可进一步包含从商业和使用者角度来看所需的其它物质，包括其它缓冲液，稀释剂，过滤物，针和注射器。此外，所述制品包含带有使用说明的包装插页，包括例如指示所述组合物的使用者将纤溶酶原组合物以及治疗伴随的疾病的其它药物给药患者。
附图简述
图1 24-25周龄的糖尿病小鼠给予纤溶酶原35天后胰岛胰高血糖素免疫组化观察结果。A为正常对照组，B为给溶媒PBS对照组，C为给纤溶酶原组。结果显示，胰高血糖素在正常对照小鼠中表达在胰岛周边α细胞区域。与给纤溶酶原组相比，给溶媒PBS对照组胰高血糖素阳性细胞(箭头标识)明显增多，阳性细胞浸润到胰岛的中央区域；给纤溶酶原组胰高血糖素阳性细胞散在的分布于胰岛周边，给纤溶酶原组与PBS组相比，胰岛形态更接近正常小鼠。说明纤溶酶原能够显著抑制胰岛α细胞增殖及胰高血糖素的分泌，修正胰岛α细胞分布紊乱，从而促进胰岛损伤的修复。
图2 26周龄的糖尿病小鼠给予纤溶酶原35天后胰岛胰高血糖素免疫组化观察结果。A为正常对照组，B为给溶媒PBS对照组，C为给纤溶酶原组，D为定量分析结果。结果显示，胰高血糖素在正常对照小鼠中表达在胰岛周边α细胞区域。与给纤溶酶原组相比，给溶媒PBS对照组胰高血糖素阳性细胞(箭头标识)明显增多，阳性细胞浸润到胰岛的中央区域，且平均光密度定量分析结果具有统计学差异(*表示P＜0.05)；给纤溶酶原组胰高血糖素阳性细胞散在的分布于胰岛周边，给纤溶酶原组与PBS组相比，其形态更接近正常小鼠。说明纤溶酶原能够显著抑制胰岛α细胞增殖及胰高血糖素的分泌，修正胰岛α细胞分布紊乱，从而促进胰岛损伤的修复。
图3给予纤溶酶原28天后PLG活性正常小鼠在T1DM模型中胰岛胰高血糖素免疫组化观察结果。A为空白对照组，B为给溶媒PBS对照组，C为给纤溶酶原组，D为定量分析结果。结果显示，给溶媒PBS对照组胰高血糖素阳性表达明显多于给溶纤溶酶原组，且平均光密度定量分析结果统计差异显著(*表示P＜0.05)。说明纤溶酶原能够显著减少糖尿病小鼠胰岛α细胞分泌胰高血糖素，促进胰岛损伤的修复。
图4 24-25周龄糖尿病小鼠给予纤溶酶原10、31天后血糖检测结果。结果显示，给纤溶酶原组小鼠的血糖明显低于给溶媒PBS对照组，且统计差异 显著(*表示P＜0.05，**表示P＜0.01)。此外，随着给药时间的延长，给溶媒PBS对照组小鼠血糖有升高趋势，而给纤溶酶原组血糖逐渐降低。说明纤溶酶原具有降血糖的作用。
图5给予纤溶酶原对糖尿病鼠血清果糖胺浓度的影响。检测结果显示，给予纤溶酶原后血清果糖胺的浓度明显降低，与给药前相比，统计差异极显著(**表示P＜0.01)。说明纤溶酶原能显著降低糖尿病小鼠的血糖。
图6 26周龄的糖尿病小鼠给予纤溶酶原35天后血清果糖胺检测结果。检测结果显示，给纤溶酶原组血清果糖胺的浓度明显低于给溶媒PBS对照组，统计差异接近显著(P＝0.06)。说明纤溶酶原能显著降低糖尿病小鼠的血糖水平。
图7 26周龄糖尿病小鼠给予纤溶酶原35天后血浆糖化血红蛋白检测结果。结果显示，给纤溶酶原组小鼠糖化血红蛋白的OD值明显低于给溶媒PBS对照组，且统计差异极显著(**表示P＜0.01)。说明纤溶酶原具有降低糖尿病小鼠血糖的作用。
图8 26周龄糖尿病小鼠给予纤溶酶原10天后IPGTT检测结果。结果显示，腹腔注射葡萄糖后，给纤溶酶原组小鼠血糖水平低于给溶媒PBS对照组，且与给溶媒PBS对照组相比给纤溶酶原组糖耐受曲线更加接近正常小鼠组。说明纤溶酶原能明显改善糖尿病小鼠糖耐受能力。
图9 T1DM模型PLG活性正常小鼠给予纤溶酶原10天后禁食后血糖检测结果。结果显示，给溶媒PBS对照组小鼠血糖明显高于给纤溶酶原组，且统计差异极显著(***表示P＜0.001)。说明纤溶酶原能显著降低PLG活性正常小鼠在T1DM模型中的血糖水平。
图10 T1DM模型PLG活性正常小鼠给予纤溶酶原28天后IPGTT检测结果。结果显示，给溶媒PBS对照组小鼠注射葡萄糖后血糖浓度明显高于给纤溶酶原组，且与给溶媒PBS对照组相比给纤溶酶原组糖耐受曲线更加接近正常小鼠。说明纤溶酶原能提高PLG活性正常小鼠在T1DM模型中的糖耐受能力。
图11显示T1DM模型小鼠给予纤溶酶原20天后血糖检测结果。结果显示，给溶媒PBS对照组小鼠血糖明显高于给纤溶酶原组小鼠，且统计差异显著(P＝0.04)。说明纤溶酶原能促进T1DM小鼠葡萄糖分解能力，从而降低血糖。
图12 26周龄糖尿病小鼠给予纤溶酶原35天后血清胰岛素检测结果。结果显示，给纤溶酶原组血清胰岛素水平明显高于给溶媒PBS对照组，且统计差异显著(*表示P＜0.05)。说明纤溶酶原能有效促进胰岛素的分泌。
图13 24-25周龄糖尿病小鼠给予纤溶酶原31天后胰脏的HE染色图片及胰岛面积比。A、B为给溶媒PBS对照组，C、D为给纤溶酶原组，E为胰岛面积定量分析结果。结果显示，给溶媒PBS对照组大部分的胰岛发生萎缩，萎缩的胰岛细胞被腺泡(↓标示)所代替，胰岛边缘的腺泡增生，致胰岛与腺泡之间分界不清；给纤溶酶原组大部分的胰岛较之于对照组面积大，且胰岛内未有腺泡增生，只有少数的胰岛内残存有少量的腺泡，胰岛与腺泡之间边界清晰。比较给纤溶酶原组和对照组的胰岛占胰腺的面积比发现，给药组比对照组大近乎一倍。说明纤溶酶原能促进24-25周龄糖尿病小鼠胰岛损伤的修复，从而通过修复损伤的胰岛治疗糖尿病。
图14 24-25周龄糖尿病小鼠给予纤溶酶原31天后胰岛天狼星红染色观察结果。A为给溶媒PBS对照组，B为给纤溶酶原组，C为定量分析结果。结果显示，给纤溶酶原组小鼠胰岛胶原沉积(箭头标识)明显少于给溶媒PBS对照组，且统计差异显著(*表示P＜0.05)。说明纤溶酶原能改善糖尿病动物胰岛的纤维化。
图15 24-25周龄糖尿病小鼠给予纤溶酶原31天后胰岛Caspase-3免疫组化染色观察结果。A为给溶媒PBS对照组，B为给纤溶酶原组。结果显示，给纤溶酶原组Caspase-3的表达(箭头标识)明显低于给溶媒PBS对照组。说明纤溶酶原能减少胰岛细胞的凋亡，保护糖尿病小鼠胰脏组织。
图16 18周龄糖尿病小鼠给予纤溶酶原35天后胰岛胰岛素免疫组化染色结果。A为给溶媒PBS对照组，B为给纤溶酶原组，C为定量分析结果。结果显示，给纤溶酶原组胰岛素的表达(箭头标识)明显高于给溶媒PBS对照组，且统计差异接近显著(P＝0.15)。说明纤溶酶原能够促进胰岛功能修复，促进胰岛素的产生和分泌。
图17 24-25周龄糖尿病小鼠给予纤溶酶原35天后胰岛的胰岛素免疫组化染色观察结果。A为给溶媒PBS对照组，B为给纤溶酶原组，C为定量分析结果。结果显示，给纤溶酶原组胰岛素的表达(箭头标识)明显高于给溶媒PBS对照组，且统计差异显著(*表示P＜0.05)。说明纤溶酶原能促进胰岛功能修复，促进胰岛素的产生和分泌。
图18 26周龄糖尿病小鼠给予纤溶酶原35天后胰岛的胰岛素免疫组化染色结果。A为给溶媒PBS对照组，B为给纤溶酶原组，C为定量分析结果。结果显示，给纤溶酶原组胰岛素的表达(箭头标识)明显高于给溶媒PBS对照组，且统计差异极显著(**表示P＜0.01)。说明纤溶酶原能有效促进胰岛功能修复，促进胰岛素的产生和分泌。
图19 24-25周龄糖尿病小鼠给予纤溶酶原31天后胰腺组织NF-κB免疫组化染色观察结果。A为正常对照组，B为给溶媒PBS对照组，C为给纤溶酶原组，D为定量分析结果。结果显示，给纤溶酶原组NF-κB的表达(箭头标识)明显高于给溶媒PBS对照组，且统计差异显著(*表示P＜0.05)。说明纤溶酶原能促进多向核转录因子NF-κB的表达，从而促进24-25周龄糖尿病小鼠胰岛炎症的修复。
图20 18周龄的糖尿病小鼠给予纤溶酶原35天后胰岛胰高血糖素免疫组化观察结果。A为正常对照组，B为给溶媒PBS对照组，C为给纤溶酶原组，D为定量分析结果。结果显示，胰高血糖素在正常对照小鼠中表达在胰岛周边α细胞区域。与给纤溶酶原组相比，给溶媒PBS对照组胰高血糖素阳性细胞(箭头标识)明显增多，胰高血糖素阳性细胞浸润到胰岛的中央区域，且平均光密度定量分析结果统计差异极显著(**表示P＜0.01)；给纤溶酶原组胰高血糖素阳性细胞散在的分布于胰岛周边，给纤溶酶原组与PBS组相比，胰岛形态更接近正常小鼠。说明纤溶酶原能够显著抑制胰岛α细胞增殖及胰高血糖素的分泌，修正胰岛α细胞分布紊乱，从而促进胰岛损伤的修复。
图21 18周龄的糖尿病小鼠给予纤溶酶原35天后胰岛IRS-2免疫组化观察结果。A为正常对照组，B为给溶媒PBS对照组，C为给纤溶酶原组，D为定量分析结果。结果显示，给溶媒PBS对照组小鼠胰岛IRS-2阳性表达(箭头标识)明显少于给纤溶酶原组，且统计差异极显著(**表示P＜0.01)；给纤溶酶原组IRS-2表达水平较给溶媒PBS组更接近正常对照组小鼠。说明纤溶酶原能有效增加胰岛细胞IRS-2的表达，改善胰岛素信号转导，减少糖尿病小鼠胰岛β细胞损伤。
图22 24-25周龄的糖尿病小鼠给予纤溶酶原31天后胰岛IRS-2免疫组化观察结果。A为正常对照组，B为给溶媒PBS对照组，C为给纤溶酶原组，D为定量分析结果。结果显示，给溶媒PBS对照组小鼠胰岛IRS-2阳 性表达(箭头标识)明显少于给纤溶酶原组，且统计差异显著(*表示P＜0.05)；给纤溶酶原组IRS-2表达水平较给溶媒PBS组更接近正常对照组小鼠。说明纤溶酶原能有效增加胰岛细胞IRS-2的表达，改善胰岛素信号转导，减少糖尿病小鼠胰岛β细胞损伤。
图23 26周龄的糖尿病小鼠给予纤溶酶原35天后胰岛IRS-2免疫组化观察结果。A为正常对照组，B为给溶媒PBS对照组，C为给纤溶酶原组，D为定量分析结果。结果显示，给溶媒PBS对照组小鼠胰岛IRS-2阳性表达(箭头标识)明显少于给纤溶酶原组；给纤溶酶原组IRS-2表达水平较给溶媒PBS组更接近正常对照组小鼠。说明纤溶酶原能有效增加胰岛细胞IRS-2的表达，改善胰岛素信号转导，减少糖尿病小鼠胰岛β细胞损伤。
图24给予纤溶酶原28天后PLG活性正常T1DM小鼠胰岛IRS-2免疫组化观察结果。A为正常对照组，B为给溶媒PBS对照组，C为给纤溶酶原组。结果显示，给溶媒PBS对照组小鼠胰岛IRS-2阳性表达(箭头标识)明显少于给纤溶酶原组；给纤溶酶原组IRS-2表达水平较给溶媒PBS组更接近正常对照组小鼠。说明纤溶酶原能有效增加胰岛细胞IRS-2的表达，改善胰岛素信号转导，减少PLG活性正常T1DM小鼠胰岛β细胞损伤。
图25 26周龄的糖尿病小鼠给予纤溶酶原35天后胰岛中性粒细胞免疫组化观察结果。A为正常对照组，B为给溶媒PBS对照组，C为给纤溶酶原组。结果显示，给纤溶酶原组阳性表达细胞(箭头标识)少于给溶媒PBS对照组，且给纤溶酶原组比给溶媒PBS组更接近正常对照组。说明纤溶酶原能减少中性粒细胞的浸润。
图26 T1DM模型中PLG活性受损小鼠给予纤溶酶原28天后胰岛中性粒细胞免疫组化观察结果。A为空白对照组，B为给溶媒PBS对照组，C为给纤溶酶原组。结果显示，给纤溶酶原组阳性表达细胞(箭头标识)少于给溶媒PBS对照组，且给纤溶酶原组比给溶媒PBS组更接近空白对照组。说明纤溶酶原纤溶酶原能减少PLG活性受损小鼠在T1DM模型中胰岛中性粒细胞的浸润。
图27 PLG活性正常小鼠在T1DM模型中给予纤溶酶原28天后胰岛中性粒细胞免疫组化观察结果。A为空白对照组，B为给溶媒PBS对照组，C为给纤溶酶原组。结果显示，给纤溶酶原组阳性表达细胞(箭头标识)少 于给溶媒PBS对照组，且给纤溶酶原组比给溶媒PBS组更接近空白对照组。说明纤溶酶原能促进PLG活性正常小鼠在T1DM模型中胰岛中性粒细胞的浸润。
图28 T1DM模型中PLG活性受损小鼠给予纤溶酶原28天后胰岛胰岛素免疫组化观察结果。A为空白对照组，B为给溶媒PBS对照组，C为给纤溶酶原组。免疫组化结果显示，给纤溶酶原组胰岛素阳性表达(箭头标识)明显多于给溶媒PBS对照组，且给纤溶酶原组比给溶媒PBS组更接近空白对照组。说明纤溶酶原能促进在T1DM模型中的PLG活性受损小鼠胰岛素的合成与分泌。
图29 PLG活性正常小鼠在T1DM模型中给予纤溶酶原28天后胰岛胰岛素免疫组化观察结果。A为空白对照组，B为给溶媒PBS对照组，C为给纤溶酶原组。免疫组化结果显示，给纤溶酶原组胰岛素阳性表达(箭头标识)明显多于给溶媒PBS对照组，且给纤溶酶原组比给溶媒PBS组更接近空白对照组。说明纤溶酶原促进T1DM模型中PLG活性正常小鼠胰岛素的合成与表达。
图30 PLG活性受损小鼠在T1DM模型中给予纤溶酶原28天后胰岛NF-κB免疫组化观察结果。A为空白对照组，B为给溶媒PBS对照组，C为给纤溶酶原组。结果显示，给纤溶酶原组NF-κB的表达(箭头标识)明显高于给溶媒PBS对照组。说明纤溶酶原能促进炎症修复因子NF-κB的表达，从而促进胰岛炎症的修复。
图31 18周龄的糖尿病小鼠给予纤溶酶原35天后胰岛NF-κB免疫组化观察结果。A为给溶媒PBS对照组，B为给纤溶酶原组。实验结果显示，给纤溶酶原组NF-κB的表达(箭头标识)明显高于给溶媒PBS对照组。说明纤溶酶原能促进多向核转录因子NF-κB的表达，从而促进相对年轻(18周龄)糖尿病小鼠胰岛炎症的修复。
图32 26周龄的糖尿病小鼠给予纤溶酶原35天后胰岛NF-κB免疫组化观察结果。A为正常对照组，B为给溶媒PBS对照组，C为给纤溶酶原组。本发明实验结果显示，给纤溶酶原组NF-κB的表达(箭头标识)明显高于给溶媒PBS对照组。说明纤溶酶原能促进多向核转录因子NF-κB的表达，从而促进相对年老(26周龄)糖尿病小鼠胰岛炎症的修复。
图33 24-25周龄的糖尿病小鼠给予纤溶酶原31天后胰岛TNF-α免疫组化观察结果。A为正常对照组，B为给溶媒PBS对照组，C为给纤溶酶原组。研究结果显示，给纤溶酶原组TNF-α的阳性表达(箭头标识)明显高于给溶媒PBS对照组，且给纤溶酶原组比给溶媒PBS组更接近正常对照组。说明纤溶酶原能促进TNF-α的表达，从而促进24-25周龄糖尿病小鼠胰岛损伤修复。
图34 26周龄的糖尿病小鼠给予纤溶酶原31天后胰岛TNF-α免疫组化观察结果。A为正常对照组，B为给溶媒PBS对照组，C为给纤溶酶原组。研究结果显示，给纤溶酶原组TNF-α的阳性表达(箭头标识)明显高于给溶媒PBS对照组，且给纤溶酶原组比给溶媒PBS组更接近正常对照组。说明纤溶酶原能促进TNF-α的表达，从而促进26周龄糖尿病小鼠胰岛损伤修复。
图35显示PLG活性受损小鼠T1DM模型中给予纤溶酶原28天后胰岛TNF-α免疫组化观察结果。A为给溶媒PBS对照组，B为给纤溶酶原组。研究结果显示，给纤溶酶原组TNF-α的阳性表达(箭头标识)明显高于给溶媒PBS对照组。说明纤溶酶原能促进TNF-α的表达，从而促进PLG活性受损小鼠T1DM模型中胰岛损伤修复。
图36显示PLG活性受损T1DM模型小鼠给予纤溶酶原28天后胰岛IgM免疫组化观察结果。A为空白对照组，B为给溶媒PBS对照组，C为给纤溶酶原组。本实验研究结果显示，给纤溶酶原组IgM的阳性表达(箭头标识)明显低于给溶媒PBS对照组，且给纤溶酶原组比给溶媒PBS组更接近正常对照组。说明纤溶酶原能降低IgM的表达，从而减少PLG活性受损小鼠在T1DM模型中胰岛损伤。
图37 24-25周龄的糖尿病小鼠给予纤溶酶原31天后胰岛TUNEL染色观察结果。A为正常对照组，B为给溶媒PBS对照组，C为给纤溶酶原组。本实验结果显示，给纤溶酶原组的阳性细胞数(箭头标识)明显少于给溶媒PBS对照组。正常对照组TUNEL阳性染色极低。正常对照组凋亡率约为8％，给溶媒PBS组凋亡率约为93％，给纤溶酶原组凋亡率为约16％。说明纤溶酶原组能显著减少糖尿病小鼠胰岛细胞的凋亡。
图38显示T1DM模型小鼠给予纤溶酶原20天后血清胰岛素检测结果。结果显示，给溶媒PBS对照组小鼠血清胰岛素浓度明显低于给纤溶酶原组小 鼠，且统计差异接近显著(P＝0.08)。说明纤溶酶原能促进T1DM小鼠胰岛素的分泌。
实施例
实施例1纤溶酶原减少24-25周龄糖尿病小鼠胰岛α细胞的增殖，恢复胰岛α细胞的正常分布和降低胰高血糖素的分泌
24-25周龄db/db雄性小鼠11只以及db/m雄性小鼠5只，实验开始当天记为第0天并称重，db/db小鼠称重后随机分为两组，给纤溶酶原组5只，给溶媒PBS对照组6只，db/m小鼠作为正常对照组。第1天开始给纤溶酶原或PBS。给纤溶酶原组尾静脉注射人源纤溶酶原2mg/0.2ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS或不注射任何液体，连续给药31天。在第32天处死小鼠，取胰脏在4％多聚甲醛中固定。固定后的胰脏组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡复水后水洗1次。PAP笔圈出组织，以3％双氧水孵育15分钟，0.01MPBS洗2次，每次5分钟。5％的正常羊血清液(Vector laboratories，Inc.，USA)封闭30分钟；时间到后，弃除羊血清液，滴加兔抗小鼠胰高血糖素抗体(Abcam)4℃孵育过夜，0.01M PBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，0.01M PBS洗2次，每次5分钟。按DAB试剂盒(Vector laboratories，Inc.，USA)显色，水洗3次后苏木素复染30秒，流水冲洗5分钟。梯度酒精脱水，二甲苯透明并中性树胶封片，切片在200倍光学显微镜下观察。
胰岛α细胞合成分泌胰高血糖素，主要散在分布于胰岛周边区域。
结果显示，与给纤溶酶原组相(图1C)比给溶媒PBS对照组(图1B)胰高血糖素阳性细胞(箭头标识)明显增多，阳性细胞浸润到胰岛的中央区域；给纤溶酶原组胰高血糖素阳性细胞散在的分布于胰岛周边，给纤溶酶原组的胰岛形态比给溶媒PBS组更接近正常对照组(1A)。说明纤溶酶原能够显著抑制24-25周龄糖尿病小鼠胰岛α细胞增殖及胰高血糖素的分泌，修正胰岛α细胞分布紊乱，提示纤溶酶原能促进胰岛损伤的修复。
实施例2纤溶酶原抑制26周龄糖尿病小鼠胰岛α细胞的增殖，恢复胰岛α细胞的正常分布和降低胰高血糖素的分泌
26周龄db/db雄性小鼠9只以及db/m雄性小鼠3只，实验开始当天记为第0天并称重，db/db小鼠称重后随机分为两组，给纤溶酶原组4只，给溶媒PBS对照组5只，db/m小鼠作为正常对照组。第1天开始给纤溶酶原或PBS。给纤溶酶原组尾静脉注射人源纤溶酶原2mg/0.2ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，连续给药35天。在第36天处死小鼠，取胰脏在4％多聚甲醛中固定。固定后的胰脏组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡复水后水洗1次。PAP笔圈出组织，以3％双氧水孵育15分钟，0.01MPBS洗2次，每次5分钟。5％的正常羊血清液(Vector laboratories，Inc.，USA)封闭30分钟；时间到后，弃除羊血清液，滴加兔抗小鼠胰高血糖素抗体(Abcam)4℃孵育过夜，0.01M PBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，0.01M PBS洗2次，每次5分钟。按DAB试剂盒(Vector laboratories，Inc.，USA)显色，水洗3次后苏木素复染30秒，流水冲洗5分钟。梯度酒精脱水，二甲苯透明并中性树胶封片，切片在200倍光学显微镜下观察。
胰岛α细胞合成分泌胰高血糖素，主要散在分布于胰岛周边区域。
结果显示，与给纤溶酶原组相(图2C)比给溶媒PBS对照组(图2B)胰高血糖素阳性细胞(箭头标识)明显增多，阳性细胞浸润到胰岛的中央区域，且平均光密度定量分析结果具有统计学差异(**表示P＜0.01)(图2D)；给纤溶酶原组胰高血糖素阳性细胞散在的分布于胰岛周边，给纤溶酶原组的胰岛形态比给溶媒PBS组更接近正常对照组(2A)。说明纤溶酶原能够显著抑制26周龄糖尿病小鼠胰岛α细胞增殖及胰高血糖素的分泌，修正胰岛α细胞分布紊乱，提示纤溶酶原能促进胰岛损伤的修复。
实施例3纤溶酶原减少PLG活性正常小鼠T1DM模型中胰高血糖素的分泌
9-10周龄PLG活性正常雄性性小鼠15只，随机分为三组，空白对照组、给溶媒PBS对照组以及给纤溶酶原组，每组各5只。给溶媒PBS对照组和给纤溶酶原组小鼠禁食4小时后单次腹腔注射200mg/kg STZ(Sigma，货号S0130)诱导T1DM模型[43]，空白对照组不做处理。注射12天后开始给药并定为给药第1天，给纤溶酶原组尾静脉注射人源纤溶酶1mg/0.1ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，连续给药28天。在 第29天处死小鼠，取胰脏在4％多聚甲醛中固定。固定后的胰脏组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡复水后水洗1次。PAP笔圈出组织，以3％双氧水孵育15分钟，0.01MPBS洗2次，每次5分钟。5％的正常羊血清液(Vector laboratories，Inc.，USA)封闭30分钟；时间到后，弃除羊血清液，滴加兔抗小鼠胰高血糖素抗体(Abcam)4℃孵育过夜，0.01M PBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，0.01M PBS洗2次，每次5分钟。按DAB试剂盒(Vector laboratories，Inc.，USA)显色，水洗3次后苏木素复染30秒，流水冲洗5分钟。梯度酒精脱水，二甲苯透明并中性树胶封片，切片在200倍光学显微镜下观察。
胰岛α细胞合成分泌胰高血糖素，主要分布于胰岛周边区域。
结果显示，给溶媒PBS对照组(图3B)胰高血糖素阳性表达明显多于给纤溶酶原组(图3C)，且平均光密度定量分析结果统计差异显著(图3D)，且给纤溶酶原组比给溶媒PBS组更接近空白对照组(3A)。说明纤溶酶原能够显著减少STZ诱导的糖尿病小鼠胰岛α细胞分泌胰高血糖素。
实施例4纤溶酶原降低糖尿病小鼠血糖
24-25周龄db/db雄性小鼠8只，随机分为两组，给纤溶酶原组5只和给溶媒PBS对照组3只。实验开始当天记为第0天并称重分组，第1天开始给纤溶酶原或PBS，给纤溶酶原组尾静脉注射人源纤溶酶原2mg/0.2ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，连续给药31天。在第10、31天禁食16小时后，用血糖试纸(Roche，Mannheim，Germany)进行血糖检测。
结果显示，给纤溶酶原组小鼠的血糖明显低于给溶媒PBS对照组，且统计差异显著(*表示P＜0.05，**表示P＜0.01)。此外，随着给药时间的延长，给溶媒PBS对照组小鼠血糖有升高趋势，而给纤溶酶原组血糖逐渐降低(图4)。说明纤溶酶原具有降低糖尿病动物血糖的作用。
实施例5纤溶酶原降低糖尿病小鼠果糖胺水平
24-25周龄db/db雄性小鼠5只，给药前一天每只小鼠眼球静脉丛取血50μl用以检测血清果糖胺浓度，并记为第0天，第一天开始给予纤溶酶原，连续给药31天。第32天摘除眼球取血，检测血清果糖胺的浓度。果糖胺浓度使用果糖胺检测试剂盒(南京建成，A037-2)进行检测。
果糖胺浓度反映1～3周内血糖的平均水平。结果显示，给予纤溶酶原后血清果糖胺的浓度明显降低，与给药前相比统计差异极显著(图5)。说明纤溶酶原能有效降低糖尿病动物血糖。
实施例6纤溶酶原降低26周龄糖尿病小鼠血清果糖胺水平
26周龄db/db雄性小鼠9只，实验开始当天记为第0天并称重，根据体重随机分为两组，给纤溶酶原组4只，给溶媒PBS对照组5只。给纤溶酶原组尾静脉注射人源纤溶酶原2mg/0.2mL/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS。第1天开始给纤溶酶原或PBS，连续给药35天。在第36天处死小鼠，检测血清果糖胺的浓度。果糖胺浓度使用果糖胺检测试剂盒(南京建成，A037-2)进行检测。
检测结果显示，给纤溶酶原组血清果糖胺的浓度明显低于给溶媒PBS对照组，统计差异接近显著(P＝0.06)(图6)。说明纤溶酶原能降低26周龄糖尿病小鼠的血糖果糖胺。
实施例7纤溶酶原降低糖尿病小鼠糖化血红蛋白水平
26周龄db/db雄性小鼠9只，实验开始当天记称重后根据体重随机分为两组，给纤溶酶原组4只，给溶媒PBS对照组5只。第1天开始给纤溶酶原或PBS，给纤溶酶原组尾静脉注射人源纤溶酶原2mg/0.2ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，连续给药35天。在第35天小鼠禁食16小时，第36天摘眼球取血，用以检测血浆糖化血红蛋白的浓度。
糖化血红蛋白含量通常可以反映患者近8～12周的血糖控制情况。结果显示，给纤溶酶原组小鼠糖化血红蛋白的浓度明显低于给溶媒PBS对照组，且统计差异显著(图7)。说明纤溶酶原能有效降低糖尿病动物血糖水平。
实施例8纤溶酶原改善糖尿病小鼠糖耐受能力
26周龄db/db雄性小鼠9只以及db/m小鼠3只。实验开始当天，db/db小鼠称重后并根据体重随机分为两组，给纤溶酶原组4只和给溶媒PBS对照组5只，db/m小鼠作为正常对照组。第1天开始给纤溶酶原或PBS，给纤溶酶原组尾静脉注射人源纤溶酶原2mg/0.2ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，连续给药10天。第11天小鼠禁食16小时后，每只小鼠按5g/kg体重腹腔注射5％葡萄糖溶液，在0，30，60，90，120，180分钟用血糖试纸(Roche，Mannheim，Germany)检测血糖浓度。
腹腔糖耐受检测(Intraperitoneal glucose tolerance test，IPGTT)可检测机体对葡萄糖的耐受能力。现有技术已知糖尿病患者糖耐量是下降的。
实验结果显示，腹腔注射葡萄糖后给纤溶酶原组小鼠血糖水平要低于给溶媒PBS对照组，且与给溶媒PBS对照组相比给纤溶酶原组糖耐受曲线更加接近正常小鼠组(图8)。说明纤溶酶原能明显改善糖尿病小鼠糖耐受能力。
实施例9纤溶酶原降低PLG活性正常小鼠在T1DM模型中血糖水平
9-10周龄PLG活性正常雄性小鼠10只，随机分为两组，给溶媒PBS对照组以及给纤溶酶原组，每组各5只。两组小鼠禁食4小时后单次腹腔注射200mg/kg链脲佐菌素(STZ)(sigma S0130)诱导T1DM[43]。STZ注射12天后开始给药并记为给药第1天，给纤溶酶原组尾静脉注射人源纤溶酶1mg/0.1ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，连续给药10天。在第11天小鼠禁食6小时后，用血糖试纸(Roche，Mannheim，Germany)测定血糖。
结果显示，给溶媒PBS对照组小鼠血糖明显高于给纤溶酶原组小鼠，且统计差异极显著(图9)。说明纤溶酶原能显著降低PLG活性正常小鼠T1DM模型的血糖水平。
实施例10纤溶酶原改善T1DM模型小鼠糖耐受水平
9-10周龄PLG活性正常雄性性小鼠15只，随机分为三组，空白对照组、给溶媒PBS对照组以及给纤溶酶原组，每组各5只。给溶媒PBS对照组和给纤溶酶原组小鼠禁食4小时后单次腹腔注射200mg/kg STZ(sigma S0130)诱导T1DM[43]，空白对照组不做处理。STZ注射12天后开始给药并记为给药第1天，给纤溶酶原组尾静脉注射人源纤溶酶1mg/0.1ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，连续给药28天。第28天小鼠禁食6小时后，按照5g/kg体重腹腔注射5％葡萄糖溶液，在注射后0、15、30、60、90分钟用血糖试纸(Roche，Mannheim，Germany)检测血糖浓度。
腹腔糖耐受检测(Intraperitoneal glucose tolerance test，IPGTT)可检测机体对葡萄糖的耐受能力。现有技术已知糖尿病患者糖耐量下降。
结果显示，给溶媒PBS对照组小鼠注射葡萄糖后血糖浓度明显高于给纤溶酶原组，且与给溶媒PBS对照组相比，给纤溶酶原组糖耐受曲线更加接近 正常小鼠(图10)。说明纤溶酶原能提高PLG活性正常小鼠T1DM模型的糖耐受能力。
实施例11纤溶酶原提高T1DM模型小鼠葡萄糖分解能力
9-10周龄C57雄性小鼠8只，随机分为两组，给溶媒PBS对照组以及给纤溶酶原组，每组各4只。给溶媒PBS对照组和给纤溶酶原组小鼠禁食4小时后单次腹腔注射200mg/kg链脲佐菌素(STZ)(sigma S0130)诱导T1DM[43]。STZ注射12天后开始给药并定为给药第1天，给纤溶酶原组尾静脉注射人源纤溶酶1mg/0.1ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS。连续给药19天，在第20天小鼠禁食6小时后，以2g/kg体重灌胃20％的葡萄糖，60分钟后，眼眶静脉丛采血并离心取上清，以葡萄糖测定试剂盒(上海荣盛361500)测定血糖。
结果显示，给溶媒PBS对照组小鼠血糖明显高于给纤溶酶原组小鼠血糖，且统计差异显著(P＝0.04)(图11)。说明纤溶酶原能提高T1DM小鼠葡萄糖分解能力，从而降低血糖。
实施例12纤溶酶原促进糖尿病小鼠胰岛素分泌功能
26周龄db/db雄性小鼠9只，实验开始当天记为第0天，称重并根据体重随机分为两组，给纤溶酶原组4只，给溶媒PBS对照组5只。第1天开始给纤溶酶原或PBS，给纤溶酶原组尾静脉注射人源纤溶酶原2mg/0.2ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，连续给药35天。在第35天小鼠禁食16小时后，在第36天摘眼球取血，离心取上清，运用胰岛素检测试剂盒(Mercodia AB)按照使用说明检测血清胰岛素水平。
检测结果显示，给纤溶酶原组血清胰岛素水平明显高于给溶媒PBS对照组，且统计差异显著(图12)。说明纤溶酶原能显著提高糖尿病小鼠胰岛素的分泌。
实施例13纤溶酶原对糖尿病小鼠胰腺的保护作用
24-25周龄db/db雄性小鼠7只，实验开始当天记为第0天并称重，根据体重随机分为两组，给纤溶酶原组4只和给溶媒PBS对照组3只。，第1天开始给纤溶酶原或PBS，给纤溶酶原组尾静脉注射人源纤溶酶原2mg/0.2ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，连续给药31天。在第32天处死小鼠，取胰脏在4％多聚甲醛中固定。固定后的胰脏组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡复水并 用苏木素和伊红染色(HE染色)，1％盐酸酒精分化，氨水返蓝，并酒精梯度脱水封片，切片在200和400倍光学显微镜下观察。
结果显示，给溶媒PBS对照组(图13A，13B)大部分的胰岛发生萎缩，萎缩的胰岛细胞被腺泡(箭头标示)所代替，胰岛边缘的腺泡增生，致胰岛与腺泡之间分界不清；给纤溶酶原组(图13C，13D)大部分的胰岛较之于对照组面积大，且胰岛内未有腺泡增生，只有少数的胰岛内残存少量的腺泡，胰岛与腺泡之间边界清晰。比较给药组和对照组的胰岛占胰腺的面积比发现，给药组比对照组大近乎一倍(图13E)。说明纤溶酶原可促进糖尿病小鼠胰岛损伤的修复，提示纤溶酶原有可能通过促进胰岛损伤的修复，从而从根本上治愈糖尿病。
实施例14纤溶酶原减少糖尿病小鼠胰岛胶原沉积
24-25周龄db/db雄性小鼠16只，实验开始当天记为第0天并称重，根据体重随机分为两组，给纤溶酶原组10只，给溶媒PBS对照组6只。第1天开始给纤溶酶原或PBS，给纤溶酶原组尾静脉注射人源纤溶酶原2mg/0.2ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，连续给药31天。在第32天处死小鼠，取胰脏在4％多聚甲醛中固定。固定后的胰脏组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡至水后水洗1次，以0.1％天狼星红染色60分钟后，流水冲洗，苏木素染色1分钟，流水冲洗，1％盐酸酒精和氨水分化返蓝，流水冲洗，烘干后封片，切片在200倍光学显微镜下观察。
天狼星红染色可使胶原持久染色，作为病理切片特殊染色方法，天狼星红染色可以特异显示胶原组织。
染色结果显示，给纤溶酶原组小鼠(图14B)胰岛胶原沉积(箭头标识)明显低于给溶媒PBS对照组(图14A)，且统计差异显著(图14C)。说明纤溶酶原能降低糖尿病动物胰岛的纤维化。
实施例15纤溶酶原减少糖尿病小鼠胰岛细胞凋亡
24-25周龄db/db雄性小鼠6只，实验开始当天记为第0天并称重，根据体重随机分为两组，给纤溶酶原组4只和给溶媒PBS对照组2只。第1天开始给纤溶酶原或PBS，给纤溶酶原组尾静脉注射人源纤溶酶原2mg/0.2ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，连续给药31天。在第32天处死小鼠，取胰脏在4％多聚甲醛中固定。固定后的胰脏组织经酒精梯度脱 水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡复水后水洗1次。以3％双氧水孵育15分钟，水洗2次，每次5分钟。5％的正常羊血清液(Vector laboratories，Inc.，USA)封闭1小时；之后，弃除羊血清液，用PAP笔圈出组织。兔抗小鼠Caspase-3(Abcam)4℃孵育过夜，PBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，PBS洗2次，每次5分钟。按DAB试剂盒(Vector laboratories，Inc.，USA)显色，水洗3次后苏木素复染30秒，流水冲洗5分钟。梯度脱水透明并封片，切片在200倍光学显微镜下观察。
Caspase-3是细胞凋亡过程中最主要的终末剪切酶，其表达越多表明处于凋亡状态的细胞越多[44]。
本发明的实验结果显示，给纤溶酶原组(图15B)Caspase-3的表达(箭头标识)明显低于给溶媒PBS对照组(图15A)。说明纤溶酶原能够减少胰岛细胞的凋亡。
实施例16纤溶酶原促进18周龄进糖尿病小鼠胰岛素的表达和分泌
18周龄db/db雄性小鼠8只，实验开始当天记为第0天并称重，根据体重随机分为两组，给纤溶酶原组和给溶媒PBS对照组，每组各4只。第1天开始给纤溶酶原或PBS，给纤溶酶原组尾静脉注射人源纤溶酶原2mg/0.2ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，连续给药31天。在第36天处死小鼠，取胰脏在4％多聚甲醛中固定。固定后的胰脏组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡复水后水洗1次。以3％双氧水孵育15分钟，水洗2次，每次5分钟。5％的正常羊血清液(Vector laboratories，Inc.，USA)封闭1小时；之后，弃除羊血清液，用PAP笔圈出组织。兔抗小鼠胰岛素抗体(Abcam)4℃孵育过夜，PBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，PBS洗2次，每次5分钟。按DAB试剂盒(Vector laboratories，Inc.，USA)显色，水洗3次后苏木素复染30秒，流水冲洗5分钟。梯度脱水透明并封片，切片在显微镜下200倍下观察。
结果显示，给纤溶酶原组(图16B)胰岛素的表达(箭头标识)明显高于给溶媒PBS对照组(图16A)，且统计差异接近显著(P＝0.15)(图16C)。说明纤溶酶原能够促进胰岛功能修复，促进胰岛素的表达和分泌。
实施例17纤溶酶原促进24-25周龄糖尿病小鼠胰岛素的表达和分泌
24-25周龄db/db雄性小鼠8只，实验开始当天记为第0天并称重，根据体重随机分为两组，给纤溶酶原组5只和给溶媒PBS对照组3只。第1天开始给纤溶酶原或PBS，给纤溶酶原组尾静脉注射人源纤溶酶原2mg/0.2ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，连续给药31天。在第32天处死小鼠，取胰脏在4％多聚甲醛中固定。固定后的胰脏组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡复水后水洗1次。以3％双氧水孵育15分钟，水洗2次，每次5分钟。5％的正常羊血清液(Vector laboratories，Inc.，USA)封闭1小时；之后，弃除羊血清液，用PAP笔圈出组织。兔抗小鼠胰岛素抗体(Abcam)4℃孵育过夜，PBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，PBS洗2次，每次5分钟。按DAB试剂盒(Vector laboratories，Inc.，USA)显色，水洗3次后苏木素复染30秒，流水冲洗5分钟。梯度脱水透明并封片，切片在显微镜下200倍下观察。
结果显示，给纤溶酶原组胰岛素的表达(箭头标识)明显高于给溶媒PBS对照组，且统计差异显著(P＝0.02)(图17)。说明纤溶酶原能有效修复胰岛功能，促进胰岛素的表达和分泌。
实施例18纤溶酶原促进糖尿病小鼠胰岛素合成分泌功能的修复
26周龄db/db雄性小鼠9只，实验开始当天记为第0天并称重，根据体重随机分为两组，给纤溶酶原组4只给溶媒PBS对照组5只。第1天开始给纤溶酶原或PBS，给纤溶酶原组尾静脉注射人源纤溶酶原2mg/0.2ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，连续给药35天。在第35天小鼠禁食16小时后，在第36天处死小鼠，取胰脏在4％多聚甲醛中固定。固定后的胰脏组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡复水后水洗1次。以3％双氧水孵育15分钟，水洗2次，每次5分钟。5％的正常羊血清液(Vector laboratories，Inc.，USA)封闭1小时；之后，弃除羊血清液，用PAP笔圈出组织。兔抗小鼠胰岛素抗体(Abcam)4℃孵育过夜，PBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，PBS洗2次，每次5分钟。按DAB试剂盒(Vector laboratories，Inc.，USA)显色，水洗3次后苏木素复染30秒，流水冲洗5分钟。梯度脱水透明并封片，切片在显微镜下200倍下观察。
结果显示，给纤溶酶原组胰岛素的表达(箭头标识)明显高于给溶媒PBS对照组，且统计差异极显著(P＝0.005)(图18)。说明纤溶酶原能有效修复糖尿病鼠胰岛功能，提高胰岛素的表达和分泌。
实施例19纤溶酶原促进24-25周龄糖尿病小鼠胰岛多向核转录因子NF-κB的表达
24-25周龄db/db雄性小鼠10只，实验开始当天记为第0天并称重，根据体重随机分为两组，给纤溶酶原组4只和给溶媒PBS对照组6只，另取4只db/m作为正常对照组，正常对照组不做处理。第1天开始给纤溶酶原或PBS，给纤溶酶原组尾静脉注射人源纤溶酶原2mg/0.2ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，连续给药31天。在第32天处死小鼠，取胰脏在4％多聚甲醛中固定。固定后的胰脏组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡复水后水洗1次。以3％双氧水孵育15分钟，水洗2次，每次5分钟。5％的正常羊血清液(Vector laboratories，Inc.，USA)封闭1小时；之后，弃除羊血清液，用PAP笔圈出组织。兔抗小鼠NF-κB(Abcam)4℃孵育过夜，PBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，PBS洗2次，每次5分钟。按DAB试剂盒(Vector laboratories，Inc.，USA)显色，水洗3次后苏木素复染30秒，流水冲洗5分钟。梯度脱水透明并封片，切片在显微镜下200倍下观察。
NF-κB为转录因子蛋白家族成员，在炎症修复过程中发挥着重要作用[45]。
本发明实验结果显示，给纤溶酶原组NF-κB的表达(箭头标识)明显高于给溶媒PBS对照组，且统计差异显著(图19)。说明纤溶酶原能促进多向核转录因子NF-κB的表达。
实施例20纤溶酶原减少18周龄糖尿病小鼠胰岛α细胞的增殖，恢复胰岛α细胞的正常分布和降低胰高血糖素的分泌
18周龄db/db雄性小鼠8只以及db/m雄性小鼠3只，实验开始当天记为第0天并称重，db/db小鼠根据体重随机分为两组，给纤溶酶原组和给溶媒PBS对照组，每组各4只，db/m小鼠作为正常对照组。第1天开始给纤溶酶原或PBS。给纤溶酶原组尾静脉注射人源纤溶酶原2mg/0.2ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，连续给药35天。在第36天 处死小鼠，取胰脏在4％多聚甲醛中固定。固定后的胰脏组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡复水后水洗1次。PAP笔圈出组织，以3％双氧水孵育15分钟，0.01MPBS洗2次，每次5分钟。5％的正常羊血清液(Vector laboratories，Inc.，USA)封闭30分钟；时间到后，弃除羊血清液，滴加兔抗小鼠胰高血糖素抗体(Abcam)4℃孵育过夜，0.01M PBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，0.01M PBS洗2次，每次5分钟。按DAB试剂盒(Vector laboratories，Inc.，USA)显色，水洗3次后苏木素复染30秒，流水冲洗5分钟。梯度酒精脱水，二甲苯透明并中性树胶封片，切片在200倍光学显微镜下观察。
胰岛α细胞合成分泌胰高血糖素，主要散在分布于胰岛周边区域。
结果显示，与给纤溶酶原组(图20C)相比，给溶媒PBS对照组(图20B)胰高血糖素阳性细胞(箭头标识)明显增多，阳性细胞浸润到胰岛的中央区域，且平均光密度定量分析结果具有统计学差异(**表示P＜0.01)(图20D)；给纤溶酶原组胰高血糖素阳性细胞散在的分布于胰岛周边，给纤溶酶原组的胰岛形态比给溶媒PBS组更接近正常对照组(20A)。说明纤溶酶原能够显著抑制18周龄糖尿病小鼠胰岛α细胞增殖及胰高血糖素的分泌，修正胰岛α细胞分布紊乱，提示纤溶酶原促进胰岛损伤的修复。
实施例21纤溶酶原促进18周龄糖尿病小鼠胰岛胰岛素受体底物2(IRS-2)的表达
18周龄db/db雄性小鼠7只以及db/m雄性小鼠3只，实验开始当天记为第0天并称重，db/db小鼠根据体重随机分为两组，给纤溶酶原组3只，给溶媒PBS对照组4只，db/m小鼠作为正常对照组。第1天开始给纤溶酶原或PBS。给纤溶酶原组尾静脉注射人源纤溶酶原2mg/0.2ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，连续给药35天。在第36天处死小鼠，取胰脏在4％多聚甲醛中固定。固定后的胰脏组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡复水后水洗1次。PAP笔圈出组织，以3％双氧水孵育15分钟，0.01MPBS洗2次，每次5分钟。5％的正常羊血清液(Vector laboratories，Inc.，USA)封闭30分钟；时间到后，弃除羊血清液，滴加兔抗小鼠IRS-2抗体(Abcam)4℃孵育过夜，0.01M PBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam) 二抗室温孵育1小时，0.01M PBS洗2次，每次5分钟。按DAB试剂盒(Vector laboratories，Inc.，USA)显色，水洗3次后苏木素复染30秒，流水冲洗5分钟。梯度酒精脱水，二甲苯透明并中性树胶封片，切片在200倍光学显微镜下观察。
胰岛素受体底物2((Insulin Receptor Substrate-2，IRS-2)是一种能够被激活的胰岛素受体酪氨酸激酶作用的底物，是胰岛素信号转导途径中重要分子，且对胰岛β细胞的生存非常重要。IRS-2在胰岛β细胞表达增加时对其具有保护效应，且对功能性胰岛β细胞的维持至关重要[46，47]。
IRS-2免疫组化结果显示，给溶媒PBS对照组小鼠(图21B)胰岛IRS-2阳性表达(箭头标识)明显少于给纤溶酶原组(图21C)，且统计差异极显著(图21D)，且给纤溶酶原组比给溶媒PBS组更接近空白对照组(21A)。说明纤溶酶原能有效增加18周龄糖尿病小鼠胰岛细胞IRS-2的表达。
实施例22纤溶酶原促进24-25周龄糖尿病小鼠胰岛IRS-2的表达
24-25周龄db/db雄性小鼠11只以及db/m雄性小鼠5只，实验开始当天记为第0天并称重，db/db小鼠根据体重随机分为两组，给纤溶酶原组5只，给溶媒PBS对照组6只，db/m小鼠作为正常对照组。第1天开始给纤溶酶原或PBS。给纤溶酶原组尾静脉注射人源纤溶酶原2mg/0.2ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS或不注射任何液体，连续给药31天。在第32天处死小鼠，取胰脏在4％多聚甲醛中固定。固定后的胰脏组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡复水后水洗1次。PAP笔圈出组织，以3％双氧水孵育15分钟，0.01MPBS洗2次，每次5分钟。5％的正常羊血清液(Vector laboratories，Inc.，USA)封闭30分钟；时间到后，弃除羊血清液，滴加兔抗小鼠IRS-2抗体(Abcam)4℃孵育过夜，0.01M PBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，0.01M PBS洗2次，每次5分钟。按DAB试剂盒(Vector laboratories，Inc.，USA)显色，水洗3次后苏木素复染30秒，流水冲洗5分钟。梯度酒精脱水，二甲苯透明并中性树胶封片，切片在200倍光学显微镜下观察。
IRS-2免疫组化结果显示，给溶媒PBS对照组小鼠(图22B)胰岛IRS-2阳性表达(箭头标识)明显少于给纤溶酶原组(图22C)，且统计差异显著(图22D)，且给纤溶酶原组比给溶媒PBS组更接近正常对照组(22A)。说明 纤溶酶原能有效增加24-25周龄糖尿病小鼠胰岛细胞IRS-2的表达。
实施例23纤溶酶原促进26周龄糖尿病小鼠胰岛IRS-2的表达
26周龄db/db雄性小鼠9只以及db/m雄性小鼠3只，实验开始当天记为第0天并称重，db/db小鼠根据体重随机分为两组，给纤溶酶原组4只，给溶媒PBS对照组5只，db/m小鼠作为正常对照组。第1天开始给纤溶酶原或PBS。给纤溶酶原组尾静脉注射人源纤溶酶原2mg/0.2ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，连续给药35天。在第36天处死小鼠，取胰脏在4％多聚甲醛中固定。固定后的胰脏组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡复水后水洗1次。PAP笔圈出组织，以3％双氧水孵育15分钟，0.01MPBS洗2次，每次5分钟。5％的正常羊血清液(Vector laboratories，Inc.，USA)封闭30分钟；时间到后，弃除羊血清液，滴加兔抗小鼠IRS-2抗体(Abcam)4℃孵育过夜，0.01M PBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，0.01M PBS洗2次，每次5分钟。按DAB试剂盒(Vector laboratories，Inc.，USA)显色，水洗3次后苏木素复染30秒，流水冲洗5分钟。梯度酒精脱水，二甲苯透明并中性树胶封片，切片在200倍光学显微镜下观察。
IRS-2免疫组化结果显示，给溶媒PBS对照组小鼠(图23B)胰岛IRS-2阳性表达(箭头标识)明显少于给纤溶酶原组(图23C)；给纤溶酶原组IRS-2表达水平接近正常对照组小鼠(图23A)。说明纤溶酶原能有效增加26周龄糖尿病小鼠胰岛细胞IRS-2的表达。
实施例24纤溶酶原促进PLG活性正常T1DM小鼠胰岛IRS-2的表达
9-10周龄PLG活性正常雄性性小鼠15只，随机分为三组，空白对照组、给溶媒PBS对照组以及给纤溶酶原组，每组各5只。给溶媒PBS对照组和给纤溶酶原组小鼠禁食4小时后单次腹腔注射200mg/kg STZ(Sigma，货号S0130)诱导I型糖尿病[43]，空白对照组不做处理。注射12天后开始给药并定为给药第1天，给纤溶酶原组尾静脉注射人源纤溶酶1mg/0.1ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，连续给药28天。在第29天处死小鼠，取胰脏在4％多聚甲醛中固定。固定后的胰脏组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡复水后水洗1次。PAP笔圈出组织，以3％双氧水孵育15分钟，0.01MPBS洗 2次，每次5分钟。5％的正常羊血清液(Vector laboratories，Inc.，USA)封闭30分钟；时间到后，弃除羊血清液，滴加兔抗小鼠IRS-2抗体(Abcam)4℃孵育过夜，0.01M PBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，0.01M PBS洗2次，每次5分钟。按DAB试剂盒(Vector laboratories，Inc.，USA)显色，水洗3次后苏木素复染30秒，流水冲洗5分钟。梯度酒精脱水，二甲苯透明并中性树胶封片，切片在200倍光学显微镜下观察。
IRS-2免疫组化结果显示，给溶媒PBS对照组小鼠(图24B)胰岛IRS-2阳性表达(箭头标识)明显少于给纤溶酶原组(图24C)，且给纤溶酶原组比给溶媒PBS组更接近空白对照组(24A)。说明纤溶酶原能有效增加9-10周龄PLG活性正常小鼠胰岛细胞IRS-2的表达。
实施例25纤溶酶原减少24-26周龄糖尿病小鼠胰岛中性粒细胞的浸润
24-26周龄db/db雄性小鼠9只以及db/m小鼠3只，db/db小鼠随机分为两组，给纤溶酶原组4只，给溶媒PBS对照组5只，db/m小鼠作为正常对照组。实验开始当天记为第0天称重分组，实验第二天开始给纤溶酶原或PBS并记为第1天。给纤溶酶原组尾静脉注射人源纤溶酶原2mg/0.2ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，连续给药35天。在第36天处死小鼠，取胰脏在4％多聚甲醛中固定。固定后的胰脏组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡复水后水洗1次。PAP笔圈出组织，以3％双氧水孵育15分钟，0.01MPBS洗2次，每次5分钟。5％的正常羊血清液(Vector laboratories，Inc.，USA)封闭30分钟；时间到后，弃除羊血清液，滴加兔抗小鼠中性粒细胞抗体(Abcam)4℃孵育过夜，0.01M PBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，0.01M PBS洗2次，每次5分钟。按DAB试剂盒(Vector laboratories，Inc.，USA)显色，水洗3次后苏木素复染30秒，流水冲洗5分钟。梯度酒精脱水，二甲苯透明并中性树胶封片，切片在200倍光学显微镜下观察。
中心粒细胞是非特异性细胞免疫系统中重要成员，当炎症发生时，它们被趋化性物质吸引到炎症部位。
中心粒细胞免疫组化结果显示，给纤溶酶原组(图25C)阳性表达细胞少于给溶媒PBS对照组(图25B)，且给纤溶酶原组比给溶媒PBS组更接近正常对照组(25A)。
实施例26纤溶酶原减少PLG活性受损小鼠在T1DM模型中胰岛中性粒细胞的浸润
9-10周龄PLG活性受损雄性小鼠10只，随机分为三组，空白对照组3只，给PBS对照组3只，给纤溶酶原组4只。给溶媒PBS对照组和给纤溶酶原组小鼠禁食4小时后单次腹腔注射200mg/kg STZ(sigma S0130)诱导I型糖尿病[43]，空白对照组不做处理。注射12天后开始给药并定为给药第1天，给纤溶酶原组尾静脉注射人源纤溶酶1mg/0.1ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，连续给药28天。在第29天处死小鼠，取胰脏在4％多聚甲醛中固定。固定后的胰脏组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡复水后水洗1次。PAP笔圈出组织，以3％双氧水孵育15分钟，0.01MPBS洗2次，每次5分钟。5％的正常羊血清液(Vector laboratories，Inc.，USA)封闭30分钟；时间到后，弃除羊血清液，滴加兔抗小鼠中性粒细胞抗体(Abcam)4℃孵育过夜，0.01M PBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，0.01M PBS洗2次，每次5分钟。按DAB试剂盒(Vector laboratories，Inc.，USA)显色，水洗3次后苏木素复染30秒，流水冲洗5分钟。梯度酒精脱水，二甲苯透明并中性树胶封片，切片在400倍光学显微镜下观察。
中心粒细胞免疫组化结果显示，给纤溶酶原组(图26C)阳性表达细胞(箭头标识)少于给溶媒PBS对照组(图26B)，且给纤溶酶原组比给溶媒PBS组更接近空白对照组(26A)。
实施例27纤溶酶原减少PLG活性正常小鼠在T1DM模型中胰岛中性粒细胞的浸润
9-10周龄PLG活性正常雄性性小鼠11只，随机分为三组，空白对照组3只，给溶媒PBS对照组4只，给纤溶酶原组4只。给溶媒PBS对照组和给纤溶酶原组小鼠禁食4小时后单次腹腔注射200mg/kg STZ(sigmaS0130)诱导I型糖尿病[43]，空白对照组不做处理。注射12天后开始给药并定为给药第1天，给纤溶酶原组尾静脉注射人源纤溶酶1mg/0.1ml/只/天， 给溶媒PBS对照组尾静脉注射同体积的PBS，连续给药28天。在第29天处死小鼠，取胰脏在4％多聚甲醛中固定。固定后的胰脏组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡复水后水洗1次。PAP笔圈出组织，以3％双氧水孵育15分钟，0.01MPBS洗2次，每次5分钟。5％的正常羊血清液(Vector laboratories，Inc.，USA)封闭30分钟；时间到后，弃除羊血清液，滴加兔抗小鼠中心粒细胞抗体(Abcam)4℃孵育过夜，0.01M PBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，0.01M PBS洗2次，每次5分钟。按DAB试剂盒(Vector laboratories，Inc.，USA)显色，水洗3次后苏木素复染30秒，流水冲洗5分钟。梯度酒精脱水，二甲苯透明并中性树胶封片，切片在400倍光学显微镜下观察。
中心粒细胞免疫组化结果显示，给纤溶酶原组(图27C)阳性表达细胞(箭头标识)少于给溶媒PBS对照组(图27B)，且给纤溶酶原组比给溶媒PBS组更接近空白对照组(27A)。
实施例28溶酶原促进在T1DM模型中的PLG活性受损小鼠胰岛素的合成与分泌
9-10周龄PLG活性受损雄性性小鼠10只，随机分为三组，空白对照组3只，给PBS对照组3只，给纤溶酶原组4只。给溶媒PBS对照组和给纤溶酶原组小鼠禁食4小时后单次腹腔注射200mg/kg STZ(sigma S0130)诱导I型糖尿病[43]，空白对照组不做处理。注射12天后开始给药并定为给药第1天，给纤溶酶原组尾静脉注射人源纤溶酶1mg/0.1ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，连续给药28天。在第29天处死小鼠，取胰脏在4％多聚甲醛中固定。固定后的胰脏组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡复水后水洗1次。PAP笔圈出组织，以3％双氧水孵育15分钟，0.01MPBS洗2次，每次5分钟。5％的正常羊血清液(Vector laboratories，Inc.，USA)封闭30分钟；时间到后，弃除羊血清液，滴加兔抗小鼠胰岛素抗体(Abcam)4℃孵育过夜，0.01M PBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，0.01M PBS洗2次，每次5分钟。按DAB试剂盒(Vector laboratories，Inc.，USA)显色，水洗3次后苏木素复染30秒，流水冲洗 5分钟。梯度酒精脱水，二甲苯透明并中性树胶封片，切片在200倍光学显微镜下观察。
免疫组化结果显示，给纤溶酶原组(图28C)胰岛素阳性表达(箭头标识)明显多于给溶媒PBS对照组(图28B，且给纤溶酶原组比给溶媒PBS组更接近空白对照组(28A))。说明纤溶酶原能促进在T1DM模型中的PLG活性受损小鼠胰岛素的合成与分泌。
实施例29纤溶酶原促进T1DM模型中PLG活性正常小鼠胰岛素的合成与表达
9-10周龄PLG活性正常雄性性小鼠11只，随机分为三组，空白对照组3只，给溶媒PBS对照组4只，给纤溶酶原组4只。给溶媒PBS对照组和给纤溶酶原组小鼠禁食4小时后单次腹腔注射200mg/kg STZ(sigma S0130)诱导I型糖尿病[43]，空白对照组不做处理。注射12天后开始给药并定为给药第1天，给纤溶酶原组尾静脉注射人源纤溶酶1mg/0.1ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，连续给药28天。在第29天处死小鼠，取胰脏在4％多聚甲醛中固定。固定后的胰脏组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡复水后水洗1次。PAP笔圈出组织，以3％双氧水孵育15分钟，0.01MPBS洗2次，每次5分钟。5％的正常羊血清液(Vector laboratories，Inc.，USA)封闭30分钟；时间到后，弃除羊血清液，滴加兔抗小鼠胰岛素抗体(Abcam)4℃孵育过夜，0.01M PBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，0.01M PBS洗2次，每次5分钟。按DAB试剂盒(Vector laboratories，Inc.，USA)显色，水洗3次后苏木素复染30秒，流水冲洗5分钟。梯度酒精脱水，二甲苯透明并中性树胶封片，切片在200倍光学显微镜下观察。
免疫组化结果显示，给纤溶酶原组(图29C)胰岛素阳性表达(箭头标识)明显多于给溶媒PBS对照组(图29B)，且给纤溶酶原组比给溶媒PBS组更接近空白对照组(29A)。说明纤溶酶原促进T1DM模型中PLG活性正常小鼠胰岛素的合成与表达。
实施例30纤溶酶原促进PLG活性受损小鼠T1DM模型中胰岛多向核转录因子NF-κB的表达
9-10周龄PLG活性受损雄性性小鼠10只，随机分为三组，空白对照组3只，给PBS对照组3只，给纤溶酶原组4只。给溶媒PBS对照组和给纤溶酶原组小鼠禁食4小时后单次腹腔注射200mg/kg STZ(sigma S0130)诱导I型糖尿病[43]，空白对照组不做处理。注射12天后开始给药并定为给药第1天，给纤溶酶原组尾静脉注射人源纤溶酶1mg/0.1ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，连续给药28天。在第29天处死小鼠，取胰脏在4％多聚甲醛中固定。固定后的胰脏组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡复水后水洗1次。PAP笔圈出组织，以3％双氧水孵育15分钟，0.01MPBS洗2次，每次5分钟。5％的正常羊血清液(Vector laboratories，Inc.，USA)封闭30分钟；时间到后，弃除羊血清液，滴加兔抗小鼠NF-κB抗体(Cell Signal)4℃孵育过夜，0.01M PBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，0.01M PBS洗2次，每次5分钟。按DAB试剂盒(Vector laboratories，Inc.，USA)显色，水洗3次后苏木素复染30秒，流水冲洗5分钟。梯度酒精脱水，二甲苯透明并中性树胶封片，切片在200倍光学显微镜下观察。
NF-κB作为一种多向核转录因子，激活后参与细胞增殖、细胞凋亡及炎症和免疫等多种基因的调节[24]。
实验结果显示，给纤溶酶原组(图30C)NF-κB的表达(箭头标识)明显高于给溶媒PBS对照组(图30B)。说明纤溶酶原能促进多向核转录因子NF-κB的表达。
实施例31纤溶酶原促进18周龄糖尿病小鼠胰岛多向核转录因子NF-κB的表达
18周龄db/db雄性小鼠7只，实验开始当天记为第0天并称重，根据体重随机分为两组，给纤溶酶原组3只，给溶媒PBS对照组4只。第1天开始给纤溶酶原或PBS，给纤溶酶原组尾静脉注射人源纤溶酶原2mg/0.2ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，连续给药35天。在第36天处死小鼠，取胰脏在4％多聚甲醛中固定。固定后的胰脏组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡复水后水洗1次。PAP笔圈出组织，以3％双氧水孵育15分钟，0.01MPBS洗2次，每次5分钟。5％的正常羊血清液(Vector  laboratories，Inc.，USA)封闭30分钟；时间到后，弃除羊血清液，滴加兔抗小鼠NF-κB抗体(Cell Signal)4℃孵育过夜，0.01M PBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，0.01M PBS洗2次，每次5分钟。按DAB试剂盒(Vector laboratories，Inc.，USA)显色，水洗3次后苏木素复染30秒，流水冲洗5分钟。梯度酒精脱水，二甲苯透明并中性树胶封片，切片在200倍光学显微镜下观察。
本发明实验结果显示，给纤溶酶原组(图31B)NF-κB的表达(箭头标识)明显高于给溶媒PBS对照组(图31A)。说明纤溶酶原能促进多向核转录因子NF-κB的表达。
实施例32纤溶酶原抑制26周龄糖尿病小鼠多向核转录因子NF-κB的表达
26周龄db/db雄性小鼠9只以及db/m雄性小鼠3只，实验开始当天记为第0天并称重，db/db小鼠根据体重随机分为两组，给纤溶酶原组4只，给溶媒PBS对照组5只，db/m小鼠作为正常对照组。第1天开始给纤溶酶原或PBS并记为第1天，给纤溶酶原组尾静脉注射人源纤溶酶原2mg/0.2ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，连续给药35天。在第36天处死小鼠，取胰脏在4％多聚甲醛中固定。固定后的胰脏组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡复水后水洗1次。PAP笔圈出组织，以3％双氧水孵育15分钟，0.01MPBS洗2次，每次5分钟。5％的正常羊血清液(Vector laboratories，Inc.，USA)封闭30分钟；时间到后，弃除羊血清液，滴加兔抗小鼠NF-κB抗体(Cell Signal)4℃孵育过夜，0.01M PBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，0.01M PBS洗2次，每次5分钟。按DAB试剂盒(Vector laboratories，Inc.，USA)显色，水洗3次后苏木素复染30秒，流水冲洗5分钟。梯度酒精脱水，二甲苯透明并中性树胶封片，切片在200倍光学显微镜下观察。
实验结果显示，给纤溶酶原组(图32C)NF-κB的表达(箭头标识)明显高于给溶媒PBS对照组(图32B)，且给纤溶酶原组比给溶媒PBS组更接近正常对照组(32A)。说明纤溶酶原能促进相对年老(26周龄)糖尿病小鼠多向核转录因子NF-κB的表达。
实施例33纤溶酶原促进24-25周龄糖尿病小鼠胰岛TNF-α的表达
24-25周龄db/db雄性小鼠11只以及db/m雄性小鼠5只，实验开始当天记为第0天并称重，db/db小鼠根据体重随机分为两组，给纤溶酶原组5只，给溶媒PBS对照组6只，db/m小鼠作为正常对照组。第1天开始给纤溶酶原或PBS，给纤溶酶原组尾静脉注射人源纤溶酶原2mg/0.2ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS或不注射任何液体，连续给药31天。在第32天处死小鼠，取胰脏在4％多聚甲醛中固定。固定后的胰脏组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡复水后水洗1次。PAP笔圈出组织，以3％双氧水孵育15分钟，0.01MPBS洗2次，每次5分钟。5％的正常羊血清液(Vector laboratories，Inc.，USA)封闭30分钟；时间到后，弃除羊血清液，滴加兔抗小鼠TNF-α抗体(Abcam)4℃孵育过夜，0.01M PBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，0.01M PBS洗2次，每次5分钟。按DAB试剂盒(Vector laboratories，Inc.，USA)显色，水洗3次后苏木素复染30秒，流水冲洗5分钟。梯度酒精脱水，二甲苯透明并中性树胶封片，切片在200倍光学显微镜下观察。
肿瘤坏死因子α(Tumor Necrosis Factor-α，TNF-α)主要由活化的单核/巨噬细胞产生，是一种重要的促炎症因子[48]。
本实验研究结果显示，给纤溶酶原组(图33C)TNF-α的阳性表达明显高于给溶媒PBS对照组(图33B)，且给纤溶酶原组比给溶媒PBS组更接近正常对照组(33A)。说明纤溶酶原能促进24-25周龄糖尿病小鼠TNF-α的表达。
实施例34纤溶酶原抑制26周龄糖尿病小鼠胰岛TNF-α的表达
26周龄db/db雄性小鼠9只以及db/m雄性小鼠3只，实验开始当天记为第0天并称重，db/db小鼠根据体重随机分为两组，给纤溶酶原组4只，给溶媒PBS对照组5只，db/m小鼠作为正常对照组。第1天开始给纤溶酶原或PBS。给纤溶酶原组尾静脉注射人源纤溶酶原2mg/0.2ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS或不注射任何液体，连续给药35天。在第36天处死小鼠，取胰脏在4％多聚甲醛中固定。固定后的胰脏组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡复水后水洗1次。PAP笔圈出组织，以3％双氧水孵育15分钟，0.01MPBS洗2次，每次5分钟。5％的正常羊血清液(Vector  laboratories，Inc.，USA)封闭30分钟；时间到后，弃除羊血清液，滴加兔抗小鼠TNF-α抗体(Abcam)4℃孵育过夜，0.01M PBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，0.01M PBS洗2次，每次5分钟。按DAB试剂盒(Vector laboratories，Inc.，USA)显色，水洗3次后苏木素复染30秒，流水冲洗5分钟。梯度酒精脱水，二甲苯透明并中性树胶封片，切片在200倍光学显微镜下观察。
研究结果显示，给纤溶酶原组(图34C)TNF-α的阳性表达明显高于给溶媒PBS对照组(图34B)，且给纤溶酶原组比给溶媒PBS组更接近正常对照组(34A)。说明纤溶酶原能26周龄糖尿病小鼠促进TNF-α的表达。
实施例35纤溶酶原促进PLG活性受损小鼠在T1DM模型中胰岛TNF-α的表达
9-10周龄PLG活性受损雄性性小鼠7只，随机分为两组，给PBS对照组3只，给纤溶酶原组4只。两组小鼠禁食4小时后单次腹腔注射200mg/kg STZ(sigma S0130)诱导I型糖尿病[43]。注射12天后开始给药并定为给药第1天，给纤溶酶原组尾静脉注射人源纤溶酶1mg/0.1ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，连续给药28天。在第29天处死小鼠，取胰脏在4％多聚甲醛中固定。固定后的胰脏组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡复水后水洗1次。PAP笔圈出组织，以3％双氧水孵育15分钟，0.01MPBS洗2次，每次5分钟。5％的正常羊血清液(Vector laboratories，Inc.，USA)封闭30分钟；时间到后，弃除羊血清液，滴加兔抗小鼠抗体TNF-α(Abcam)4℃孵育过夜，0.01M PBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，0.01M PBS洗2次，每次5分钟。按DAB试剂盒(Vector laboratories，Inc.，USA)显色，水洗3次后苏木素复染30秒，流水冲洗5分钟。梯度酒精脱水，二甲苯透明并中性树胶封片，切片在200倍光学显微镜下观察。
本实验研究结果显示，给纤溶酶原组(图35B)TNF-α的阳性表达明显高于给溶媒PBS对照组(图35A)。说明纤溶酶原能促进PLG活性受损小鼠T1DM模型TNF-α的表达。
实施例36纤溶酶原减轻PLG活性受损小鼠在T1DM模型中胰岛损伤
9-10周龄PLG活性受损雄性性小鼠10只，随机分为三组，空白对照组3只，给PBS对照组3只，给纤溶酶原组4只。给溶媒PBS对照组和给纤溶酶原组小鼠禁食4小时后单次腹腔注射200mg/kg STZ(sigma S0130)诱导I型糖尿病[43]，空白对照组不做处理。注射12天后开始给药并定为给药第1天，给纤溶酶原组尾静脉注射人源纤溶酶1mg/0.1ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS，连续给药28天。在第29天处死小鼠，取胰脏在4％多聚甲醛中固定。固定后的胰脏组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡复水后水洗1次。PAP笔圈出组织，以3％双氧水孵育15分钟，0.01MPBS洗2次，每次5分钟。5％的正常羊血清液(Vector laboratories，Inc.，USA)封闭30分钟；时间到后，弃除羊血清液，滴加山羊抗鼠IgM(HRP)抗体(Abcam)室温孵育1小时，0.01M PBS洗2次，每次5分钟。按DAB试剂盒(Vector laboratories，Inc.，USA)显色，水洗3次后苏木素复染30秒，流水冲洗5分钟。梯度酒精脱水，二甲苯透明并中性树胶封片，切片在200倍光学显微镜下观察。
IgM抗体在清除凋亡和坏死细胞过程中发挥着重要作用，组织器官损伤局部IgM抗体的水平与损伤程度呈正相关[49，50]。因此，检测组织器官局部IgM抗体的水平能够反映该组织器官的损伤情况。
研究结果显示，给纤溶酶原组(图36C)IgM的阳性表达明显低于给溶媒PBS对照组(图36B)给纤溶酶原组比给溶媒PBS组更接近空白对照组(36A)。说明纤溶酶原能降低IgM的表达，提示纤溶酶原能减轻PLG活性受损小鼠T1DM模型中的胰岛损伤。
实施例37纤溶酶原减少24-25周龄糖尿病小鼠胰岛细胞的凋亡
24-25周龄db/db雄性小鼠11只以及db/m雄性小鼠5只，实验开始当天记为第0天并称重，db/db小鼠根据体重随机分为两组，给纤溶酶原组5只，给溶媒PBS对照组6只，db/m小鼠作为正常对照组。第1天开始给纤溶酶原或PBS，给纤溶酶原组尾静脉注射人源纤溶酶原2mg/0.2ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS或不注射任何液体，连续给药31天。在第32天处死小鼠，取胰脏在4％多聚甲醛中固定。固定后的胰脏组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，切片脱蜡复水后水洗1次。PAP笔圈出组织，滴加蛋白酶K工作液 覆盖组织，室温孵育7min，0.01M PBS洗3次，每次3分钟。滴加TUNEL试剂盒(罗氏)试剂1和试剂2混合液体(5∶45)，于37℃恒温孵育40min，0.01M PBS洗3次，每次3分钟。滴加甲醇配制的3％双氧水溶液(过氧化氢∶甲醇＝1∶9)室温避光孵育20分钟，0.01M PBS洗3次，每次3分钟。滴加tunel试剂盒试剂3，37℃恒温孵育30min，0.01M PBS洗3次，DAB试剂盒(Vector laboratories，Inc.，USA)显色，水洗3次后苏木素复染30秒，流水冲洗5分钟。梯度酒精脱水，二甲苯透明并中性树胶封片，切片在200倍光学显微镜下观察。
TUNEL染色可以用来检测组织细胞在凋亡晚期过程中细胞核DNA的断裂情况。
本实验结果显示，给纤溶酶原组(图37C)的阳性细胞数(箭头标识)明显少于给溶媒PBS对照组(图37B)。正常对照组TUNEL阳性染色极低(图37A)。正常对照组凋亡率约为8％，给溶媒PBS组凋亡率约为93％，给纤溶酶原组凋亡率约为16％。说明纤溶酶原组能显著减少糖尿病小鼠胰岛细胞的凋亡。
实施例38纤溶酶原改善T1DM模型小鼠胰岛素分泌
9-10周龄C57雄性小鼠6只，随机分为两组，给溶媒PBS对照组和给纤溶酶原组各3只。两组小鼠禁食4小时后单次腹腔注射200mg/kg链脲佐菌素(STZ)(sigma S0130)诱导T1DM[43]。STZ注射12天后开始给药并定为给药第1天，给纤溶酶原组尾静脉注射人源纤溶酶1mg/0.1ml/只/天，给溶媒PBS对照组尾静脉注射同体积的PBS。连续给药20天，在第21天小鼠禁食6小时后，眼球静脉丛取血，离心取上清，运用胰岛素检测试剂盒(Mercodia AB)，按照使用说明检测血清胰岛素浓度。
结果显示，给溶媒PBS对照组小鼠胰岛素浓度明显低于给纤溶酶原组小鼠，且统计差异接近显著(P＝0.08)(图38)。说明纤溶酶原能促进T1DM小鼠胰岛素的分泌。
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Claims (48)

  1. 一种降低糖尿病受试者胰高血糖素分泌的方法，包括给药受试者有效量的纤溶酶原。
  2. 权利要求1的方法，其中所述纤溶酶原还降低糖尿病受试者胰高血糖素的表达。
  3. 权利要求1或2的方法，其中所述糖尿病为T1DM或T2DM。
  4. 权利要求1-3任一项的方法，其中所述纤溶酶原降低糖尿病受试者进食后的胰高血糖素分泌。
  5. 权利要求1-4任一项的方法，其中所述纤溶酶原降低糖尿病受试者禁食状态下的胰高血糖素分泌。
  6. 权利要求1-5任一项的方法，其中所述纤溶酶原在糖尿病受试者血糖升高状态下降低胰高血糖素的分泌，使血糖回复到正常或接近正常水平。
  7. 权利要求1-6任一项的方法，其中所述纤溶酶原在降低受试者胰高血糖素的表达和/或分泌的同时，促进所述胰岛素表达和/或分泌。
  8. 权利要求7的方法，其中所述纤溶酶原通过降低受试者胰高血糖素的表达和/或分泌的同时，促进所述胰岛素表达和/或分泌，实现使受试者血糖回复到正常或接近正常水平。
  9. 权利要求1-8任一项的方法，其中所述纤溶酶原促进胰岛素受体底物2(IRS-2)的表达。
  10. 一种促进糖尿病受试者胰岛素分泌的方法，包括给药受试者有效量的纤溶酶原。
  11. 权利要求10的方法，其中所述纤溶酶原还促进糖尿病受试者胰岛素的表达。
  12. 权利要求10或11的方法，其中所述糖尿病为T1DM或T2DM。
  13. 权利要求10-12任一项的方法，其中所述纤溶酶原促进糖尿病受试者进食后的胰岛素分泌。
  14. 权利要求10-12任一项的方法，其中所述纤溶酶原促进糖尿病受试者禁食状态下的胰岛素分泌。
  15. 权利要求10-14任一项的方法，其中所述纤溶酶原促进糖尿病受试者应答血糖升高刺激的胰岛素分泌，使血糖回复到正常或接近正常水平。
  16. 权利要求10-15任一项的方法，其中所述纤溶酶原在促进所述胰岛素表达和/或分泌的同时，降低受试者胰高血糖素的表达和/或分泌。
  17. 权利要求16的方法，其中所述纤溶酶原通过促进所述胰岛素表达和/或分泌的同时，降低受试者胰高血糖素的表达和/或分泌，实现使受试者血糖回复到正常或接近正常水平。
  18. 一种降低糖尿病受试者血糖的方法，包括给药受试者有效量的纤溶酶原。
  19. 权利要求18的方法，其中所述血糖选自如下的一项或多项：血清葡萄糖水平、血清果糖胺水平、血清糖化血红蛋白水平。
  20. 权利要求19的方法，其中所述血糖为血清葡萄糖水平。
  21. 权利要求18-20任一项的方法，其中所述糖尿病为T1DM或T2DM。
  22. 一种提高糖尿病受试者糖耐量的方法，包括给药受试者有效量的纤溶酶原。
  23. 权利要求22的方法，其中所述糖尿病为T2DM。
  24. 一种促进糖尿病受试者餐后血糖下降的方法，包括给药受试者有效量的纤溶酶原。
  25. 权利要求24的方法，其中所述纤溶酶原在受试者餐前30分钟至1.5小时给予。
  26. 权利要求25的方法，其中所述纤溶酶原在受试者餐前30分钟至1小时给予。
  27. 一种促进糖尿病受试者对葡萄糖的利用的方法，包括给药受试者有效量的纤溶酶原。
  28. 权利要求1-27任一项的方法，其中所述纤溶酶原可与一种或多种其它药物或治疗方法联用。
  29. 权利要求28的方法，其中所述纤溶酶原可与一种或多种选自如下的药物联用：抗糖尿病药物、抗心脑血管疾病药物、抗血栓药物、抗高血压药物，抗血脂药物、抗凝药物、抗感染药物。
  30. 权利要求1-29任一项的方法，其中所述纤溶酶原与序列2、6、8、10或12具有至少75％、80％、85％、90％、95％、96％、97％、98％或99％的序列同一性，并且仍然具有纤溶酶原活性。
  31. 权利要求1-30任一项的方法，所述纤溶酶原是在序列2、6、8、10或12的基础上，添加、删除和/或取代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个氨基酸，并且仍然具有纤溶酶原活性的蛋白质。
  32. 权利要求1-31任一项的方法，所述纤溶酶原是包含纤溶酶原活性片段、并且仍然具有纤溶酶原活性的蛋白质。
  33. 权利要求1-32任一项的方法，所述纤溶酶原选自Glu-纤溶酶原、Lys-纤溶酶原、小纤溶酶原、微纤溶酶原、delta-纤溶酶原或它们的保留纤溶酶原活性的变体。
  34. 权利要求1-33任一项的方法，所述纤溶酶原为天然或合成的人纤溶酶原、或其仍然保留纤溶酶原活性的变体或片段。
  35. 权利要求1-33任一项的方法，所述纤溶酶原为来自灵长类动物或啮齿类动物的人纤溶酶原直向同系物或其仍然保留纤溶酶原活性的变体或片段。
  36. 权利要求1-35任一项的方法，所述纤溶酶原的氨基酸如序列2、6、8、10或12所示。
  37. 权利要求1-36任一项的方法，其中所述纤溶酶原是人天然纤溶酶原。
  38. 权利要求1-37任一项的方法，其中所述受试者是人。
  39. 权利要求1-38任一项的方法，其中所述受试者缺乏或缺失纤溶酶原。
  40. 权利要求1-39任一项的方法，所述缺乏或缺失是先天的、继发的和/或局部的。
  41. 一种用于权利要求1-40任一项的方法的纤溶酶原。
  42. 一种药物组合物，其包含药学上可接受的载剂和用于权利要求1-40中任一项所述方法的纤溶酶原。
  43. 一种预防性或治疗性试剂盒，其包含：(i)用于权利要求1-40中任一项所述方法的纤溶酶原和(ii)用于递送所述纤溶酶原至所述受试者的构件(means)。
  44. 根据权利要求43所述的试剂盒，其中所述构件为注射器或小瓶。
  45. 权利要求43或44的试剂盒，其还包含标签或使用说明书，该标签或使用说明书指示将所述纤溶酶原投予所述受试者以实施权利要求1-40中任一项所述方法。
  46. 一种制品，其包含：
    含有标签的容器；和
    包含(i)用于权利要求1-40中任一项所述方法的纤溶酶原或包含纤溶酶原的药物组合物，其中所述标签指示将所述纤溶酶原或组合物投予所述受试者以实施权利要求1-40中任一项所述方法。
  47. 权利要求43-45中任一项的试剂盒或权利要求46的制品，还包含另外的一个或多个构件或容器，该构件或容器中含有其他药物。
  48. 权利要求47的试剂盒或制品，其中所述其他药物选自下组：抗糖尿病药物、抗心脑血管疾病药物、抗血栓药物、抗高血压药物，抗血脂药物、抗凝药物、抗感染药物。
PCT/CN2017/089066 2016-12-15 2017-06-19 一种使胰高血糖素、胰岛素恢复正常平衡的方法 WO2018107706A1 (zh)

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