CN108210906A

CN108210906A - Treat drug of coronary atherosclerosis and its complication and application thereof

Info

Publication number: CN108210906A
Application number: CN201710466253.1A
Authority: CN
Inventors: 李季男
Original assignee: Shenzhen Life Science Research Institute Co Ltd
Current assignee: Shenzhen Life Science Research Institute Co Ltd
Priority date: 2016-12-15
Filing date: 2017-06-19
Publication date: 2018-06-29
Also published as: CN108210902A; HK1257239A1; CN108210900A; HK1257583A1; HK1257233A1; HK1257236A1; CN108210898A; CN108210904A; HK1257240A1; HK1257234A1; HK1257237A1; CN108210896A; CN108210895A; CN108210914A

Abstract

The present invention relates to a kind of methods for preventing and/or treating subject's coronary atherosclerosis and its associated disease, plasminogen including administration subject prevention and/or therapeutically effective amount, wherein the subject suffers from, suspects with coronary atherosclerosis and its associated disease or with the risk for suffering from coronary atherosclerosis and its associated disease.The invention further relates to for preventing and/or treat the drug comprising plasminogen, pharmaceutical composition, product, the kit of subject's coronary atherosclerosis and its associated disease.

Description

Treat drug of coronary atherosclerosis and its complication and application thereof

Technical field

The present invention relates to effect of the plasminogen in terms of preventing and/or treating atherosclerosis and its associated disease, And then provide completely new therapeutic strategy for prevention and/or treatment atherosclerosis and its associated disease.

Background technology

The main reason for atherosclerosis (atherosclerosis, AS) is coronary heart disease, cerebral infarction, peripheral vascular disease. Lesion basis of the lipidosis for atherosclerosis, its main feature is that involvement arterial disease generally first has since inner membrance Lipid and the accumulation of compound carbohydrate, bleeding and thrombosis, and then proliferation of fibrous tissue and calcinosis, and have arterial media by Gradually change in quality and calcification, artery wall thickening is caused to be hardened, lumen of vessels it is narrow.Lesion often involves big-and-middle muscular artery, once it develops to It is enough to block lumen of artery, then the tissue or organ that the artery is supplied are by ischemic or necrosis.

Atherosclerosis is most normal in the common pathologic basis of numerous cardiovascular and cerebrovascular diseases and disease of cardiovascular system The disease seen seriously endangers human health.The occurrence and development of atherosclerosis include lipid invasion, platelet activation, thrombus Formation, inner film injury, inflammatory reaction, oxidative stress, vascular smooth muscle cells (VSMC) activation, Basic selective material metabolism and blood vessel Rebuild etc.^[1].Though experienced the research in a nearly century, many scholars also proposed the different theories about AS pathogenesis, such as Lipid penetrate into theory, macrophage receptor missing theory, cause the smooth muscle theory of mutation, damage response theory, inflammatory reaction theory, Hemodynamic theory and immunological theory etc., but any theory individually cannot comprehensively explain the occurrence and development of AS.In recent years Come, the experimental study data of a large amount of cells and molecular level extend people's Human Umbilical Vein Endothelial Cells, VSMC, mononuclear macrophage and The understanding of the normal homeostasis of blood platelet is further recognized so as to have to the effect in terms of its formation and pathogenesis in AS Know.

1. the effect of vascular endothelial cell damage

The study found that the damage of some time function of vascular endothelium is just formed already before atherosclerotic plaque occurs. Esper etc.^[2]Report, endothelial cell can generate a large amount of molecules with two-way function, can reach promotion and depression effect Balance.When endothelial cell loses the ability for maintaining this fine balance, lipid and leucocyte (mainly monocyte and T leaching Bar cell) endothelium will be invaded, so as to cause the generation of inflammatory reaction and the formation of fat line.The dysfunction of endothelial cell, Activation and morphology damage can cause monocyte in blood, the variation of film VSMC and most end form in blood platelet and vascular wall Into AS.Its specific mechanism is as follows：(1) the permeability increase of endothelial cell is the main start-up procedure of AS, is that lipid entrance is dynamic The subendothelial earliest pathological change of astillen^[3]；(2) make blood platelet and monocyte sticks increase.Ott etc.^[4]Report, a work( Can the endothelial cell of imbalance may promote sticking for monocyte due to the Expression of Cell Adhesion Molecules increase on its surface, so as to Promote to penetrate into AS patches from circulating containing germy monocyte；(3) a variety of growth factors are secreted, as monocyte becomes Change protein 21 (MPC21), fibroblast growth factor, transforming growth factor (TGF) and platelet derived growth factor (PDGF) etc., so as to which monocyte be attracted to assemble and attach to endothelium, and subendothelial gap is moved into, the street cleaner through its surface Receptor, CD36 receptors and Fc are receptor-mediated, and huge uptake enters under inner membrance the lipid aoxidized, form the bubble of monocyte source property Foam cell.Boos etc.^[3]Research points out, the morbidity indication and its serious journey that endothelial injuries degree to a certain extent can be as AS The novel index of degree.

2. hematoblastic effect

After arterial endothelial cell damage, it can promote on platelet adhesion reaction to impaired endothelial cell, so as to promote PDGF's Release, leads to the continuous hyperplasia of myointimal cell, eventually leads to collage synthesis, forms AS patches.In the final rank of AS thrombosis Section, it is hematoblastic stick, activate and assemble can lead to arterial occlusion and secondary ischemic^[5].Blood platelet and endothelial cell, connective Tissue interaction, it is significant to the generation of local tube wall AS.Effect of the blood platelet in AS is mainly manifested in：(1) appoint The endothelial injuries of what form can be such that blood platelet largely sticks and be gathered in endothelium part, activate blood coagulation system, cause thrombus shape Into.(2) it secretes and discharges various active substance, such as PDGF, platelet factor 4, beta-thromboglobulin are to VSMC and monokaryon Cell is respectively provided with strong chemotactic effect, participates in VSMC and swims out of, is proliferated and modifies aortic tunica intima, monocyte is attracted to stick Attached endothelium.There is scholar to point out that PDGF has chemotactic into fibration and promotes the respective antigenic determinant of mononuclear cell proliferation, It causes to play an important role during AS.(3) venous endothelial cell can generate nitric oxide and prostacyclin, and constantly be released in intrapulmonary It puts, adjusts hematoblastic function.

3. lipid is in atherosclerosis generation, developing effect

Numerous studies prove, the pathological change and blood lipid level of AS, and particularly plasma cholesterol and serum triglyceride level is close Cut phase is closed^[6].There is scholar's proposition, the deposition of lipid and aliphatic acid is important in endothelial dysfunction and AS forming processes Pathomechanism.The study found that compared with natural arterial, have the artery ApoC1 of AS patches and the albumen of apo E and Gene expression is significantly raised, this may be the reason of AS is formed and is not merely a kind of result^[7].It has been gained public acceptance that, Mechanism of action of the hyperlipidemia in AS morbidities mainly makes the permeability of endothelial cell in addition to directly endothelial cell damage is caused Increase, it is related that the oxidative modification of this and low-density lipoprotein (LDL) becomes oxidative low density lipoprotein (ox-LDL).Work as ox- When LDL passes through undamaged endothelium, blood plasma LDL is transported to subcutaneous clearance and carries out oxidative modification.LDL causes macrophage The hyperplasia of cleaning reaction and middle film VSMC form atheromatous plaque.Above variation can eventually lead to endarterium fat line, fibrous plaque And/or the formation of atheromatous plaque.

4. the effect of mononuclear macrophage

It is loaded research shows that containing in AS patches and including monocyte, the macrophage of cells of monocytic origin, ox-LDL The inflammatory reactions cellular infiltration such as macrophage (i.e. foam cells) and T lymphocytes^[8].Effect of the monocytes/macrophages in AS It may be summarized to be：(1) phagocytosis:The foam cells of lesion early stage enters endothelium mostly from the monocyte in blood, the latter Under be changed into macrophage, the specific receptor on surface can be combined with ox-LDL, so as to take in a large amount of cholesterol, become foam Cell.(2) inflammatory reaction and immune response are participated in：Above-mentioned phagocytosis can by extracellular matrix discharge inflammatory reaction because Son and induce distinctive inflammatory reaction.In the infiltration of the intralesional visible T lymphocytes of AS, while compared with non-ruptured patch, Contain more macrophages in the fibrous cap of the AS patches of rupture.(3) breeder reaction is participated in：Macrophage can be released after being activated Cytokine profiles and growth factor are put, the migration of film VSMC and hyperplasia in promotion.Meanwhile Expression of Macrophages various metals Protease and serine stretch protein enzyme make extracellular substrate degeneration, and patch is unstable or even has the tendency that rupture^[9]。

The effect of 5.VSMC

By research in recent years, it was recognized that middle film VSMC proliferation, migration enter the synthesis of inner membrance and stromatin, It is to participate in the key link that AS progressive stages lesion is formed, and play an important role in AS and the intimal thickening of restenosis^[10].AS spots The morbidity of block and restenosis and progress include the complicated interaction between cells of vascular wall, cell factor, inflammatory reaction, chemotactic The factor, growth factor have therefrom played important function.Ldl receptor mediate phagocytosis lipids of the VSMC of migration through its surface is formed VSMC sources foam cell, participates in the formation of lesion.Meanwhile the inner membrance VSMC of these hyperplasia can also synthesize collagen, elasticity Albumen and glycoprotein etc. and the free lipid of macrophage phagocytosis LDL releases, make the intimal thickening of lesion be hardened, promote hardening The formation of patch.In this regard, people make many effort to inhibit the accumulation of above-mentioned cell, and taken in terms of stent postoperative restenosis Obtained very ten-strike^[11]。

Diabetes are close with relationship between atherosclerosis, show as diabetic and the time of atherosclerosis occur Early, degree weight and poor prognosis, and atherosclerosis is the major causes of death of diabetic.

The blood vessel that the characteristics of coronary artery pathological change of clinical discovery diabetic, mainly lesion was involved compared with It is more, coronary artery stenosis is serious, lesion more diffuses seriously, mechanism more be considered that metabolism of blood glucose causes Atherosclerosis extremely Change, with further in-depth study, more the result shows that, diabetes cause atherosclerosis to be not single factors institute It causes, but through a variety of ways and more complicated mechanism is come the generation and development that induce and promote atherosclerosis, example Such as macrophage polarization, the up-regulation of macrophage migration inhibition factor approach, glycosylation end products approach, scavenger receptor, pancreas islet Element is resisted, Ubiquitin-proteasome system (ubiquitin proteasome system, UPS) activates, platelet derived growth The factor (platelet-derived growth factor, PDGF) activated pathway etc.^[12]。

In the tissues such as diabetes B patient's body white adipose, polarize there are macrophage unbalance, it is huge to show as M1 types Phagocyte increases.M1 type macrophage Major Secretories TNF-α, IL-6, MCP 1 etc. play pro-inflammatory effect, The generation that not only induced insulin is resisted of above-mentioned cell factor but also promotion atherosclerosis^[13]。

Macrophage migration inhibition factor (macrophagemigration inhibitory factor, MIF) is one The important immune factor with inflammatory reaction of participation of kind.In diabetic, the expression of MIF increases, and may be closed with diabetes And atherosclerosis is related^[14].It causes atherosclerosis mechanism as follows：(1) MIF makes macrophage be soaked in inflammation part Profit, activation, accelerate the phagocytosis of lipid, the generation of reducing foam cell.Studies have found that macrophage absorbs oxidized low density fat Albumen raises MIF, and corresponding MIF can increase the intake of OxLDL ELISA, promotes foam wanshing.(2)MIF It can be made to express MCP 1 and cell-cell adhesion point respectively with activating vascular endothelial cell and smooth muscle cell Son 1 increases mononuclear macrophage migration, so as to accelerate atherosclerosis.Resisted in various artery models using MIF Body can weaken the marker of subendothelial macrophage, foam cells, macrophage activation.

In diabetic, glycosylation end products (advanced glycation end products, AGEs) with can To promote the generation of atherosclerotic lesion and progress.Glucose and the non-enzymatic of protein and lipoprotein sugar in AGEs arterial walls Glycosylation reaction product accelerates atherosclerosis after being combined with corresponding receptor by following mechanism：(1) long term hyperglycemia can make AGEs generates increase, and AGEs can modify protein, nucleic acid and lipid, and reactive oxygen species is made to generate increase, enhances oxidative stress, AGEs can increase neutrophil NADPH oxidase work while the generation by increasing polymorphonuclear Property, promote blood vessel oxidative stress, increase the incidence of diabetic's angiocardiopathy^[15].(2) AGEs increases adhesion molecule table It reaches, medullary system can increase the expression of vascular adhesion molecules 1 with AGEs receptors on non-myeloid cell surface, accelerate diabetes related Atherosclerosis^[16]。

Insulin resistance (insulin resistance, IR) is the target tissue exogenous or endogenous of insulin action Property insulin sensibility and reactivity decline.Diabetes B is often associated with insulin resistance.Insulin resistance passes through as follows Mechanism can accelerate diabetic artherosclerosis：(1) insulin resistance accelerates macrophage apoptosis：The study found that diabetes The atherosclerotic plaque core of patient progress's phase is significantly increased compared with non-diabetic patients^[17].Diabetes development phase artery congee Sample hardens lesion, and due to macrophage insulin resistance, the Apoptosis that er stress is induced significantly increases, and promotes spot Block core increases.(2) finding that the activation of CX3CL1/CX3CL1 axis is apparent in insulin resistance and metabolic syndrome patient increases, It is proportionate with atherosclerosis acceleration.The activation of the axis is found in animal model increases the unstability of patch.Insulin It resists and accelerates atherosclerosis by activating CX3CL1/CX3CL1 axis induction of vascular Vascular Smooth Muscle Cell Apoptosis^[18].Macrophage Vit D3 receptor, which knocks out, promotes insulin resistance, accelerates atherosclerosis^[19]。

The Endogenous oxidative of diabetic stress enhance so that the UPS of macrophage is by excessive activation^[20].The mistake of UPS Degree activation promotes the expression of the diabetic inflammation factor (such as the vascular cell adhesion factor 1 and Intercellular surface adhesion molecule-1) and divides It secretes, causes vascular endothelial cell irreversible damage, lead to atherosclerosis^[21]。

In addition, the AGEs of diabetic, angiotensinⅡ, Endothelin, inflammation and hyperlipidemia state increase PDGF ways The activity of diameter, and the increase of PDGF pathway activities has the function of to promote inflammatory reaction.PDGF has up-regulation connective tissue growth The effect of factor expression promotes endothelial cell and fibroblastic migration, sticks and be proliferated, aggravates the hair of atherosclerosis It is raw^[22]。

So far, mainly there are blood lipid-lowering medicine, antiplatelet drug, blood pressure lowering to the drug therapy of atherosclerosis Drug, vasodilator drug, hypoglycemic drug, anticoagulation medicine, thrombolytic agent.We are surprised to find fibrinolytic under study for action Proenzyme can cut down the lipid accumulation of ductus arteriosus wall, deposition, reduce proliferation of fibrous tissue, repair atherosclerosis to vascular wall Damage, improve atherosclerosis caused by histoorgan ischemic injuries and the related diseases caused by histoorgan ischemic Disease.

Summary of the invention

The present invention relates to prevent and/or treat subject's coronary atherosclerosis and its associated disease.

On the one hand, the present invention relates to for preventing and/or treat subject's coronary atherosclerosis and its associated disease Method, including administration subject prevention and/or therapeutically effective amount plasminogen, wherein the subject suffers from, suspects trouble There are coronary atherosclerosis and its associated disease or with the risk for suffering from coronary atherosclerosis and its associated disease.This Invention further relates to plasminogen for preventing and/or treating the purposes of subject's coronary atherosclerosis and its associated disease. The invention further relates to plasminogens to prepare for preventing and/or treat subject's coronary atherosclerosis and its associated disease Drug, pharmaceutical composition, product, the purposes in kit.Further, the invention further relates to for preventing and/or treat Subject's coronary atherosclerosis and its plasminogen of associated disease.The invention further relates to for prevent and/or treat by Examination person's coronary atherosclerosis and its drug comprising plasminogen, pharmaceutical composition, product, the kit of associated disease.

In some embodiments, the coronary atherosclerosis associated disease is included due to coronary atherosclerosis Coronary heart diseases and angina pectoris, myocardial infarction, arrhythmia cordis, the heart failure of initiation.In some embodiments, the atherosclerosis For with diabetes complicated atherosclerosis.In some embodiments, the plasminogen passes through one chosen from the followings Or multinomial prevention and/or treatment atherosclerosis；It reduces experimenter's serum total cholesterol level, reduce experimenter's serum glycerine Three esters are horizontal, reduce experimenter's serum low-density lipoprotein white level, raising experimenter's serum hdl level.At some In embodiment, the plasminogen prevents and/or treats coronary artery by reducing the lipidosis of subject artery's tube wall Atherosis.In some embodiments, the plasminogen passes through one or more preventions chosen from the followings and/or treatment Coronary atherosclerosis：The fat of subject's liver is transported and is reduced in the fat metabolism for promoting liver, the fat for promoting liver Deposition.

On the other hand, the present invention relates to for preventing and/or treat subject caused by coronary atherosclerosis Tissue, organ ischemia's damage and its associated disease method, include to subject using a effective amount of plasminogen.This hair It is bright to further relate to plasminogen for preventing and/or treating tissue, organ of the subject caused by coronary atherosclerosis to lack Blood damages and its purposes of associated disease.The invention further relates to plasminogen prepare for prevent and/or treat subject by In caused by coronary atherosclerosis tissue, organ ischemia's damage and its drug of associated disease, pharmaceutical composition, product, Purposes in kit.Further, the invention further relates to for preventing and/or treat subject since coronary artery is athero- hard The plasminogen of tissue, organ ischemia's damage and its associated disease caused by changing.The invention further relates to for preventing and/or treat Tissue of the subject caused by coronary atherosclerosis, organ ischemia's damage and its associated disease comprising plasminogen Drug, pharmaceutical composition, product, kit.

In some embodiments, the tissue of the subject, organ ischemia's damage cause for coronary atherosclerosis Myocardial damage.In some embodiments, the associated disease is coronary heart diseases and angina pectoris, the heart caused by cardiac insufficiency Flesh infarct, arrhythmia cordis or heart failure.In some embodiments, the tissue, organ ischemia's damage or associated disease are brain Ischemic injuries or its associated disease.In some embodiments, the illness for cerebral ischemia, cerebral thrombus, encephalatrophy, cerebral hemorrhage or Cerebral embolism.In some embodiments, the associated disease is renal insufficiency, hypertension, glomerulus fibrosis, renal failure or urine Toxication.

It yet still another aspect, the present invention relates to for preventing and/or treat subject caused by coronary atherosclerosis Coronary artery thrombosis and its associated disease method, including to subject apply a effective amount of plasminogen.The present invention is also Be related to plasminogen for prevent and/or treat coronary artery thrombosis of the subject caused by coronary atherosclerosis and its The purposes of associated disease.The invention further relates to plasminogens to prepare for preventing and/or treat subject due to coronary artery Purposes in the drug of coronary artery thrombosis and its associated disease caused by atherosis, pharmaceutical composition, product, kit. Further, the invention further relates to coronal dynamic caused by coronary atherosclerosis for preventing and/or treating subject The plasminogen of arteries and veins thrombus and its associated disease.The invention further relates to for preventing and/or treat subject due to coronary artery The drug comprising plasminogen of coronary artery thrombosis and its associated disease caused by atherosis, pharmaceutical composition, product, examination Agent box.

In some embodiments, the illness includes coronary heart diseases and angina pectoris, myocardial infarction, arrhythmia cordis, heart failure, brain Ischemic, cerebral thrombus, encephalatrophy, cerebral hemorrhage, cerebral embolism, cerebral infarction, renal insufficiency, hypertension, glomerulus fibrosis, renal failure, Uremia, bowel necrosis, intermittent claudication, gangrene.

It yet still another aspect, the present invention relates to the coronary atherosclerosiies prevented and/or treatment sub-ject is concurrent Method, including to subject apply a effective amount of plasminogen.The invention further relates to plasminogens for preventing and/or controlling Treat the purposes of the concurrent coronary atherosclerosis of sub-ject.The invention further relates to plasminogens to prepare for preventing And/or in the drug of the concurrent coronary atherosclerosis for the treatment of sub-ject, pharmaceutical composition, product, kit Purposes.Further, the invention further relates to for preventing and/or treat the concurrent coronary atherosclerosis of sub-ject Plasminogen.The invention further relates to for preventing and/or treat the concurrent coronary atherosclerosis of sub-ject Drug, pharmaceutical composition, product, kit comprising plasminogen.

It yet still another aspect, the present invention relates to the coronary heart diseases and angina pectoris, the cardiac muscle stalks that prevent and/or treat diabetic subjects Extremely, arrhythmia cordis or the method for heart failure, including applying a effective amount of plasminogen to subject.The invention further relates to fibrinolysins It is originally intended to prevent and/or treat the purposes of the coronary heart diseases and angina pectoris of diabetic subjects, myocardial infarction, arrhythmia cordis or heart failure. The invention further relates to plasminogens to prepare coronary heart diseases and angina pectoris, cardiac muscle stalk for preventing and/or treating diabetic subjects Extremely, the purposes in the drug of arrhythmia cordis or heart failure, pharmaceutical composition, product, kit.Further, the invention further relates to For preventing and/or treating the coronary heart diseases and angina pectoris of diabetic subjects, myocardial infarction, arrhythmia cordis or the fibrinolysin of heart failure It is former.The invention further relates to for preventing and/or treat the coronary heart diseases and angina pectoris of diabetic subjects, myocardial infarction, arrhythmia cordis Or the drug comprising plasminogen, pharmaceutical composition, product, the kit of heart failure.

It yet still another aspect, the present invention relates to the sides for preventing and/or treating subject artery's atherosis and its associated disease Method, including applying a effective amount of plasminogen to subject.The invention further relates to plasminogen for prevent and/or treat by The purposes of examination person's atherosclerosis and its associated disease.The invention further relates to plasminogens to prepare for preventing and/or control Purposes in the drug for the treatment of subject artery's atherosis and its associated disease, pharmaceutical composition, product, kit.Further Ground, the invention further relates to for preventing and/or treat the plasminogen of subject artery's atherosis and its associated disease.This hair It is bright further relate to for prevent and/or treat subject artery's atherosis and its associated disease the drug comprising plasminogen, Pharmaceutical composition, product, kit.

In some embodiments, the atherosclerosis include atherosclerosis of aorta, coronary atherosclerosis, Cerebral atherosclerosis, renal artery atherosclerosis, superior mesenteric atherosclerosis, lower limb atherosclerosis.In some implementations In scheme, the atherosclerosis associated disease include due to atherosclerosis cause tissue, organ ischemia and cause Associated disease, the coronary heart diseases and angina pectoris caused including coronary atherosclerosis, myocardial infarction, arrhythmia cordis, heart failure；Brain moves Cerebral ischemia caused by pulse atherosclerosis, cerebral thrombus, encephalatrophy, cerebral hemorrhage, cerebral embolism；The renal function that renal artery atherosclerosis causes Incomplete, hypertension, glomerulus fibrosis, renal failure, uremia；Abdominal pain, digestion after the heavy meal that superior mesenteric atherosclerosis causes Bad, constipation, is had blood in stool at intestinal wall necrosis；The intermittent claudication of lower limb atherosclerosis initiation, gangrene.

In some embodiments, the atherosclerosis is selected from：Coronary atherosclerosis, cerebral atherosclerosis And renal artery atherosclerosis.In some embodiments, the Atherosclerosis turns to and diabetes complicated Atherosclerosis Change.In some embodiments, the plasminogen is athero- by one or more preventions chosen from the followings and/or treatment artery Hardening；It is low close to reduce experimenter's serum total cholesterol level, reduction experimenter's serum triglyceride levels, reduction experimenter's serum Spend lipoprotein levels, raising experimenter's serum hdl level.In some embodiments, the plasminogen passes through Reduce the lipidosis prevention of subject artery's tube wall and/or treatment atherosclerosis.In some embodiments, the fibre Lyase original passes through one or more preventions chosen from the followings and/or treatment atherosclerosis：Promote the fat metabolism of liver, promote Into the fat transport of liver and the fat deposition of reduction subject's liver.

It yet still another aspect, the present invention relates to prevent and/or treat tissue, device of the subject caused by atherosclerosis The method of official's ischemic injuries and its associated disease, including applying a effective amount of plasminogen to subject.The invention further relates to Plasminogen is used to preventing and/or treating tissue, organ ischemia damage and its phase of the subject caused by atherosclerosis The purposes of related disorders.The invention further relates to plasminogens to prepare for preventing and/or treat subject due to Atherosclerosis Change the purposes in caused tissue, the drug of organ ischemia's damage and its associated disease, pharmaceutical composition, product, kit.Into One step, the invention further relates to for preventing and/or treat tissue of the subject caused by atherosclerosis, organ ischemia Damage and its plasminogen of associated disease.The invention further relates to for preventing and/or treat subject due to Atherosclerosis Tissue, the drug comprising plasminogen of organ ischemia's damage and its associated disease caused by changing, pharmaceutical composition, product, examination Agent box.

In some embodiments, the tissue of the subject, organ ischemia's damage are myocardial damage, cerebral injury or kidney damage Wound.In some embodiments, the illness is coronary heart diseases and angina pectoris, myocardial infarction, arrhythmia cordis, heart failure, cerebral ischemia, brain Thrombus, encephalatrophy, cerebral hemorrhage or cerebral embolism, renal insufficiency, hypertension, glomerulus fibrosis, renal failure or uremia.

It yet still another aspect, the present invention relates to prevent and/or treat arterial blood of the subject caused by atherosclerosis The method of bolt and its associated disease, including applying a effective amount of plasminogen to subject.The invention further relates to plasminogens For preventing and/or treating the purposes of arterial thrombus and its associated disease of the subject caused by atherosclerosis.This hair It is bright further relate to plasminogen prepare for prevent and/or treat arterial thrombus of the subject caused by atherosclerosis and Purposes in the drug of its associated disease, pharmaceutical composition, product, kit.Further, the invention further relates to for preventing And/or the plasminogen of arterial thrombus and its associated disease of the treatment subject caused by atherosclerosis.The present invention is also It is related to fine for preventing and/or treating including for arterial thrombus and its associated disease of the subject caused by atherosclerosis Drug, pharmaceutical composition, product, the kit of lyase original.

It yet still another aspect, the present invention relates to the sides of atherosclerosis prevented and/or treatment sub-ject is concurrent Method, including applying a effective amount of plasminogen to subject.The invention further relates to plasminogen for prevent and/or treat by The purposes of the diabetes complicated atherosclerosis of examination person.The invention further relates to plasminogens to prepare for preventing and/or control Treat the purposes in the drug of the concurrent atherosclerosis of sub-ject, pharmaceutical composition, product, kit.Further Ground, the invention further relates to for preventing and/or treat the plasminogen of the concurrent atherosclerosis of sub-ject.This hair It is bright further relate to for prevent and/or treat the concurrent atherosclerosis of sub-ject the drug comprising plasminogen, Pharmaceutical composition, product, kit.

In some embodiments, the atherosclerosis is chosen from the followings one or more：Atherosclerosis of aorta, Coronary atherosclerosis, cerebral atherosclerosis, renal artery atherosclerosis, superior mesenteric atherosclerosis, artery of lower extremity congee Sample hardens.

It yet still another aspect, the present invention relates to treatment diabetes or the method for atherosclerosis subject's hyperlipidemia, Including applying a effective amount of plasminogen to subject.It is used to treat diabetes the invention further relates to plasminogen or artery is athero- Harden the purposes of subject's hyperlipidemia.The invention further relates to plasminogens to prepare for treating diabetes or Atherosclerosis Change the purposes in the drug of subject's hyperlipidemia, pharmaceutical composition, product, kit.Further, the invention further relates to In the plasminogen for the treatment of diabetes or atherosclerosis subject's hyperlipidemia.The invention further relates to for treating diabetes Or the drug comprising plasminogen, pharmaceutical composition, product, the kit of atherosclerosis subject's hyperlipidemia.

In some embodiments, the subject has as follows one or more：Serum total cholesterol level raising, Serum triglyceride level raising, the raising of serum low-density LP level, high density lipoprotein level of serum reduce.At some In embodiment, the hyperlipidemia one or more is improved by chosen from the followings；The total courage of experimenter's serum is reduced to consolidate Alcohol level reduces experimenter's serum triglyceride levels, reduces experimenter's serum low-density lipoprotein white level, raising subject's blood Aloof from politics and material pursuits density lipoprotein levels.

It yet still another aspect, the present invention relates to for preventing or cutting down the method for subject artery's tube wall lipidosis, wherein The subject is susceptible to suffer from atherosclerosis or has suffered from atherosclerosis, a effective amount of the method includes being applied to subject Plasminogen.It is used to prevent or cut down the purposes of subject artery's tube wall lipidosis the invention further relates to plasminogen.This hair It is bright further relate to plasminogen prepare for prevent or cut down the drug of subject artery's tube wall lipidosis, pharmaceutical composition, Purposes in product, kit.Further, the invention further relates to for preventing or cut down subject artery's tube wall lipidosis Plasminogen.The invention further relates to for prevent or cut down the drug of subject artery's tube wall lipidosis, pharmaceutical composition, Product, kit.

In some embodiments, the subject for being susceptible to suffer from atherosclerosis is with primary or secondary fat The subject of metabolic disorder.In some embodiments, the subject for being susceptible to suffer from atherosclerosis be with liver diseases, Kidney trouble, obesity, hyperlipidemia or diabetes subject.In some embodiments, the plasminogen can with by The one or more other medicines or therapy that examination person needs are administered in combination.In some embodiments, the other medicines include： Blood lipid-lowering medicine, antiplatelet drug, blood-pressure drug, vasodilator drug, hypoglycemic drug, anticoagulation medicine, thrombus dissolving Drug, hepatic, antiarrhythmic drug, cardiac drug, diuretic, anti-infectives, antiviral drugs, immunological regulation Drug, inflammation adjust class drug, antitumor drug, hormone medicine, thyroxine.In some embodiments, the drug Including blood lipid-lowering medicine：Statins；Fibrates；Niacin；Cholestyramine；Clofibrate；Unrighted acid such as yishouning, lipid level and Heart arteries and veins is found pleasure in；Sodium alginate；Antiplatelet drug：Aspirin；Persantine；Clopidogrel；Xi Luota；Vasodilator drug： Hydralazine；Nitroglycerin and isosorbide dinitrate；Sodium nitroprussiate；α nitre receptor blocking pharmacon such as prazosin；Alpha receptor blocking agent such as phentolamine； β draws receptor agonist such as albuterol；Captopril, enalapril；Nifepine, thiazolone；Willow Gamma Amino Butyric Acid, minoxidil, forefront Parathyrine, atrial natriuretic peptide；Thrombolytic agent：Urokinase and streptokinase；Tissue-type plasminogen activator；Single chain urokinase type plasminogen activator type fibrinolysin Activator；TNK- tissue-type plasminogen activators；Anticoagulation medicine：Heparin；Enoxaparin；Nagqu heparin；Bivalirudin.

In some embodiments, the plasminogen and sequence 2,6,8,10 or 12 have at least 75%, 80%, 85%th, 90%, 95%, 96%, 97%, 98% or 99% sequence identity, and still there is activities of endothelial tissue plasminogen.One In a little embodiments, plasminogen is on the basis of sequence 2,6,8,10 or 12, and addition deletes and/or replaces 1-100,1- 90、1-80、1-70、1-60、1-50、1-45、1-40、1-35、1-30、1-25、1-20、 1-15、1-10、1-5、1-4、1-3、 1-2,1 amino acid, and still there is the protein of activities of endothelial tissue plasminogen.

In some embodiments, the plasminogen is comprising activities of endothelial tissue plasminogen segment and still with fibrinolytic The protein of zymogen activity.In some embodiments, the plasminogen be selected from Glu- plasminogens, Lys- plasminogens, Miniplasminogen, Microplasminogen, delta- plasminogens or their variant for retaining activities of endothelial tissue plasminogen.In some realities Apply in scheme, the plasminogen for natural or synthetic human plasminogen or its still retain the variant of activities of endothelial tissue plasminogen Or segment.In some embodiments, the plasminogen is the human plasminogen from primate or rodent Directly to homologue or its still retain the variant of activities of endothelial tissue plasminogen or segment.In some embodiments, the plasminogen Amino acid as shown in sequence 2,6,8,10 or 12.In some embodiments, the plasminogen is naive plasminogen.

In some embodiments, the subject is people.In some embodiments, the subject lacks or lacks Plasminogen.In some embodiments, the shortage or missing are inborn, secondary and/or local.

In some embodiments, described pharmaceutical composition includes pharmaceutically acceptable supporting agent and for preceding method Plasminogen.In some embodiments, the kit can be preventative or therapeutic agent box, it includes：(i) it is used for The plasminogen of preceding method and (ii) are for delivering the plasminogen to the component (means) of the subject.At some In embodiment, the component is syringe or bottle.In some embodiments, the kit also includes label or use The plasminogen is administered the subject to implement aforementioned either method by specification, the label or operation instructions instruction.

In some embodiments, the product includes：Container containing label；With comprising (i) for preceding method Plasminogen or the pharmaceutical composition comprising plasminogen, wherein label instruction administers the plasminogen or composition The subject is to implement aforementioned either method.

In some embodiments, the kit or product, should also comprising other one or more components or container Contain other drugs in component or container.In some embodiments, the other drugs are selected from the group：Blood lipid-lowering medicine resists Antiplatelet drug, blood-pressure drug, vasodilator drug, hypoglycemic drug, anticoagulation medicine, thrombolytic agent, hepatic, Antiarrhythmic drug, cardiac drug, diuretic, anti-infectives, antiviral drugs, immunoregulation medicament, inflammation are adjusted Class drug, antitumor drug, hormone medicine, thyroxine.

In some embodiments of preceding method, the plasminogen can preferably be passed through by administered either systemically or locally Following approach application：Intravenously, intramuscular, subcutaneous administration fibrinolysin was treated originally.In some embodiments of preceding method In, the plasminogen is applied with appropriate peptide carrier or combination of stabilizers.In some embodiments of preceding method, institute State plasminogen with daily 0.0001-2000mg/kg, 0.001-800mg/kg, 0.01-600mg/kg, 0.1-400mg/kg, 1-200mg/kg, 1-100mg/kg, 10-100mg/kg (being calculated with per kilogram of body weight) or 0.0001-2000mg/cm2,0.001- 800mg/cm2、0.01-600mg/cm2、 0.1-400mg/cm2、1-200mg/cm2、1-100mg/cm2、10-100mg/cm2 The dosage of (being calculated with body surface area every square centimeter) is applied, and is preferably at least repeated once, preferably at least application daily.

The present invention clearly covers all combinations for belonging to the technical characteristic between embodiment of the present invention, and these groups Technical solution after conjunction clearly discloses in this application, just as above-mentioned technical proposal is individually and clearly disclosing. In addition, the present invention also clearly covers the combination between each embodiment and its element, the technical solution after the combination is at this It is clearly disclosed in text.

Detailed description of the invention

" atherosclerosis " is a kind of chronic, gradual arterial disease, the fats portion deposited in artery during morbidity Or all block blood flow.When otherwise smooth, solid endarterium is roughening, thickens, and by fat, fibrin, calcium and thin When born of the same parents' clast blocks, just there is atherosclerosis.Atherosclerosis is a progressive process.When the Lipid Concentration in blood When greatly increasing, fatty streaks will be formed along arterial wall.These stripeds can cause metabolism of lipid and cholesterol to deposit, these precipitations It is attached on originally smooth endarterium, so as to form brief summary.Then the cicatricial tissue of fibrosis is grown below these brief summaries, Lead to doped calcium.The calcium of deposition evolves as the hard film of chalk shape (being known as atheromatous plaque) that can not be removed.Intra-arterial The permanent film of this layer of portion can hinder normal expansion and the contraction of artery, so as to slow down endarterial blood flow velocity, so as to very Clot is easily formed, interfere or blood is prevented to flow through artery.

Only for atherosclerosis, the imperceptible any symptom of people.Only when with some internal vitals After connected artery is blocked, this disease can be just found.The symptom because caused by being obstructed the artery in the organ is more apparent.Example Such as, if heart blood supply arterial portions are obstructed, people may feel angina pectoris；But if be blocked completely, it is possible to lead Cause heart disease (dead by the heart tissue for artery blood supply of being obstructed).If atherosclerosis influences brain artery, Ren Menhui Feel dizziness, blurred vision and syncope, in some instances it may even be possible to cause apoplexy (it is dead by the brain tissue for artery blood supply of being obstructed, so as to cause Neurotrosis is such as paralysed by the limbs that dead brain tissue controls).The artery for leading to kidney portion is obstructed and is also possible to lead to renal failure It exhausts.The blood vessel for leading to eye is obstructed and may cause to blind.Artery of extremity blocks the lesion that may lead to each limbs.

Fibrinolysin is the key component of plasminogen activating system system (PA systems).It is a kind of protease of wide spectrum, can Several components of hydrolyzed cellular epimatrix (ECM), gather including fibrin, gelatin, fibronectin, laminin and albumen Sugar^[23].In addition, fibrinolysin, which can activate some metalloprotein enzyme precursors (pro-MMPs), forms active metalloproteinases (MMPs).Therefore fibrinolysin is considered as an important upstream regulation object of extracellular proteolysis effect^[24,25].Fibrinolysin is The PAs of two kinds of physiologicals is passed through by plasminogen：Tissue-type plasminogen activator (tPA) or urokinase type plasminogen activation What agent (uPA) proteolysis was formed.Due to plasminogen, relative level is higher in blood plasma and other body fluid, conventionally PA The adjusting of system is mainly realized by the synthesis of PAs and activity level.The synthesis of PA system components is strictly adjusted by different factors Section, such as hormone, growth factor and cell factor.In addition, also there are fibrinolysins and the specific physiological inhibitor of PAs.Fibrinolysin Major inhibitors are α 2- antiplasmins (α fibrinolysin (inhibitor.The master of fibrinolysin).The activity of PAs is simultaneously by uPA's and tPA Plasminogen activator inhibitor-1 (PAI-1) inhibits and the main plasminogen activator agent inhibitor -2 (PAI-2) for inhibiting uPA It adjusts.Certain cell surfaces have the uPA specific cell surface receptors (uPAR) of direct hydrolysis activity^[26,27]。

Plasminogen is a single chain glycoprotein, is made of 791 amino acid, molecular weight is about 92kDa^[28,29].Fibrinolytic Proenzyme is mainly synthesized in liver, is largely present in extracellular fluid.Content of plasminogen is about 2 μM in blood plasma.Therefore plasminogen It is a huge potential source of proteolytic activity in tissue and body fluid^[30,31].There are two kinds of molecule shapes for plasminogen Formula：Glutamic acid-plasminogen (Glu-plasminogen) and lysine-plasminogen (Lys-plasminogen).Natural point It secretes with the plasminogen of uncracked form tool there are one amino terminal (N- ends) glutamic acid, therefore is referred to as glutamic acid-fibrinolytic Proenzyme.However, in the presence of fibrinolysin, glutamic acid-plasminogen is hydrolyzed at Lys76-Lys77 as lysine-fibrinolysin It is former.Compared with glutamic acid-plasminogen, lysine-plasminogen has higher affinity with fibrin, and can be with higher Rate activated by PAs.The Arg560-Val561 peptide bonds of the plasminogen of both forms can be cut by uPA or tPA, be caused The formation of the dichain proteins enzyme fibrinolysin of disulfide bond connection^[32].The amino terminus portion of plasminogen includes five homologous tricyclics, I.e. so-called kringles, carboxy-terminal sections include protease domain.Some kringles contain mediation plasminogen with Fibrin and its lysine-binding site of inhibitor α 2-AP specificity interaction.One plasminogen of latest find is The segment of 38kDa is effective inhibitor of angiogenesis including kringles1-4.This segment is named as blood vessel suppression Element can be generated by several protease hydrolytic plasminogens.

The main substrate of fibrinolysin is fibrin, and fibrinous dissolving is to prevent the pass that pathologic thrombus is formed Key^[33].Fibrinolysin also has the substrate specificity of several components to ECM, including laminin, fibronectin, proteoglycans And gelatin, show that fibrinolysin also plays an important role in ECM reconstructions^[29,34,35].Indirectly, fibrinolysin can also pass through conversion Certain protease precursors are active protease come the other components for the ECM that degrades, including MMP-1, MMP-2, MMP-3 and MMP-9. It is thus proposed that fibrinolysin may be an important upstream regulator of extracellular proteolysis^[36].In addition, fibrinolysin Ability with the growth factor for activating certain potential forms^[37-39].In vitro, fibrinolysin can also hydrolyze the component of complement system And discharge chemotactic complement fragment.

" fibrinolysin " is a kind of very important enzyme being present in blood, and fibrin clot can be hydrolyzed to fiber egg White catabolite and d-dimer.

" plasminogen " is the zymogen forms of fibrinolysin, according to the sequence in swiss prot, by the day containing signal peptide Right people source plasminogen amino acid sequence (sequence 4) calculating is made of 810 amino acid, and molecular weight is about 90kD, mainly in liver The dirty middle glycoprotein that synthesizes and can recycle in blood, encodes the cDNA sequence of the amino acid sequence as shown in sequence 3.Overall length Plasminogen include seven structural domains：The Pan Apple (PAp) of serine protease domain, N-terminal positioned at C-terminal Structural domain and 5 Kringle structural domains (Kringle1-5).With reference to the sequence in swiss prot, signal peptide includes residual Base Met1-Gly19, PAp include residue Glu20-Val98, and Kringle1 includes residue Cys103-Cys181, Kringle2 packet It includes residue Glu184-Cys262, Kringle3 and includes residue Cys275-Cys352, Kringle4 includes residue Cys377- Cys454, Kringle5 include residue Cys481-Cys560.According to NCBI data, serine protease domain includes residue Val581-Arg804。

Glu- plasminogens are the plasminogens of Native full-length, are made of 791 amino acid and (do not contain 19 amino acid Signal peptide), the cDNA sequence of the sequence is encoded as shown in sequence 1, and amino acid sequence is as shown in sequence 2.In vivo, also exist A kind of is that hydrolysis is so as to the Lys- plasminogens formed at the 76-77 amino acids of Glu- plasminogens, such as 6 institute of sequence Show, encode the cDNA sequence of the amino acid sequence as shown in sequence 5.Delta- plasminogens (proenzyme a- ammonia sminogen) are complete Long plasminogen has lacked the segment of Kringle2-Kringle5 structures, only containing Kringle1 and serine protease Domain^[40,41], there is the amino acid sequences (sequence 8) of document report delta- plasminogens^[41], encode the amino acid sequence CDNA sequences such as sequence 7.Miniplasminogen (Mini-plasminogen) is made of Kringle5 and serine protease domain, Have document report it include residue Val443-Asn791 (using do not contain signal peptide Glu- plasminogen sequences Glu residues as Initial amino acid)^[42], amino acid sequence encodes cDNA sequence such as 9 institute of sequence of the amino acid sequence as shown in sequence 10 Show.And Microplasminogen (Micro-plasminogen) only contains serine protease domain, there is its amino acid of document report Sequence include residue A la543-Asn791 (using do not contain signal peptide Glu- plasminogen sequences Glu residues for originate ammonia Base acid)^[43], also there is patent document CN102154253A to report that its sequence includes residue Lys531-Asn791 (not contain letter The Glu residues of the Glu- plasminogen sequences of number peptide are initial amino acid), this patent sequence reference patent document CN102154253A, amino acid sequence encode the cDNA sequence of the amino acid sequence as shown in sequence 11 as shown in sequence 12.

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

In this application, content or active ratio of the meaning of the plasminogen " shortage " for plasminogen in subject's body Normal person is low, down to the normal physiological function for being enough to influence the subject；The meaning of the plasminogen " missing " is tested The content of plasminogen or activity are substantially less than normal person or even activity in person's body or expression is atomic, are only provided by external source It could maintain normal physiological function.

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

In cyclic process, plasminogen uses the nonactive conformation of closing, but when being bound to thrombus or cell surface, Under the mediation of plasminogen activator (plasminogen activator, PA), it is changed into the activity in open conformation Fibrinolysin.Fibrin clot further can be hydrolyzed to fibrin degradation product (FDP) and D- dimerization by active fibrinolysin Body, and then thrombus.Wherein the PAp structural domains of plasminogen include the weight that plasminogen is maintained to be in nonactive closing conformation Determinant is wanted, and KR structural domains can then be combined with the lysine residue being present on receptor and substrate.It is known a variety of to make For the enzyme of plasminogen activator, including：Tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), Kallikrein and Hageman factor (Hageman factor (HF)) etc..

" activities of endothelial tissue plasminogen segment " is molten to be referred in plasminogen protein, can be combined concurrently with the target sequence in substrate Wave the active fragment of proteolysis function.Technical solution the present invention relates to plasminogen is covered with activities of endothelial tissue plasminogen segment Instead of the technical solution of plasminogen.Activities of endothelial tissue plasminogen segment of the present invention is the serine stretch protein comprising plasminogen The protein in enzyme domain, it is preferable that activities of endothelial tissue plasminogen segment of the present invention includes sequence 14, has at least with sequence 14 80%th, the protein of the amino acid sequence of 90%, 95%, 96%, 97%, 98%, 99% homology.Therefore, it is of the present invention Plasminogen include containing the activities of endothelial tissue plasminogen segment and still maintain the albumen of the activities of endothelial tissue plasminogen.

At present, plasminogen in blood and its activity determination method are included：To tissue plasminogen activator's activity Detection (t-PAA), Plasma Tissue-Type Plasminogen Activitor antigen detection (t-PAAg), to plasma tissue activities of endothelial tissue plasminogen Detection (plgA), the detection (plgAg) of plasma tissue plasminogen antigen, Plasma Tissue-Type Plasminogen Activitor mortifier live The detection of property, the detection of Plasma Tissue-Type Plasminogen Activitor mortifier antigen, plasma fibrin lyase-antifibrin The compound analyte detection of lyase (PAP).The detection method of most common of which is Chromogenic assay：To by inspection blood plasma in plus streptokinase (SK) And chromophoric substrate, the PLG in by inspection blood plasma are transformed into PLM under the action of SK, the latter acts on chromophoric substrate, then uses and divides Light photometric determination, absorbance increase directly proportional to activities of endothelial tissue plasminogen.In addition can also be used immuno-chemical method, gel electrophoresis, Immunoturbidimetry, radioimmunodiffusion etc. are measured the activities of endothelial tissue plasminogen in blood.

" ortholog thing or lineal homologue (ortholog) " refers to the homologue between different plant species, both same including albumen Source object also includes DNA homology object, also referred to as ortholog, Paralog object.It is referred specifically in different plant species by same ancestors Gene evolution and come albumen or gene.The plasminogen of the present invention includes the natural plasminogen of people, further includes from not Plasminogen ortholog thing infraspecific, that there is activities of endothelial tissue plasminogen or lineal homologue.

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

" separation " plasminogen refers to the plasminogen protein for detaching and/or recycling from its natural surroundings.In some realities It applies in scheme, the plasminogen can purify (1) and extremely be more than the 90%, purity (by weight) more than 95% or more than 98%, As determined by by Lowry methods, such as more than 99% (by weight), (2) are to being enough by using rotating cup sequence analysis To homogeney, which is by making for the degree of at least 15 residues of instrument acquisition N-terminal or internal amino acid sequence or (3) With the sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Coomassie blue or silver staining under reproducibility or non-reducing conditions (SDS-PAGE) it determines.The plasminogen of separation also include by biotechnology from recombinant cell prepare, and pass through to The plasminogen of few purification step separation.

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

It is defined as introducing notch if necessary about " amino acid sequence identity percentage (%) " with reference to peptide sequence It is candidate after realizing largest percentage sequence identity, and when any conservative replacement not being considered as a part for sequence identity The percentage of the amino acid residue identical with the amino acid residue in reference peptide sequence in sequence.To measure percent amino acid The comparison of sequence identity purpose can be realized, such as using publicly available with the various ways in the range of art technology Computer software, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.Those skilled in the art can determine Surely the suitable parameter of aligned sequences is used for, any algorithm needed including realizing maximum contrast to compared sequence.However, For the purposes of the present invention, amino acid sequence identity percent value is to compare computer program ALIGN-2 using sequence to generate 's.

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

Score X/Y multiplies 100

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

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

Term " individual ", " subject " and " patient " is used interchangeably herein, and refers to mammal, including but not limited to Mouse (rat, mouse), non-human primates, people, dog, cat, ungulate (such as horse, ox, sheep, pig, goat) etc..

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

The preparation of plasminogen of the present invention

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

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

Suitable integration portion of the expression vector usually in host organisms as episome or as host chromosome DNA Divide and replicate.In general, expression vector contain selection marker (such as amicillin resistance, hygromycin resistance, tetracyclin resistance, Kalamycin resistance or neomycin resistance) to help to be detected those cells that external source is converted with desired DNA sequence dna.

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

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

Outside microorganism, mammalian cell (such as the mammalian cell cultivated in cell culture in vitro) also may be used For expressing and generating the anti-Tau antibody (such as polynucleotides of the anti-Tau antibody of encoding schemes) of the present invention.Referring to Winnacker,From Genes to Clones, VCH Publishers,N.Y.,N.Y.(1987).Suitable mammal Host cell includes Chinese hamster ovary celI system, various Cos cell lines, HeLa cells, myeloma cell line and inverted B cell or miscellaneous Hand over knurl.Expression control sequence, such as replication orgin, promoter and enhancer can be included for the expression vector of these cells (Queen etc., Immunol.Rev.89:49 (1986)) and required machining information site, such as ribosome bind site, RNA splice sites, polyadenylation site and transcription terminator sequences.The example of suitable expression control sequence is white exempts from Promoter derived from epidemic disease globulin gene, SV40, adenovirus, bovine papilloma virus, cytomegalovirus etc..Referring to Co etc., J.Immunol.148:1149(1992)。

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

Pharmaceutical formulation

Can be by by plasminogen with the desired purity and optional pharmaceutical carrier, excipient or stabilizer (Remington's Pharmaceutical Sciences, 16 editions, Osol, A. ed. (1980)) are mixed to form lyophilized preparation Or aqueous solution prepares treatment preparaton.Acceptable carrier, excipient, stabilizer are nontoxic to receptor under dosage used and concentration Property, and including buffer such as phosphate, citrate and other organic acids；Antioxidant includes ascorbic acid and methionine； Preservative (such as stearyl dimethyl benzyl ammonium chloride；Hexamethonium chloride；Benzalkonium chloride (benzalkonium Chloride), benzethonium chloride；Phenol, butanol or benzyl alcohol；Alkyl parabens such as methyl or propyl p-hydroxybenzoate Ester；Catechol；Resorcinol；Cyclohexanol；3- amylalcohols；Metacresol)；Low molecular weight polypeptide (less than about 10 residues)；Albumen Matter such as seralbumin, gelatin or immunoglobulin；Hydrophilic polymer such as polyvinylpyrrolidone；Amino acid such as glycine, paddy Glutamine, asparagine, histidine, arginine or lysine；Monosaccharide, disaccharides and other carbohydrate include glucose, sweet Dew sugar or dextrin；Chelating agent such as EDTA；Carbohydrate such as sucrose, mannitol, fucose or sorbierite；Into salt counter ion such as sodium；Metal Compound (such as zinc-albumen composition)；And/or nonionic surfactant, such as TWEENTM, PLURONICSTM or poly- second Glycol (PEG).It is preferred that the anti-VEGF antibodies preparaton being lyophilized, described in WO 97/04801, it includes herein as ginseng It examines.

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

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

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

The plasminogen of the present invention can prepare sustained release preparation.The appropriate example of sustained release preparation includes with definite shape and contains There are the half penetrating matrix of solid hydrophobic polymers of glycoprotein, such as film or microcapsules.Sustained-release matrix example includes polyester, hydrogel (such as poly- (2- hydroxyethyl-methacrylates) (Langer, J.Biomed.Mater.Res., 15:167-277(1981)； Langer,Chem. Tech.,12:98-105 (1982)) or it is poly- (vinyl alcohol), polyactide (United States Patent (USP) 3773919, EP 58, 481), the copolymer (Sidman, etc. Biopolymers 22 of Pidolidone and γ ethyl-L-glutamates:547 (1983)), Nondegradable ethylene vinyl acetate (ethylene-vinyl acetate) (Langer, etc. ibid) can The poly lactic coglycolic acid of degradation such as Lupron DepotTM are (by poly lactic coglycolic acid and leucyl proline (leuprolide) microsphere of the injectable of acetic acid esters composition) and poly- D- (-) -3- hydroxybutyric acids.Polymer such as ethylene-second Vinyl acetate and lactic-co-glycolic acid energy sustained release molecule 100 days or more, and the time of some hydrogels release proteins compared with It is short.Can protein stabilized rational strategy be made to design according to Related Mechanism.For example, if it find that the mechanism of cohesion is to pass through sulphur Intermolecular S -- S is formed for Disulfide interchange, then can by modifying sulfhydryl residue, from acid solution be lyophilized, control humidity, Stabilization is realized using suitable additive and the specific polymer matrix composition of exploitation.

Administration and dosage

Can be by different modes, such as by intravenous, in peritonaeum, subcutaneously, encephalic is intrathecal, intra-arterial (such as via Arteria carotis), it is intramuscular to realize the application of pharmaceutical composition of the present invention.

Include sterile aqueous or non-aqueous solution, suspension and emulsion for the prepared product of parenteral administration.It is non-aqueous molten The example of agent is propylene glycol, polyethylene glycol, vegetable oil such as olive oil and injectable organic ester, such as ethyl oleate.Aqueous carrier packet Water, alcohol/aqueous solution, emulsion or suspension are included, including brine and buffer medium.It is molten that parenteral medium includes sodium chloride Liquid, woods grignard dextrose, dextrose and sodium chloride or fixing oil.Intravenous vehicles include liquid and nutritional supplement, electrolysis Matter supplement, etc..There may also be preservatives and other additives, such as, antimicrobial, antioxidant, chelating Agent and inert gas, etc..

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

Product or medicine box

One embodiment of the invention is related to a kind of product or medicine box, and it includes available for treating as caused by diabetes The plasminogen or fibrinolysin of the present invention of cardiovascular disease and its associated disease.The product preferably includes a container, label or Package insert.Appropriate container has bottle, bottle, syringe etc..Container can be made of a variety of materials such as glass or plastics.It is described Container contains composition, and the composition can effectively treat the disease of the present invention or illness and (such as described with sterile access port Container can be intravenous solution packet or bottle, and containing can be by the plug that hypodermic needle penetrates).In the composition at least A kind of activating agent is plasminogen/fibrinolysin.On the container or appended label illustrates the composition for treating this hair The bright cardiovascular disease as caused by diabetes and its associated disease.The product can further include containing pharmaceutically acceptable buffer solution Second container, the brine of such as phosphate-buffered, Ringer's solution and glucose solution.It can further include from business With other materials required from the point of view of user's angle, including other buffer solutions, diluent, filtrate, needle and syringe.In addition, The product includes the package insert with operation instruction, including for example indicating the user of the composition by plasminogen group It closes object and treats the other medicines administered patient of adjoint disease.

Brief description

Fig. 1 ApoE Atherosclerosis Model mouse give plasminogen substantially aorta oil red O stain figure after 10 days Piece.The results show that plasminogen group mouse aorta bow, aorta pectoralis and abdominal aorta Lipid Plaque (arrow logo) face Product, which is significantly less than, gives solvent PBS control group, and it is 36.0% to account for vessel area ratio to solvent PBS control group lipid, to plasminogen Group is 29.6%.Illustrate that plasminogen can reduce ApoE Atherosclerosis Model rat aortas atheromatous plaque deposition, promote dynamic The reparation of pulse atherosclerosis vascular damaged.

Fig. 2 ApoE Atherosclerosis Model mouse give PBS or plasminogen after 20 days substantially aorta oil red O contaminate Chromatic graph piece.The results show that plasminogen group mouse aorta bow, aorta pectoralis and abdominal aorta Lipid Plaque (arrow mark Know) area is significantly less than and gives solvent PBS control group.It is 48.1% to account for vessel area ratio to solvent PBS control group lipid, to fibre Lyase original group is 39.4%.Illustrate that plasminogen can reduce ApoE Atherosclerosis Model rat aortas atheromatous plaque deposition, Promote the reparation of atherosclerotic blood vessel damage.

It is representative that Fig. 3 ApoE Atherosclerosis Model mouse give 30 days posterior sinus of Valsalva oil red O stains of plasminogen Picture.A is gives solvent PBS control group, and B is gives plasminogen group.The results show that give plasminogen group mouse aorta sinus fat Deposition (arrow logo) is considerably less than and gives solvent PBS control group.Illustrate that plasminogen can improve deposition of the fat in aortic sinus.

Fig. 4 ApoE Atherosclerosis Model mouse give plasminogen 30 days aorta posterior valve HE dyeing representative diagrams Piece.A, C is gives solvent PBS control group, and B, D is give plasminogen group.The results show that give plasminogen group (Fig. 4 B, 4D) mouse Aorta petal plaque deposition (arrow logo), which is considerably less than, gives solvent PBS control group (Fig. 4 A, 4C), and aortic valve fusion journey The former is spent less than the latter.Illustrate that plasminogen can improve the damage of Atherosclerosis Model mouse aorta valve.

Fig. 5 ApoE Atherosclerosis Model mouse give plasminogen 30 days aorta posterior oil red O stain representative diagrams Piece.A is gives solvent PBS control group, and B is gives plasminogen group.As a result plasminogen group aorta oil red O stained areas are shown to (arrow logo), which is significantly less than, gives solvent PBS control group.Illustrate that plasminogen can significantly reduce Atherosclerosis Model mouse The deposition of aorta lipid improves the damage of aorta inner wall.

26 week old diabetic mices of Fig. 6 give plasminogen serum middle-high density lipoprotein cholesterol (HDL-C) after 35 days Content detection result.The results show that after people source fibrinolysin source is injected 35 days, to HDL-C in plasminogen group mice serum Content, which is significantly higher than, gives solvent PBS control groups, and significantly (* represents P to statistical discrepancy<0.05).Illustrate that injecting plasminogen can promote Into the raising of serum HDL-C level.

Fig. 7 24-25 week old diabetic mices give plasminogen low density lipoprotein cholesterol in serum after 31 days (LDL-C) content detection result.The results show that diabetic mice continuously injected people source fibrinolysin source after 31 days, to fibrinolytic LDL-C contents in proenzyme group mice serum, which are less than, gives solvent PBS control group.Illustrate that plasminogen can reduce LDL-C in serum Content.

Fig. 8 ApoE Atherosclerosis Model mouse give the variation of plasminogen weight after 30 days.The results show that it gives Give plasminogen after 30 days mouse weight without substantially changeing, illustrate administration processing to the bodies of ApoE Atherosclerosis Model mouse It has no significant effect again.

Fig. 9 ApoE Atherosclerosis Model mouse give the testing result of plasminogen serum total cholesterol after 30 days. Solvent PBS control group is given the results show that being significantly lower than to plasminogen group mouse total cholesterol concentration, and statistical discrepancy significantly (* Represent P<0.05).Illustrate that plasminogen can reduce the content of T-CHOL in ApoE Atherosclerosis Model mice serums.

Figure 10 ApoE Atherosclerosis Model mouse give plasminogen serum triglyceride testing result after 30 days. Solvent PBS control group is given the results show that being significantly lower than to plasminogen group mouse triglyceride concentration, and statistical discrepancy significantly (* Represent P<0.05).Illustrate that plasminogen can reduce the content of triglycerides in ApoE Atherosclerosis Model mice serums.

Figure 11 ApoE Atherosclerosis Model mouse give plasminogen serum LDL cholesterol after 30 days (LDL-C) testing result.Solvent PBS control group is given the results show that being significantly lower than to plasminogen group mouse LDL-C concentration, And significantly (* represents P to statistical discrepancy<0.05).Illustrate that plasminogen can be reduced in ApoE Atherosclerosis Model mice serums The content of LDL-C.

Figure 12 ApoE Atherosclerosis Model mouse give plasminogen heart organ coefficient statistical result after 30 days. Solvent PBS control group is given the results show that being significantly lower than to plasminogen group mouse heart organ coefficient.Illustrate that plasminogen can change Cardiac compensation caused by kind ApoE Atherosclerosis Models mouse heart damage is loose.

Figure 13 ApoE Atherosclerosis Model mouse give plasminogen liver oil red O stain representative diagram after 30 days Piece.A is gives solvent PBS control group, and for B to give plasminogen group, C is quantitative analysis results.The results show that give plasminogen group Mouse liver fat deposition (arrow logo), which is considerably less than, gives solvent PBS control group, and significantly (* represents P to statistical discrepancy< 0.05).Illustrate that plasminogen can improve Atherosclerosis Model mouse liver fat deposition.

Figure 14 ApoE Atherosclerosis Model mouse give plasminogen after 30 days heart IgM immunostainings it is representative Picture.A is gives solvent PBS control group, and B is gives plasminogen group.The results show that the sun to plasminogen group mouse heart IgM Property expression (arrow logo) be considerably less than give solvent PBS control group, illustrate that plasminogen can promote the heart caused by atherosclerosis The reparation of dirty damage.

Figure 15 ApoE Atherosclerosis Model mouse give plasminogen after 30 days heart sirius red stains it is representative Picture.A is gives solvent PBS control group, and B is gives plasminogen group.The results show that deposition (the arrow to plasminogen group collagen Mark) it is considerably less than and gives solvent PBS control group, illustrate that plasminogen can mitigate ApoE Atherosclerosis Models mouse heart fibre Dimensionization.

26 week old diabetic mices of Figure 16 give plasminogen ventricle oil red O stain representativeness picture after 35 days.A be to Solvent PBS control group, B is gives plasminogen group.The results show that plasminogen group mouse ventricle lipidosis (arrow logo) Considerably less than give solvent PBS control group.Illustrate that plasminogen can reduce diabetic mice ventricle lipidosis, promote ventricle The reparation of damage.

Figure 17 ApoE Atherosclerosis Model mouse give plasminogen 30 days posterior sinus of Valsalva sirius red stains generations Table picture.A, C is gives solvent PBS control group, and B, D is give plasminogen group.As a result it is shown to plasminogen group aortic sinus The area of blood vessel collagen deposition (arrow logo), which is significantly less than, gives solvent PBS control group, illustrates that plasminogen can Cut down artery congee hardening model mouse aorta sinus Fibrosis levels.

Figure 18 24-25 week old diabetic mices give plasminogen 31 days aorta posterior HE dyeing pictures.A is to solvent PBS control group, B is gives plasminogen group.The results show that there is foam cells to deposit (arrow to solvent PBS control group vascular wall Mark), middle elastic film is disorganized, and tube wall is concave-convex uneven；Plasminogen group middle elastic membrane structure rule is given, in wave Shape.Show that inject plasminogen has certain repair to the Aortic injury caused by diabetes.

Figure 19 24-25 week old diabetic mices give plasminogen serum cTnT content detection result after 31 days. The results show that plasminogen group cardiac muscle troponin I concentration significantly lower than giving solvent PBS control group, and statistical difference is heteropolar Significantly (* * represent P<0.01).Illustrate that plasminogen can remarkably promote the reparation of diabetes later stage mouse cardiac muscle damage.

3% cholesterol hyperlipemia model mouse of Figure 20 give plasminogen 10 days and serum high-density fat egg after 20 days White cholesterol detection result.The results show that it is apparently higher than after giving plasminogen to plasminogen group mice serum HDL-C concentration Give solvent PBS control group, and heteropolar significantly (the * * tables of the two equal statistical difference of high-density lipoprotein concentration after being administered 10 days and 20 days Show P<0.01).Illustrate that plasminogen can effectively improve containing for hyperlipemia model mice serum middle-high density lipoprotein cholesterol It is disorderly to improve hyperlipemia model lipid of mice for amount.

Figure 21 shows that 3% cholesterol hyperlipemia model mouse gives 20 days atherosclerosis indexes of plasminogen and calculates As a result.Result of calculation is shown, is significantly lower than to plasminogen group rat aorta atherosis index and is given solvent PBS control group, and Statistical discrepancy is notable.Illustrate that plasminogen can reduce the risk that atherosclerosis occurs for hyperlipemia model mouse.

Figure 22 shows that 3% cholesterol hyperlipemia model mouse gives 20 days heart risk indexs of plasminogen and calculates knot Fruit.Solvent PBS control group is given, and statistical discrepancy is extremely notable the results show that being significantly less than to plasminogen group CRI.Illustrate fibre Lyase proper energy effectively reduces the risk that heart disease occurs for hyperlipemia model mouse.

Embodiment：

1 plasminogen of embodiment improves ApoE atherosclerosis mouse aortas Lipid Plaque deposition

6 week old male ApoE mouse 13 feed high cholesterol diet (Nantong Te Luofei, TP2031) high in fat 16 weeks to lure Lead Atherosclerosis Model^[47,48].Mouse after Cheng Mo before administration three days every 50 μ l of blood is taken to detect T-CHOL (T- CHO) content, and two groups are randomly divided into according to T-CHO contents, to solvent PBS control group 7, to plasminogen group 6.Start Administration is set to the 1st day, to 1mg/0.1ml/ pcs/day of plasminogen group mouse tail vein injection people source plasminogen, to solvent The PBS of PBS control group tail vein injection same volume.Administration 10 days, continues to feed high cholesterol diet high in fat during administration, and the 11st Its two groups of mouse are each to take an execution at random, and aorta is taken to fix 24-48 hours in 4% paraformaldehyde, and oil red O is carried out after splitting It substantially dyes, aorta is taken pictures in 7 times of Microscopic observations of Stereo microscope.

Oil red O stain can show lipidosis, reflect the severity of damage^[49].Coloration result (Fig. 1) is shown, to fibre Lyase original group mouse aorta bow, aorta pectoralis and abdominal aorta Lipid Plaque (arrow logo) area are significantly less than to solvent PBS control group, it is 36.0% to account for vessel area ratio to solvent PBS control group lipids, is 29.6% to plasminogen group.Experiment It proves that plasminogen can reduce ApoE Atherosclerosis Model rat aortas atheromatous plaque deposition, promotes atherosclerosis It repairs.

2 plasminogen of embodiment reduces ApoE atherosclerosis mouse aortas Lipid Plaque deposition

6 week old male ApoE mouse 13 feed high cholesterol diet (Nantong Te Luofei, TP2031) high in fat 16 weeks to lure Lead Atherosclerosis Model^[47,48].Mouse after Cheng Mo before administration three days every 50 μ l of blood is taken to detect T-CHOL (T- CHO) content, and two groups are randomly divided into according to T-CHO contents, to solvent PBS control group 7, to plasminogen group 6.Start Administration is set to the 1st day, to 1mg/0.1mL/ pcs/day of plasminogen group mouse tail vein injection people source plasminogen, to solvent The PBS of PBS control group tail vein injection same volume.Administration 20 days, continues to feed high cholesterol diet high in fat during administration, and the 21st Its two groups of mouse are each to take an execution at random, and aorta is taken to fix 24-48 hours in 4% paraformaldehyde, and oil red O is carried out after splitting It substantially dyes, aorta is taken pictures in 7 times of Microscopic observations of Stereo microscope.

The arch of aorta, aorta pectoralis and abdominal aorta Lipid Plaque (arrow logo) area, which are significantly less than, gives solvent PBS Control group, it is 48.1% to account for vessel area ratio to solvent PBS control group lipid, is 39.4% (Fig. 2) to plasminogen group.It says Bright plasminogen can reduce ApoE Atherosclerosis Model rat aorta atheromatous plaques, promote the reparation of atherosclerosis.

3 plasminogen of embodiment improves ApoE atherosclerosis mouse aorta sinus lipidosis

6 week old male ApoE mouse 13 feed high cholesterol diet (Nantong Te Luofei, TP2031) high in fat 16 weeks to lure Lead Atherosclerosis Model^[47,48].Mouse after Cheng Mo before administration three days every 50 μ l of blood is taken to detect T-CHOL (T- CHO) content, and two groups are randomly divided into according to T-CHO contents, to solvent PBS control group 7, to plasminogen group 6.Start Administration is set to the 1st day, to 1mg/0.1mL/ pcs/day of plasminogen group mouse tail vein injection people source plasminogen, to solvent The PBS of PBS control group tail vein injection same volume.Administration 30 days, and mouse was put to death in the 31st day, it cores dirty in 4% poly first Aldehyde fixes 24-48 hours, is sunk to the bottom overnight respectively at 4 DEG C in 15%, 30% sucrose, OCT embeddings, 8 μm of frozen section thickness, oil Red O dyes 15min, and 75% alcohol breaks up 5 seconds, bush uniformly dyeing core 30 seconds, glycerin gelatine mounting.Slice is in 200 times of optical microphotographs Microscopic observation.

The results show that it is considerably less than to plasminogen group (Fig. 3 B) mouse aorta sinus fat deposition (arrow logo) to molten Matchmaker's PBS control group (Fig. 3 A).Illustrate that plasminogen can improve aortic sinus fat deposition in atherosclerosis.

4 plasminogen of embodiment improves the damage of ApoE atherosclerosis mouse aortas sinus

6 week old male ApoE mouse 13 feed high cholesterol diet (Nantong Te Luofei, TP2031) high in fat 16 weeks to lure Lead Atherosclerosis Model^[47,48].Mouse after Cheng Mo before administration three days every 50 μ l of blood is taken to detect T-CHOL (T- CHO) content, and two groups are randomly divided into according to T-CHO contents, to solvent PBS control group 7, to plasminogen group 6.Start Administration is set to the 1st day, to 1mg/0.1ml/ pcs/day of plasminogen group mouse tail vein injection people source plasminogen, to solvent The PBS of PBS control group tail vein injection same volume.Administration 30 days, continues to feed high cholesterol diet high in fat during administration.In It puts to death mouse within 31 days, cores dirty in 4% paraformaldehyde fixation 24-48 hours.Tissue samples after fixation are through alcohol serial dehydration With the transparent rear progress paraffin embedding of dimethylbenzene.Aortic sinus histotomy thickness be 3 μm, slice dewaxing rehydration and with haematoxylin with Eosin stains (HE dyeing), ammonium hydroxide returns blue and alcohol serial dehydration mounting after the differentiation of 1% hydrochloride alcohol, is sliced respectively at 40 times (Fig. 4 A and 4B) and 200 times of (Fig. 4 C and 4D) optical microphotograph Microscopic observations.

Coloration result is shown, (arrow logo) is deposited to plasminogen group (Fig. 4 B, 4D) mouse aorta sinus Lipid Plaque Considerably less than give solvent PBS control group (Fig. 4 A, 4C), and aortic valve fusion degree the former be less than the latter.Illustrate fibrinolysin Proper energy improves aorta petal membrane damage in atherosclerosis.

5 plasminogen of embodiment reduces ApoE atherosclerosis mouse aorta lipidosis

6 week old male ApoE mouse 13 feed high cholesterol diet (Nantong Te Luofei, TP2031) high in fat 16 weeks to lure Lead Atherosclerosis Model^[47,48].Mouse after Cheng Mo before administration three days every take 50 μ l of blood to detect T-CHOL (T-CHO) content, and two groups are randomly divided into according to T-CHO contents, to solvent PBS control group 7, to plasminogen group 6. Start administration to be set to the 1st day, to 1mg/0.1mL/ pcs/day of plasminogen group mouse tail vein injection people source plasminogen, to molten The PBS of matchmaker's PBS control group tail vein injection same volume.Administration 30 days, continues to feed high cholesterol diet high in fat during administration.In It puts to death mouse within 31st day, aorta is taken to fix 24-48 hours in 4% paraformaldehyde, respectively at 4 DEG C of mistakes in 15%, 30% sucrose Night sinks to the bottom, OCT embeddings, 8 μm of frozen section thickness, oil red O stain 15min, and 75% alcohol breaks up 5 seconds, bush uniformly dyeing core 30 Second, glycerin gelatine mounting.Slice is in 200 times of optical microphotograph Microscopic observations.

Coloration result is shown, apparent to plasminogen group (Fig. 5 B) aorta oil red O stain deposition (arrow logo) area Less than giving solvent PBS control group (Fig. 5 A).Illustrate that plasminogen can be substantially reduced ApoE Atherosclerosis Model mouse masters The deposition of artery lipid wall in the blood vessels, improves the damage of aorta.

High-density lipoprotein cholesterol in 6 plasminogen of embodiment raising diabetic mice serum

26 week old db/db male mices 20 are grouped at random, to plasminogen group 11, to solvent PBS control group 9. To 2mg/0.2ml/ pcs/day of plasminogen group tail vein injection people source plasminogen, solvent PBS control group tail vein injection is given The PBS of same volume.Continuous injection plucks eyeball and adopts whole blood after 35 days, 4 DEG C, 3500r/min is centrifuged 10 minutes, supernatant is taken to carry out high Density lipoprotein-cholesterol (HDL-C) detects.Using kit, (biological work is built up in Nanjing for the detection of high-density lipoprotein cholesterol Journey research institute, article No. A112-1), and carried out according to the kit the method.

Testing result shows that db/db mouse continuously injected people source fibrinolysin source after 35 days, in plasminogen group mice serum The content of HDL-C is higher than to solvent PBS control group (Fig. 6), and statistical discrepancy is notable.

Diabetes generally entail cardiovascular atherosclerotic^[45,46], and high-density lipoprotein is a kind of anti-Artery is athero- Hardening'sPlasma lipoprotein, be coronary heart disease protective factors, be commonly called as " blood vessel scavenger ".The testing result proves plasminogen energy The level of Serum HDL-C is improved, so as to help to improve the atherosclerosis of diabetic mice.

7 plasminogen of embodiment reduces low density lipoprotein cholesterol in diabetic mice serum

The random grouping of 24-25 week old db/db male mices 10, to plasminogen group and to solvent PBS control group each 5 Only, and 3 db/m are taken as Normal group.To plasminogen group tail vein injection people source plasminogen 2mg/0.2ml/ only/ My god, the PBS of PBS control group tail vein injection same volume, Normal group mouse does not make any processing.Start administration and be set to the 0th My god, mouse plucks eyeball and adopts whole blood after continuous injection 31 days, and 4 DEG C, 3500r/min is centrifuged 10 minutes, and supernatant is taken to carry out low-density Lipoprotein cholesterol (LDL-C) detects.Using kit, (Nanjing is built up bioengineering and is ground for the detection of low density lipoprotein cholesterol Study carefully institute, article No. A113-1), and carried out according to the detection kit the method.

Testing result shows that db/db mouse continuously injected people source fibrinolysin source after 31 days, gave plasminogen group mice serum In LDL-C contents be less than to solvent PBS control group (Fig. 7).

Low-density lipoprotein is a kind of hdl particle for delivering cholesterol and entering peripheral tissues' cell, can be oxidized to oxygen Change low-density lipoprotein, when working as low-density lipoprotein (OX-LDL) excess of low-density lipoprotein, especially oxidative modification, it takes The cholesterol of band is just accumulated on arterial wall, causes artery sclerosis.Therefore low density lipoprotein cholesterol is referred to as that " bad courage is consolidated Alcohol "^[52].The results show plasminogen can reduce the content of low density lipoprotein cholesterol in serum, so as to be conducive to Control atherosclerosis.

Influence of 8 plasminogen of embodiment to ApoE atherosclerosis mouse weights

6 week old male ApoE mouse 13 feed high cholesterol diet (Nantong Te Luofei, TP2031) high in fat 16 weeks to lure Lead Atherosclerosis Model^[47,48].Mouse after Cheng Mo before administration three days every 50 μ l of blood is taken to detect T-CHOL (T- CHO) content, and two groups are randomly divided into according to T-CHO contents, to solvent PBS control group 7, to plasminogen group 6.Start Administration is set to the 1st day, to 1mg/0.1ml/ pcs/day of plasminogen group mouse tail vein injection people source plasminogen, to solvent The PBS of PBS control group tail vein injection same volume is administered 30 days, continues to feed high cholesterol diet high in fat during administration.In to The the 1st, 31 day of medicine weighs mouse weight.

The results show that give plasminogen after 30 days mouse weight without (Fig. 8) is substantially change, illustrate administration processing to ApoE The weight of Atherosclerosis Model mouse has no significant effect.

9 plasminogen of embodiment reduces ApoE atherosclerosis lipid of mice contents

6 week old male ApoE mouse 13 feed high cholesterol diet (Nantong Te Luofei, TP2031) high in fat 16 weeks to lure Lead Atherosclerosis Model^[47,48].Mouse after Cheng Mo before administration three days every 50 μ l of blood is taken to detect T-CHOL (T- CHO) content, and two groups are randomly divided into according to T-CHO contents, to solvent PBS control group 7, to plasminogen group 6.Start Administration is set to the 1st day, to 1mg/0.1ml/ pcs/day of plasminogen group mouse tail vein injection people source plasminogen, to solvent The PBS of PBS control group tail vein injection same volume.Administration 30 days, continues to feed high cholesterol diet high in fat during administration. Mouse fasting in 30 days 16 hours extracts eyeball on the 31st day and takes blood, and centrifugation obtains supernatant, to detect serum total cholesterol (T- CHO), serum triglyceride (TG) and serum LDL cholesterol (LDL-C) content.

1. serum total cholesterol content

Using detection kit (Bioengineering Research Institute, article No. A111-1 are built up in Nanjing) and according to the detection kit The method detects serum total cholesterol content.

Testing result is shown, is significantly lower than to plasminogen group mouse total cholesterol concentration and is given solvent PBS control group, and Statistical discrepancy is significantly (Fig. 9).Illustrate that plasminogen can reduce T-CHOL in ApoE Atherosclerosis Model mice serums Content.

2. serum triglyceride content

Using TG detection kits (Bioengineering Research Institute, article No. A110-1 are built up in Nanjing) and according to the kit Illustrate to detect serum TG content using COD-PAP methods.Testing result is shown, is significantly lower than and is given to plasminogen group mouse TG concentration Solvent PBS control group, and statistical discrepancy is significantly (Figure 10).

3. serum LDL cholesterol content

Using low density lipoprotein cholesterol (LDL-C), according to detection kit, (Bioengineering Research Institute, goods are built up in Nanjing Number A113-1) and detect serum low-density LP content according to the kit the method.

Testing result is shown, is significantly lower than to plasminogen group mouse LDL-C concentration and is given solvent PBS control group, and counted Significant difference (Figure 11).Illustrate that plasminogen can reduce the content of LDL-C in ApoE Atherosclerosis Model mice serums, Improve atherosclerosis.

The above results prove that plasminogen can significantly reduce the serum total cholesterol of Atherosclerosis Model mouse, glycerine Three esters and low density lipoprotein cholesterol content improve atherosclerosis.Meanwhile by reducing serum total cholesterol, glycerine The content of three esters and low density lipoprotein cholesterol reduces atherosclerotic complications, such as atherosclerotic painstaking effort The risk of pipe disease.

10 plasminogen of embodiment improves ApoE atherosclerosis mouse heart compensatory hypertrophies

6 week old male ApoE mouse 13 feed high cholesterol diet (Nantong Te Luofei, TP2031) high in fat 16 weeks to lure Lead Atherosclerosis Model^[47,48].Mouse after Cheng Mo before administration three days every 50 μ l of blood is taken to detect T-CHOL (T- CHO) content, and two groups are randomly divided into according to T-CHO contents, to solvent PBS control group 7, to plasminogen group 6.Start Administration is set to the 1st day, to 1mg/0.1mL/ pcs/day of plasminogen group mouse tail vein injection people source plasminogen, to solvent The PBS of PBS control group tail vein injection same volume.Administration 30 days, continues to feed high cholesterol diet high in fat during administration.In to Mouse is put to death in the 31st day of medicine after weighing, dirty weigh, and calculate heart coefficient of coring.Heart coefficient (%)=cardiac weight/body Weight × 100.

The results show that it is significantly lower than to plasminogen group mouse heart coefficient to solvent PBS control group (Figure 12).Illustrate fibre It is loose that lyase proper energy mitigates Cardiac compensation of the ApoE Atherosclerosis Model mouse caused by heart injury.

11 plasminogen of embodiment improves ApoE atherosclerosis mouse liver lipidosis

6 week old male ApoE mouse 13 feed high cholesterol diet (Nantong Te Luofei, TP2031) high in fat 16 weeks to lure Lead Atherosclerosis Model^[47,48].Mouse after Cheng Mo before administration three days every 50 μ l of blood is taken to detect T-CHOL (T- CHO) content, and two groups are randomly divided into according to T-CHO contents, to solvent PBS control group 7, to plasminogen group 6.Start Administration is set to the 1st day, to 1mg/0.1mL/ pcs/day of plasminogen group mouse tail vein injection people source plasminogen, to solvent The PBS of PBS control group tail vein injection same volume.Administration 30 days, continues to feed high cholesterol diet high in fat during administration.In It puts to death mouse within 31 days, liver is taken to fix 24-48 hours in 4% paraformaldehyde, it is heavy overnight respectively at 4 DEG C in 15%, 30% sucrose Bottom, OCT embeddings, 8 μm of frozen section thickness, oil red O stain 15min, 75% alcohol break up 5 seconds, bush uniformly dyeing core 30 seconds, glycerine Gelatin mounting.Slice is in 400 times of optical microphotograph Microscopic observations.

Coloration result is shown, is considerably less than and is given to plasminogen group (Figure 13 B) mouse liver fat deposition (arrow logo) Solvent PBS control group (Figure 13 A), and quantitative analysis statistical discrepancy is significantly (Figure 13 C).Illustrate that plasminogen can improve ApoE arteries Atherosis mouse liver fat deposition.

12 plasminogen of embodiment improves the damage of ApoE atherosclerosis mouse heart

6 week old male ApoE mouse 13 feed high cholesterol diet (Nantong Te Luofei, TP2031) high in fat 16 weeks to lure Lead Atherosclerosis Model^[47,48].Mouse after Cheng Mo before administration three days every 50 μ l of blood is taken to detect T-CHOL (T- CHO) content, and two groups are randomly divided into according to T-CHO contents, to solvent PBS control group 7, to plasminogen group 6.Start Administration is set to the 1st day, to 1mg/0.1ml/ pcs/day of plasminogen group mouse tail vein injection people source plasminogen, to solvent The PBS of PBS control group tail vein injection same volume.Administration 30 days, continues to feed high cholesterol diet high in fat during administration.In It puts to death mouse within 31 days, cores dirty in 4% paraformaldehyde fixation 24-48 hours.Tissue after fixation is through alcohol serial dehydration and two Paraffin embedding is carried out after toluene is transparent.Histotomy thickness is 3 μm, is washed 1 time after slice dewaxing rehydration.PAP are irised out tissue, With 3% dioxygen water incubation 15 minutes, 0.01MPBS was washed 2 times, every time 5 minutes.5% normal sheep serum liquid (Vector Laboratories, Inc., USA) it closes 30 minutes；After time arrives, mountain sheep anti mouse IgM (HRP) is added dropwise in reject sheep blood serum liquid Antibody (Abcam) is incubated at room temperature 1 hour, and 0.01MPBS is washed 2 times, every time 5 minutes.By DAB kits (Vector Laboratories, Inc., USA) colour developing, haematoxylin is redyed 30 seconds after washing, and flowing water rinses 5 minutes.Gradient alcohol dehydration, two Toluene is transparent and neutral gum mounting, is sliced in 200 times of optical microphotograph Microscopic observations.IgM antibody is thin in removing apoptosis and necrosis Born of the same parents play an important role in the process, and the level of injuries of tissues and organs local I gM antibody is proportionate with degree of injury^[50,51]。 Therefore, the level of detection histoorgan local I gM antibody can reflect the degree of impairment of the histoorgan.Experiment is found, to fibre The positive expression of lyase original group mouse (Figure 14 B) heart IgM is considerably less than and gives solvent PBS control group (Figure 14 A).Illustrate fibrinolytic Proenzyme can be obviously improved the damage of ApoE mouse cardiac muscles.

13 plasminogen of embodiment reduces ApoE atherosclerosis mouse heart Fibrosis levels

6 week old male ApoE mouse 13 feed high cholesterol diet (Nantong Te Luofei, TP2031) high in fat 16 weeks to lure Lead Atherosclerosis Model^[47,48].Mouse after Cheng Mo before administration three days every 50 μ l of blood is taken to detect T-CHOL (T- CHO) content, and two groups are randomly divided into according to T-CHO contents, to solvent PBS control group 7, to plasminogen group 6.Start Administration is set to the 1st day, to 1mg/0.1ml/ pcs/day of plasminogen group mouse tail vein injection people source plasminogen, to solvent The PBS of PBS control group tail vein injection same volume.Administration 30 days, continues to feed high cholesterol diet high in fat during administration.In Mouse is put to death within 31 days, materials heart fixes 24-48 hours in 4% paraformaldehyde.Tissue after fixation through alcohol serial dehydration and Paraffin embedding is carried out after dimethylbenzene is transparent.Histotomy thickness is 3 μm, is washed 1 time after slice dewaxing rehydration, with 0.1% day wolf After the red saturation picric acid of star dyes 30 minutes, flowing water rinses 2min, haematoxylin dyeing 1 minute, and flowing water rinses, 1% hydrochloride alcohol Differentiation, ammonium hydroxide return indigo plant, and flowing water rinses, neutral gum mounting after drying, in 200 times of optical microphotograph Microscopic observations.

Sirius red stains can be such that collagen persistently dyes, and be the specific stain method to collagen tissue in pathological section, with Special display collagen tissue.

Coloration result is shown, is considerably less than to the deposition (arrow logo) of plasminogen group (Figure 15 B) collagen to solvent PBS control group (Figure 15 A) illustrates that plasminogen can reduce collagen in ApoE Atherosclerosis Model mouse heart tissues Deposition reduces cardiac fibrosis.

14 plasminogen of embodiment reduces diabetic mice ventricle lipidosis

Diabetes generally entail cardiovascular atherosclerotic^[45,46].Cardiovascular atherosclerotic can cause myocardium thin The ischemic injuries of born of the same parents.Oil red O stain can show lipidosis, reflect the severity of damage^[49]。

26 week old db/db male mices 9, are randomly divided into two groups, to plasminogen group 4, to solvent PBS control group 5 Only.To 2mg/0.2ml/ pcs/day of plasminogen group tail vein injection people source plasminogen, noted to solvent PBS control group tail vein The PBS of same volume is penetrated, is administered 35 days.Mouse was put to death in the 36th day, is cored dirty in 4% paraformaldehyde fixation 24-48 hours, difference It is sunk to the bottom overnight for 4 DEG C in 15%, 30% sucrose, OCT embeddings, 8 μm of frozen section thickness, oil red O stain 15min, 75% alcohol Differentiation 5 seconds, bush uniformly dyeing core 30 seconds, glycerin gelatine mounting.Slice is in 400 times of optical microphotograph Microscopic observations.

The results show that it is considerably less than to plasminogen group mouse (Figure 16 B) ventricle lipidosis (arrow logo) to solvent PBS control group (Figure 16 A).Illustrate that plasminogen can reduce diabetic mice ventricle lipidosis, promote repairing for ventricle damage It is multiple.

15 plasminogen of embodiment improves ApoE atherosclerosis mouse aorta sinus fibrosis

6 week old male ApoE mouse 13 feed high cholesterol diet (Nantong Te Luofei, TP2031) high in fat 16 weeks to lure Lead Atherosclerosis Model^[31,32].Mouse after Cheng Mo before administration three days every 50 μ l of blood is taken to detect T-CHOL (T- CHO) content, and two groups are randomly divided into according to T-CHO contents, to solvent PBS control group 7, to plasminogen group 6.Start Administration is set to the 1st day, to 1mg/0.1mL/ pcs/day of plasminogen group mouse tail vein injection people source plasminogen, to solvent The PBS of PBS control group tail vein injection same volume.Administration 30 days, and mouse was put to death in the 31st day, it cores dirty in 4% poly first Aldehyde fixes 24-48 hours, is sunk to the bottom overnight respectively at 4 DEG C in 15%, 30% sucrose, OCT embeddings, 8 μm of frozen section thickness, with After 0.1% Picro-Sirius red saturation picric acid dyes 30 minutes, flowing water rinses 2min, haematoxylin dyeing 1 minute, and flowing water rinses, 1% hydrochloride alcohol breaks up, and ammonium hydroxide returns indigo plant, and flowing water rinses, neutral gum mounting after drying, in 40 optical microphotograph Microscopic observations, figure 17C, D are respectively Figure 17 A, B black surround regional enlarged drawing pieces.

As a result be shown to plasminogen group (Figure 17 B, 17D) collagen deposition (arrow logo) area be significantly less than to Solvent PBS control group (Figure 17 A, 17C) illustrates that plasminogen can cut down artery congee hardening model mouse aorta sinus fibrosis It is horizontal.

The protective effect that 16 plasminogen of embodiment damages diabetic mice aorta inner wall

24-25 week old db/db male mices 10, experiment are denoted as the 0th day and weigh, divided at random according to weight on the day of starting It it is two groups, to solvent PBS control group and each 5 to plasminogen group.Start within 1st day that (PBS is phosphoric acid to plasminogen or PBS Buffer salt solution (Phosphate Buffer Saline) is the solvent of plasminogen herein), successive administration 31 days.To fibre Lyase original group mouse presses 2mg/0.2ml/ pcs/day of tail vein injection plasminogen, is given in the same manner to solvent PBS control group The PBS of same volume.Mouse was put to death at the 32nd day and aorta is taken to fix 24 hours in 10% neutral formalin fixer. Tissue samples after fixation carry out paraffin embedding after alcohol serial dehydration and dimethylbenzene are transparent.Histotomy thickness is 5 μm, is cut Piece dewax rehydration and use haematoxylin and eosin stains (HE dyeing), 1% hydrochloride alcohol break up after ammonium hydroxide return indigo plant and alcohol gradient take off Water seal piece is sliced in 400 times of optical microphotograph Microscopic observations.

HE coloration results show have foam cells to deposit (arrow logo) to solvent PBS control group vascular wall, middle level Elastic membrane is disorganized, and tube wall is concave-convex uneven (Figure 18 A)；Give plasminogen group middle elastic membrane structure rule, undulate (figure 18B).Show that inject plasminogen has certain repair to the damage of the active inner vessel wall caused by diabetes.

Protective effect of 17 plasminogen of embodiment to diabetic mice myocardial damage

Diabetes generally entail cardiovascular atherosclerotic^[45,46].Cardiovascular atherosclerotic can cause myocardium thin The ischemic injuries of born of the same parents.Cardiac muscle troponin I (cardiac troponin I, CTNI) is the important symbol object of myocardial damage, Serum-concentration is capable of the degree of reflecting myocardium damage^[44].This experiment observes plasminogen to the heart by detecting cardiac muscle troponin I The repair of injury of muscle.

24-25 week old db/db male mices 28, experiment are denoted as the 0th day and weigh, divided at random according to weight on the day of starting It it is two groups, to solvent PBS control group 12, to plasminogen group 16.Start within second day after grouping to plasminogen or PBS simultaneously It is denoted as the 1st day, successive administration 31 days.To plasminogen group mouse by 2mg/0.2ml/ pcs/day of tail vein injection plasminogen, give Solvent PBS control group tail vein injection gives the PBS of same volume.It extracts eyeball within 32nd day and takes blood, centrifuged with 3500r/min 15-20 minutes, and take the concentration mensuration of supernatant detection progress cardiac muscle troponin I.The results show that give plasminogen group myocardium myo The concentration of calcium protein I, which is significantly lower than, gives solvent PBS control group, and statistical discrepancy is extremely significantly (Figure 19).Illustrate plasminogen energy Remarkably promote the reparation of the myocardial damage caused by diabetic mice cardiovascular atherosclerotic.

It is dense that 18 plasminogen of embodiment improves 3% cholesterol hyperlipemia model mice serum high-density lipoprotein cholesterol Degree

9 week old male C57 mouse 16 feed 3% cholesterol high lipid food (Nantong Te Luofei) 4 weeks, induce hyperlipemia Disease^[52,53], this model is set to 3% cholesterol hyperlipemia model.Mouse after Cheng Mo continues to feed the feeding high in fat of 3% cholesterol Material.Three days every mouse take 50 μ L of blood before administration, detect T-CHOL, and divide at random according to total cholesterol concentration and weight It is two groups, every group each 8.Start administration to be denoted as the 1st day, give plasminogen group mouse tail vein injection people source plasminogen 1mg/ It 0.1ml/ pcs/day, to the PBS of solvent PBS control group tail vein injection same volume, is administered 20 days.In mouse fasting in the 10th, 20 day It 16 hours, pricks within the 11st, 21 day orbital venous plexus and takes 50 μ l of blood, centrifugation obtains supernatant, to detect serum high-density LP courage Sterol (HDL-C).By detection kit, (bio-engineering research is built up in Nanjing to high-density lipoprotein cholesterol content herein Institute, article No. A112-1) the method detection.

Testing result is shown, is apparently higher than to plasminogen group mice serum HDL-C concentration and is given solvent PBS control group, and The two HDL-C concentration after being administered 10,20 days has significant difference (Figure 20).Illustrate that plasminogen can improve hyperlipidemia mould The content of type mice serum middle-high density lipoprotein cholesterol improves hyperlipemia in mice blood fat disorder.

19 plasminogen of embodiment reduces the risk that 3% cholesterol hyperlipemia model rat aorta atherosis is formed

9 week old male C57 mouse 16 feed 3% cholesterol high lipid food (Nantong Te Luofei) 4 weeks, induce hyperlipemia Disease [52,53], this model are set to 3% cholesterol hyperlipemia model.Mouse after Cheng Mo continues to feed the feeding high in fat of 3% cholesterol Material.Three days every mouse take 50 μ l of blood before administration, detect T-CHOL (T-CHO), and according to total cholesterol concentration and weight It is randomly divided into two groups, every group each 8.Start administration to be denoted as the 1st day, give plasminogen group mouse tail vein injection people source fibrinolysin Former 1mg/0.1ml/ pcs/day, to the PBS of solvent PBS control group tail vein injection same volume.Mouse starts after administration in the 20th day Fasting, fasting 16 hours prick orbital venous plexus on the 21st day and take 50 μ L of blood, and centrifugation obtains supernatant, and total cholesterol level uses total courage Sterol detection kit (Bioengineering Research Institute, article No. A111-1 are built up in Nanjing) is detected；High-density lipoprotein cholesterol (HDL-C) using high-density lipoprotein cholesterol detection kit, (Bioengineering Research Institute, article No. A112- are built up in Nanjing to content 1) it is detected.

Atherosclerosis index is the overall target for clinically predicting atherosclerosis, it is believed that it is as to hat Clinical meaning is than the T-CHOL of individual event, triglycerides, high-density lipoprotein and low-density in terms of the estimation of worry degree of risk Lipoprotein bigger [54].Atherosclerosis index=(T-CHO-HDL-C)/HDL-C.

Result of calculation is shown, is significantly lower than to plasminogen group rat aorta atherosis index and is given solvent PBS control group, And statistical discrepancy is significantly (Figure 21).Illustrate that plasminogen can reduce the wind that atherosclerosis occurs for hyperlipemia model mouse Danger.

20 plasminogen of embodiment reduces by 3% cholesterol hyperlipemia model mouse heart onset risk

9 week old male C57 mouse 16 feed 3% cholesterol high lipid food (Nantong Te Luofei) 4 weeks, induce hyperlipemia Disease [52,53], this model are set to 3% cholesterol hyperlipemia model.Mouse after Cheng Mo continues to feed the feeding high in fat of 3% cholesterol Material.Three days every mouse take 50 μ l of blood, detection T-CHOL (T-CHO) before administration, and are divided at random according to total cholesterol concentration It is two groups, every group each 8.Start administration to be denoted as the 1st day, give plasminogen group mouse tail vein injection people source plasminogen 1mg/ 0.1ml/ pcs/day, to the PBS of solvent PBS control group tail vein injection same volume.Mouse is fasted after administration in the 20th day, Fasting 16 hours pricks orbital venous plexus on the 21st day and takes 50 μ L of blood, and centrifugation obtains supernatant, and total cholesterol level uses T-CHOL Detection kit (Bioengineering Research Institute, article No. A111-1 are built up in Nanjing) is detected；High-density lipoprotein cholesterol (HDL- C) content is carried out using high-density lipoprotein cholesterol detection kit (Bioengineering Research Institute, article No. A112-1 are built up in Nanjing) Detection.Cardiac risk index=T-CHO/HDL.

Cardiac risk index (cardiac risk index, CRI) to assess blood fat disorder induce heart disease wind Danger [54].

Solvent PBS control group is given the results show that being significantly less than to plasminogen group CRI, and statistical discrepancy extremely significantly (figure 22).Illustrate that plasminogen can effectively reduce the risk that heart disease occurs for hyperlipemia model mouse.

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Sequence table

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

<120>A kind of method for treating coronary atherosclerosis and its complication

<130> PDK03570

<160> 14

<170> PatentIn version 3.5

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

Glu Gly Val Met Arg Asn Asn

785 790

<210> 3

<211> 2433

<212> DNA

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

<400> 3

atggaacata aggaagtggt tcttctactt cttttatttc tgaaatcagg tcaaggagag 60

cctctggatg actatgtgaa tacccagggg gcttcactgt tcagtgtcac taagaagcag 120

ctgggagcag gaagtataga agaatgtgca gcaaaatgtg aggaggacga agaattcacc 180

tgcagggcat tccaatatca cagtaaagag caacaatgtg tgataatggc tgaaaacagg 240

aagtcctcca taatcattag gatgagagat gtagttttat ttgaaaagaa agtgtatctc 300

tcagagtgca agactgggaa tggaaagaac tacagaggga cgatgtccaa aacaaaaaat 360

ggcatcacct gtcaaaaatg gagttccact tctccccaca gacctagatt ctcacctgct 420

acacacccct cagagggact ggaggagaac tactgcagga atccagacaa cgatccgcag 480

gggccctggt gctatactac tgatccagaa aagagatatg actactgcga cattcttgag 540

tgtgaagagg aatgtatgca ttgcagtgga gaaaactatg acggcaaaat ttccaagacc 600

atgtctggac tggaatgcca ggcctgggac tctcagagcc cacacgctca tggatacatt 660

ccttccaaat ttccaaacaa gaacctgaag aagaattact gtcgtaaccc cgatagggag 720

ctgcggcctt ggtgtttcac caccgacccc aacaagcgct gggaactttg tgacatcccc 780

cgctgcacaa cacctccacc atcttctggt cccacctacc agtgtctgaa gggaacaggt 840

gaaaactatc gcgggaatgt ggctgttacc gtgtccgggc acacctgtca gcactggagt 900

gcacagaccc ctcacacaca taacaggaca ccagaaaact tcccctgcaa aaatttggat 960

gaaaactact gccgcaatcc tgacggaaaa agggccccat ggtgccatac aaccaacagc 1020

caagtgcggt gggagtactg taagataccg tcctgtgact cctccccagt atccacggaa 1080

caattggctc ccacagcacc acctgagcta acccctgtgg tccaggactg ctaccatggt 1140

gatggacaga gctaccgagg cacatcctcc accaccacca caggaaagaa gtgtcagtct 1200

tggtcatcta tgacaccaca ccggcaccag aagaccccag aaaactaccc aaatgctggc 1260

ctgacaatga actactgcag gaatccagat gccgataaag gcccctggtg ttttaccaca 1320

gaccccagcg tcaggtggga gtactgcaac ctgaaaaaat gctcaggaac agaagcgagt 1380

gttgtagcac ctccgcctgt tgtcctgctt ccagatgtag agactccttc cgaagaagac 1440

tgtatgtttg ggaatgggaa aggataccga ggcaagaggg cgaccactgt tactgggacg 1500

ccatgccagg actgggctgc ccaggagccc catagacaca gcattttcac tccagagaca 1560

aatccacggg cgggtctgga aaaaaattac tgccgtaacc ctgatggtga tgtaggtggt 1620

ccctggtgct acacgacaaa tccaagaaaa ctttacgact actgtgatgt ccctcagtgt 1680

gcggcccctt catttgattg tgggaagcct caagtggagc cgaagaaatg tcctggaagg 1740

gttgtagggg ggtgtgtggc ccacccacat tcctggccct ggcaagtcag tcttagaaca 1800

aggtttggaa tgcacttctg tggaggcacc ttgatatccc cagagtgggt gttgactgct 1860

gcccactgct tggagaagtc cccaaggcct tcatcctaca aggtcatcct gggtgcacac 1920

caagaagtga atctcgaacc gcatgttcag gaaatagaag tgtctaggct gttcttggag 1980

cccacacgaa aagatattgc cttgctaaag ctaagcagtc ctgccgtcat cactgacaaa 2040

gtaatcccag cttgtctgcc atccccaaat tatgtggtcg ctgaccggac cgaatgtttc 2100

atcactggct ggggagaaac ccaaggtact tttggagctg gccttctcaa ggaagcccag 2160

ctccctgtga ttgagaataa agtgtgcaat cgctatgagt ttctgaatgg aagagtccaa 2220

tccaccgaac tctgtgctgg gcatttggcc ggaggcactg acagttgcca gggtgacagt 2280

ggaggtcctc tggtttgctt cgagaaggac aaatacattt tacaaggagt cacttcttgg 2340

ggtcttggct gtgcacgccc caataagcct ggtgtctatg ttcgtgtttc aaggtttgtt 2400

acttggattg agggagtgat gagaaataat taa 2433

<210> 4

<211> 810

<212> PRT

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

<400> 4

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

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

785 790 795 800

Thr Trp Ile Glu Gly Val Met Arg Asn Asn

805 810

<210> 5

<211> 2145

<212> DNA

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

<400> 5

aaagtgtatc tctcagagtg caagactggg aatggaaaga actacagagg gacgatgtcc 60

aaaacaaaaa atggcatcac ctgtcaaaaa tggagttcca cttctcccca cagacctaga 120

ttctcacctg ctacacaccc ctcagaggga ctggaggaga actactgcag gaatccagac 180

aacgatccgc aggggccctg gtgctatact actgatccag aaaagagata tgactactgc 240

gacattcttg agtgtgaaga ggaatgtatg cattgcagtg gagaaaacta tgacggcaaa 300

atttccaaga ccatgtctgg actggaatgc caggcctggg actctcagag cccacacgct 360

catggataca ttccttccaa atttccaaac aagaacctga agaagaatta ctgtcgtaac 420

cccgataggg agctgcggcc ttggtgtttc accaccgacc ccaacaagcg ctgggaactt 480

tgtgacatcc cccgctgcac aacacctcca ccatcttctg gtcccaccta ccagtgtctg 540

aagggaacag gtgaaaacta tcgcgggaat gtggctgtta ccgtgtccgg gcacacctgt 600

cagcactgga gtgcacagac ccctcacaca cataacagga caccagaaaa cttcccctgc 660

aaaaatttgg atgaaaacta ctgccgcaat cctgacggaa aaagggcccc atggtgccat 720

acaaccaaca gccaagtgcg gtgggagtac tgtaagatac cgtcctgtga ctcctcccca 780

gtatccacgg aacaattggc tcccacagca ccacctgagc taacccctgt ggtccaggac 840

tgctaccatg gtgatggaca gagctaccga ggcacatcct ccaccaccac cacaggaaag 900

aagtgtcagt cttggtcatc tatgacacca caccggcacc agaagacccc agaaaactac 960

ccaaatgctg gcctgacaat gaactactgc aggaatccag atgccgataa aggcccctgg 1020

tgttttacca cagaccccag cgtcaggtgg gagtactgca acctgaaaaa atgctcagga 1080

acagaagcga gtgttgtagc acctccgcct gttgtcctgc ttccagatgt agagactcct 1140

tccgaagaag actgtatgtt tgggaatggg aaaggatacc gaggcaagag ggcgaccact 1200

gttactggga cgccatgcca ggactgggct gcccaggagc cccatagaca cagcattttc 1260

actccagaga caaatccacg ggcgggtctg gaaaaaaatt actgccgtaa ccctgatggt 1320

gatgtaggtg gtccctggtg ctacacgaca aatccaagaa aactttacga ctactgtgat 1380

gtccctcagt gtgcggcccc ttcatttgat tgtgggaagc ctcaagtgga gccgaagaaa 1440

tgtcctggaa gggttgtagg ggggtgtgtg gcccacccac attcctggcc ctggcaagtc 1500

agtcttagaa caaggtttgg aatgcacttc tgtggaggca ccttgatatc cccagagtgg 1560

gtgttgactg ctgcccactg cttggagaag tccccaaggc cttcatccta caaggtcatc 1620

ctgggtgcac accaagaagt gaatctcgaa ccgcatgttc aggaaataga agtgtctagg 1680

ctgttcttgg agcccacacg aaaagatatt gccttgctaa agctaagcag tcctgccgtc 1740

atcactgaca aagtaatccc agcttgtctg ccatccccaa attatgtggt cgctgaccgg 1800

accgaatgtt tcatcactgg ctggggagaa acccaaggta cttttggagc tggccttctc 1860

aaggaagccc agctccctgt gattgagaat aaagtgtgca atcgctatga gtttctgaat 1920

ggaagagtcc aatccaccga actctgtgct gggcatttgg ccggaggcac tgacagttgc 1980

cagggtgaca gtggaggtcc tctggtttgc ttcgagaagg acaaatacat tttacaagga 2040

gtcacttctt ggggtcttgg ctgtgcacgc cccaataagc ctggtgtcta tgttcgtgtt 2100

tcaaggtttg ttacttggat tgagggagtg atgagaaata attaa 2145

<210> 6

<211> 714

<212> PRT

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

<400> 6

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

Thr Trp Ile Glu Gly Val Met Arg Asn Asn

705 710

<210> 7

<211> 1245

<212> DNA

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

<400> 7

gagcctctgg atgactatgt gaatacccag ggggcttcac tgttcagtgt cactaagaag 60

cagctgggag caggaagtat agaagaatgt gcagcaaaat gtgaggagga cgaagaattc 120

acctgcaggg cattccaata tcacagtaaa gagcaacaat gtgtgataat ggctgaaaac 180

aggaagtcct ccataatcat taggatgaga gatgtagttt tatttgaaaa gaaagtgtat 240

ctctcagagt gcaagactgg gaatggaaag aactacagag ggacgatgtc caaaacaaaa 300

aatggcatca cctgtcaaaa atggagttcc acttctcccc acagacctag attctcacct 360

gctacacacc cctcagaggg actggaggag aactactgca ggaatccaga caacgatccg 420

caggggccct ggtgctatac tactgatcca gaaaagagat atgactactg cgacattctt 480

gagtgtgaag aggcggcccc ttcatttgat tgtgggaagc ctcaagtgga gccgaagaaa 540

tgtcctggaa gggttgtagg ggggtgtgtg gcccacccac attcctggcc ctggcaagtc 600

agtcttagaa caaggtttgg aatgcacttc tgtggaggca ccttgatatc cccagagtgg 660

gtgttgactg ctgcccactg cttggagaag tccccaaggc cttcatccta caaggtcatc 720

ctgggtgcac accaagaagt gaatctcgaa ccgcatgttc aggaaataga agtgtctagg 780

ctgttcttgg agcccacacg aaaagatatt gccttgctaa agctaagcag tcctgccgtc 840

atcactgaca aagtaatccc agcttgtctg ccatccccaa attatgtggt cgctgaccgg 900

accgaatgtt tcatcactgg ctggggagaa acccaaggta cttttggagc tggccttctc 960

aaggaagccc agctccctgt gattgagaat aaagtgtgca atcgctatga gtttctgaat 1020

ggaagagtcc aatccaccga actctgtgct gggcatttgg ccggaggcac tgacagttgc 1080

cagggtgaca gtggaggtcc tctggtttgc ttcgagaagg acaaatacat tttacaagga 1140

gtcacttctt ggggtcttgg ctgtgcacgc cccaataagc ctggtgtcta tgttcgtgtt 1200

tcaaggtttg ttacttggat tgagggagtg atgagaaata attaa 1245

<210> 8

<211> 414

<212> PRT

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

<400> 8

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410

<210> 9

<211> 1104

<212> DNA

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

<400> 9

gtcaggtggg agtactgcaa cctgaaaaaa tgctcaggaa cagaagcgag tgttgtagca 60

cctccgcctg ttgtcctgct tccagatgta gagactcctt ccgaagaaga ctgtatgttt 120

gggaatggga aaggataccg aggcaagagg gcgaccactg ttactgggac gccatgccag 180

gactgggctg cccaggagcc ccatagacac agcattttca ctccagagac aaatccacgg 240

gcgggtctgg aaaaaaatta ctgccgtaac cctgatggtg atgtaggtgg tccctggtgc 300

tacacgacaa atccaagaaa actttacgac tactgtgatg tccctcagtg tgcggcccct 360

tcatttgatt gtgggaagcc tcaagtggag ccgaagaaat gtcctggaag ggttgtaggg 420

gggtgtgtgg cccacccaca ttcctggccc tggcaagtca gtcttagaac aaggtttgga 480

atgcacttct gtggaggcac cttgatatcc ccagagtggg tgttgactgc tgcccactgc 540

ttggagaagt ccccaaggcc ttcatcctac aaggtcatcc tgggtgcaca ccaagaagtg 600

aatctcgaac cgcatgttca ggaaatagaa gtgtctaggc tgttcttgga gcccacacga 660

aaagatattg ccttgctaaa gctaagcagt cctgccgtca tcactgacaa agtaatccca 720

gcttgtctgc catccccaaa ttatgtggtc gctgaccgga ccgaatgttt catcactggc 780

tggggagaaa cccaaggtac ttttggagct ggccttctca aggaagccca gctccctgtg 840

attgagaata aagtgtgcaa tcgctatgag tttctgaatg gaagagtcca atccaccgaa 900

ctctgtgctg ggcatttggc cggaggcact gacagttgcc agggtgacag tggaggtcct 960

ctggtttgct tcgagaagga caaatacatt ttacaaggag tcacttcttg gggtcttggc 1020

tgtgcacgcc ccaataagcc tggtgtctat gttcgtgttt caaggtttgt tacttggatt 1080

gagggagtga tgagaaataa ttaa 1104

<210> 10

<211> 367

<212> PRT

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

<400> 10

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

<210> 11

<211> 750

<212> DNA

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

<400> 11

gccccttcat ttgattgtgg gaagcctcaa gtggagccga agaaatgtcc tggaagggtt 60

gtaggggggt gtgtggccca cccacattcc tggccctggc aagtcagtct tagaacaagg 120

tttggaatgc acttctgtgg aggcaccttg atatccccag agtgggtgtt gactgctgcc 180

cactgcttgg agaagtcccc aaggccttca tcctacaagg tcatcctggg tgcacaccaa 240

gaagtgaatc tcgaaccgca tgttcaggaa atagaagtgt ctaggctgtt cttggagccc 300

acacgaaaag atattgcctt gctaaagcta agcagtcctg ccgtcatcac tgacaaagta 360

atcccagctt gtctgccatc cccaaattat gtggtcgctg accggaccga atgtttcatc 420

actggctggg gagaaaccca aggtactttt ggagctggcc ttctcaagga agcccagctc 480

cctgtgattg agaataaagt gtgcaatcgc tatgagtttc tgaatggaag agtccaatcc 540

accgaactct gtgctgggca tttggccgga ggcactgaca gttgccaggg tgacagtgga 600

ggtcctctgg tttgcttcga gaaggacaaa tacattttac aaggagtcac ttcttggggt 660

cttggctgtg cacgccccaa taagcctggt gtctatgttc gtgtttcaag gtttgttact 720

tggattgagg gagtgatgag aaataattaa 750

<210> 12

<211> 249

<212> PRT

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

<400> 12

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Trp Ile Glu Gly Val Met Arg Asn Asn

245

<210> 13

<211> 684

<212> DNA

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

<400> 13

gttgtagggg ggtgtgtggc ccacccacat tcctggccct ggcaagtcag tcttagaaca 60

aggtttggaa tgcacttctg tggaggcacc ttgatatccc cagagtgggt gttgactgct 120

gcccactgct tggagaagtc cccaaggcct tcatcctaca aggtcatcct gggtgcacac 180

caagaagtga atctcgaacc gcatgttcag gaaatagaag tgtctaggct gttcttggag 240

cccacacgaa aagatattgc cttgctaaag ctaagcagtc ctgccgtcat cactgacaaa 300

gtaatcccag cttgtctgcc atccccaaat tatgtggtcg ctgaccggac cgaatgtttc 360

atcactggct ggggagaaac ccaaggtact tttggagctg gccttctcaa ggaagcccag 420

ctccctgtga ttgagaataa agtgtgcaat cgctatgagt ttctgaatgg aagagtccaa 480

tccaccgaac tctgtgctgg gcatttggcc ggaggcactg acagttgcca gggtgacagt 540

ggaggtcctc tggtttgctt cgagaaggac aaatacattt tacaaggagt cacttcttgg 600

ggtcttggct gtgcacgccc caataagcct ggtgtctatg ttcgtgtttc aaggtttgtt 660

acttggattg agggagtgat gaga 684

<210> 14

<211> 228

<212> PRT

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

<400> 14

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Gly Val Met Arg

225

Claims

1. a kind of method for preventing and/or treating subject's coronary atherosclerosis and its associated disease, including administration Subject prevent and/or therapeutically effective amount plasminogen, wherein the subject suffer from, suspects with coronary artery it is athero- firmly Change and its associated disease have the risk for suffering from coronary atherosclerosis and its associated disease.

2. the method for claim 1 wherein the coronary atherosclerosis associated disease is included since coronary artery is athero- hard Change the coronary heart diseases and angina pectoris caused, myocardial infarction, arrhythmia cordis, heart failure.

3. the method for claims 1 or 2, wherein the atherosclerosis is and diabetes complicated atherosclerosis.

4. the method for any one of claim 1-3, wherein the plasminogen by one or more preventions chosen from the followings and/ Or treatment atherosclerosis；Experimenter's serum total cholesterol level is reduced, experimenter's serum triglyceride levels is reduced, reduces Experimenter's serum low-density lipoprotein white level, raising experimenter's serum hdl level.

5. the method for any one of claim 1-4, wherein the plasminogen is sunk by reducing the lipid of subject artery's tube wall Product prevention and/or treatment coronary atherosclerosis.

6. the method for any one of claim 1-4, wherein the plasminogen by one or more preventions chosen from the followings and/ Or treatment coronary atherosclerosis：Subject's liver is transported and is reduced in the fat metabolism for promoting liver, the fat for promoting liver Fat deposition.

It is 7. a kind of for preventing and/or treat tissue of the subject caused by coronary atherosclerosis, organ ischemia's damage And its method of associated disease, including applying a effective amount of plasminogen to subject.

8. the method for claim 7, wherein the tissue of the subject, organ ischemia's damage cause for coronary atherosclerosis Myocardial damage.

9. the method for claim 7 or 8, wherein the associated disease is coronary heart diseases and angina pectoris, the heart caused by cardiac insufficiency Flesh infarct, arrhythmia cordis or heart failure.

10. the method for any one of claim 7-9, wherein the tissue, organ ischemia's damage or associated disease are cerebral ischemia Damage or its associated disease.