CN108210917A

CN108210917A - A kind of method and drug for preventing and treating obesity

Info

Publication number: CN108210917A
Application number: CN201711350913.6A
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: 2017-12-15
Publication date: 2018-06-29
Also published as: HK1257231A1

Abstract

The present invention relates to a kind of methods prevented and/or treat overweight/obesity and its associated disease, and fat and its associated disease a effective amount of plasminogen of subject is susceptible or suffering from including administration, to cut down exception/excess deposition of the fat in parts of body.The invention further relates to the fat drug of prevention and/or treatment and its purposes in medicine preparation.

Description

A kind of method and drug for preventing and treating obesity

Technical field

The present invention relates to a kind of methods and drug prevented and/or treat obesity and its associated disease.

Background technology

Obesity (obesity) refers to that body fat accumulates excessive and/or abnormal distribution.According to determining for the World Health Organization Justice is overweight and fat refer to may unhealthful body fat be excessive and/or abnormal accumulation.Early in 1948, WHO will be fat Be defined as a kind of disease, and increase to International Classification of Diseases (International classification of disease, ICD in).In June, 2013, association of American Medical Association (American Medical Association, AMA) is in its history It is primary to proclaim that fat is a kind of disease, medical intervention measure is needed to prevent and treat^[1].It is overweight and fat while be more The risk factor of kind of disease, including cardiovascular and cerebrovascular disease (heart disease, hypertension, dyslipidemia, cerebral apoplexy), 2 patients with type Ⅰ DM, Musculoskeletal disease (osteoarthritis etc.), disease of digestive system (gallbladder disease), sleep apnea or respiratory disorder and certain Cancer (carcinoma of endometrium, breast cancer, colon cancer) etc.^[2].The data of WHO show that 2014, the whole world was shared more than 1,900,000,000 adults It is overweight, wherein being obesity more than 600,000,000 people^[2].China's overweight rate of adult is 31.5%, obesity rates 12.2%^[3].It is overweight and fat Having become influences the important illness of residents ' health.

Clinically, mainly reflect body fat indirectly by measuring body surface.Common measurement index has constitution Index (body mass index, BMI) and waistline (waist circumference, WC).BMI is that diagnosis obesity is most heavy The index wanted, waistline can reflect the accumulation degree of stomach fat.At present the criteria for classification of the World Health Organization with BMI >= 25kg·m ²To be overweight, the kgm of BMI >=30²For obesity.What this classification was mainly formulated according to the white data in Europe, and In the case where BMI is identical, the ratio of Asia body fat mass in human body is higher than westerner, and abdominal obesity is more serious^[4]。

By studying Chinese fat related data, 2003《Chinese Adult is overweight and obesity prevention and control refer to Southern (on probation)》It proposes with BMI >=24kgm²The boundary overweight for Chinese Adult, BMI >=28kgm²For fat boundary； 2011《Chinese Adult obesity prevents Consensus of experts》It is abdominal obesity to propose male's waistline >=90cm, women waistline >=85cm Criterion.

The treatment of obesity is broadly divided into lifestyle modification, drug therapy and surgical operation therapy.Evidence-based medicine EBM at present It is first-line treatment scheme that evidence, which recommends lifestyle modification,.《Chinese Adult is overweight and obesity prevention and control guide (on probation)》Refer to Go out, when lifestyle modification is invalid, i.e., cannot make weight loss 5%, when BMI indexes are still greater than 28, it is recommended to carry out drug and is controlled It treats.It is overweight and with a kind of patient of complication (angiocardiopathy, hypertension, diabetes B etc.), it is invalid through Spinal injury, Also it is recommended to carry out drug therapy.

Slimming medicine can be divided into：CNS inhibition appetite drug, appetite-suppressing gut myoelectric activity, act on periphery interference battalion Support material absorbing and the metabolism drug that has additional nutrients.There are two stopped the big long-acting slimming drugs of CNS inhibition appetite drug three With：Rimonabant (rimonabant) and sibutramine (sibutramine) are respectively because that can increase spirit and angiocardiopathy wind It is dangerous and deactivated by EMEA requirements in October, 2008 year and in January, 2010^[5-6], after extensive use once causes it to halt There is blank in slimming drugs market.In recent years effect of the gastrointestinal hormone in terms of modulation of appetite, control blood glucose has become research hot topic, this The zoopery of a little drug moieties plays the role of obvious appetite-suppressing, lowers weight, but its safety and use are effective Dosage also needs to the research of many further clinical tests.The drug for acting on periphery is directed to two aspect target spots：1. it acts on Gastrointestinal tract reduces fat absorption lipase inhibitor and sodium-glucose co-transporters body 2 (SGLT2) inhibitor；2. act on fat Tissue reduces Fatty synthesis, promotes fat splitting：Mainly there is the enzyme on lipid metaboli access.Lipase inhibitor is by inhibiting stomach The lipase of enteron aisle and pancreas, the absorption for the middle fat that cuts down one's diet.Orlistat (Orlistat) for the with obvious effects of weight-reducing, Side effect is smaller, but U.S.'s report has 13 users that severe liver injury has occurred, and nearest FDA decisions urge its manufacturer more New product specification^[7]。

Carry out should at least monthly assessing the validity of drug and safety for first 3 months for drug therapy, later Also it should assess 1 time within every 3 months^[8].The safety of slimming medicine is the main reason for limiting its extensive use, while drug Curative effect on obesity is also limited.Therefore, drug therapy is still the supplementary means of bariatrician at present, in the help of medical practitioner Under the mode of making the life better be only and treat overweight and fat prefered method.Obesity is a kind of chronic disease, and no drug can generate Permanent curative effect on obesity, only Long-term taking medicine could keep weight.The overweight and obese patient persistently risen in face of quantity finds More safe and effective slimming medicine is the urgent hope of people.

Summary of the invention

The present invention relates to following items：

1. a kind of method prevented or treat subject's obesity, including a effective amount of plasminogen of subject is administered.

2. 1 method, wherein the plasminogen by reduce lipid around histoorgan, histoorgan and/or Exception or over-deposit the treatment obesity in abdominal cavity.

3. 2 method, wherein the plasminogen reduce lipid subcutaneous, heart, liver, lungs, kidney, blood vessel, Mesenterium, peritonaeum, body cavity, exception or over-deposit around organ.

4. 1 method, wherein the plasminogen reduces subject's blood fat, especially triglycerides and low-density lipoprotein White horizontal stretcher obesity.

5. the method for any one of 1-4, wherein the obesity is for simple obesity or secondary to caused by Other diseases It is fat.

6. a kind of method prevented or treat subject's obesity, including a effective amount of plasminogen of subject is administered, Described in obesity secondary to endocrine disturbance disease, glycometabolism disease, liver diseases, kidney trouble, angiocardiopathy, enteron aisle Disease, thyroid disease, gall-bladder or disease of biliary tract, excessive consumption of alcohol, drug effect.

7. a kind of fat method prevented and/or treatment subject disease is concurrent, a effective amount of including administration subject Plasminogen, wherein the concurrent obesity of the disease include the concurrent obesity of the concurrent obesity of endocrine system disease, metabolic disease, The concurrent obesity of the concurrent obesity of the concurrent obesity of angiocardiopathy, disease of digestive system, degenerative disease.

8. 7 method, fat include the concurrent obesity of diabetes complicated obesity, hypertension, artery congee wherein described Sample hardens the concurrent obesity of the concurrent obesity of concurrent obesity, liver diseases, osteoporosis.

9. a kind of method of complication caused by prevention or treatment are fat, including a effective amount of fibrinolytic of subject is administered Proenzyme, wherein the complication of the obesity includes cardiovascular and cerebrovascular disease, metabolic disease, musculoskeletal disease, digestive system disease Disease, sleep apnea, respiratory disorder.

10. 9 method, wherein the complication is hypertension, diabetes, coronary heart diseases and angina pectoris, myocardial infarction, the heart Restrain not normal, atherosclerosis, cerebral thrombus, cerebral hemorrhage, osteoarthritis, osteoproliferation, cholecystitis, fatty liver, hepatic sclerosis.

11. a kind of method for reducing subject artery's atherosis onset risk, including a effective amount of fibre of subject is administered Lyase is former.

12. 11 method, sent out wherein the plasminogen reduces subject artery's atherosis by treating obesity Sick risk.

13. a kind of method for reducing subject's obesity onset risk, including a effective amount of plasminogen of subject is administered To reduce exception or over-deposit of the fat around histoorgan or its, in subcutaneous or abdominal cavity.

16. purposes of the plasminogen in the drug for preparing prevention or treatment subject's obesity.

17. 16 purposes, wherein the plasminogen passes through in one or more abatement subject's bodies chosen from the followings The fat of exception or over-deposit：

1) lipid is reduced in the deposition selected from one or more following positions：Subcutaneously, heart, liver, lungs, kidney, blood Pipe, mesenterium, peritonaeum, body cavity, exception or over-deposit around organ,

2) promote liver fat removing and

3) removing of lipid in blood is promoted to reduce subject's heart disease risk.

18. a kind of method for reducing subject's blood fat, including a effective amount of plasminogen of subject is administered.

19. 18 method, wherein the plasminogen reduces serum triglyceride level, low-density lipoprotein white level.

20. a kind of method for reducing subject artery's atherosis or heart disease risk, has including administration subject The plasminogen of effect amount mitigates exception or over-deposit of the lipid in vascular wall.

21. a kind of method for treating subject's obesity promotes liver including a effective amount of plasminogen of administration subject Removing to deposition fat.

22. a kind of method for treating subject's obesity, including a effective amount of plasminogen of subject is administered, wherein described Plasminogen passes through one or more abatement subject's body fats chosen from the followings：

2) promote liver fat removing and

3) promote the removing of lipid in blood.

23. 22 method, wherein the obesity is for simple obesity or secondary to fat caused by Other diseases Disease.

24. 23 method, wherein the obesity secondary to endocrine disturbance disease, glycometabolism disease, liver diseases, Kidney trouble, angiocardiopathy, intestines problem, thyroid disease, gall-bladder or disease of biliary tract, excessive consumption of alcohol, drug effect.

25. according to the method for any one of item 1-24, wherein the plasminogen can be with one or more other medicines or controlling Treatment means are administered in combination.

26. 25 method, wherein one or more other medicines include hypertension therapeutic drug, treating diabetes With drug, treatment of atherosclerosis drug, chronic glomerulonephritis medicine, chronic pyelonephritis medicine, kidney Sick syndrome drug, renal insufficiency treatment drug, uremia therapy drug, kidney transplantation treatment drug, fat Fat liver treatment drug, cirrhosis treatment drug, bariatrician drug.

27. according to the method for item 26, wherein the other medicines include：Blood lipid-lowering medicine, antiplatelet drug, blood pressure lowering Drug, vasodilator drug, hypoglycemic drug, anticoagulation medicine, thrombolytic agent, hepatic, antiarrhythmic drug, by force Psychological treatment object, diuretic, anti-infectives, antiviral drugs, immunoregulation medicament, inflammation adjust class drug, antitumor drug, Hormone medicine, thyroxine.

28. 27 method, wherein the drug includes blood lipid-lowering medicine：Statins；Fibrates；Niacin；Cholestyramine；Peace It is appropriate bright；Unrighted acid such as yishouning, lipid level and heart arteries and veins pleasure；Sodium alginate；Antiplatelet drug：Aspirin；Pan Centime；Clopidogrel；Xi Luota；Vasodilator drug：Hydralazine；Nitroglycerin and isosorbide dinitrate；Sodium nitroprussiate；α nitre receptor hinders Disconnected agent such as prazosin；Alpha receptor blocking agent such as phentolamine；β draws receptor agonist such as albuterol；Captopril, enalapril；The heart Pain is fixed, thiazolone；Willow Gamma Amino Butyric Acid, minoxidil, prostaglandin, atrial natriuretic peptide；Thrombolytic agent：Urokinase and streptokinase；Tissue Type plasminogen activator；Single-chain Urokinase-type Plasminogen Activator；TNK- tissue-type plasminogen activators；Anticoagulant Object：Heparin；Enoxaparin；Nagqu heparin；Bivalirudin.

29. the method for any one of 1-28, wherein the plasminogen has at least with sequence 2,6,8,10 or 12 75%th, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity, and still there is fibrinolysin Former activity.

30. the method for any one of 1-29, the plasminogen is on the basis of sequence 2,6,8,10 or 12, add, It deletes and/or replaces 1-100,1-90,1-80,1-70,1-60,1-50,1- 45,1-40,1-35,1-30,1-25,1-20,1- 15th, 1-10,1-5,1-4,1-3,1-2,1 amino acid, and still there is the protein of activities of endothelial tissue plasminogen.

31. the method for any one of 1-30, the plasminogen is comprising activities of endothelial tissue plasminogen segment and still has The protein of activities of endothelial tissue plasminogen.

32. the method for any one of 1-31, the plasminogen is selected from Glu- plasminogens, Lys- plasminogens, small fibre Lyase original, Microplasminogen, delta- plasminogens or their variant for retaining activities of endothelial tissue plasminogen.

33. the method for any one of 1-32, the plasminogen for natural or synthetic human plasminogen or its still protect Stay the variant or segment of activities of endothelial tissue plasminogen.

34. the method for any one of 1-33, the plasminogen is from the people of primate or rodent fibre Lyase original directly to homologue or its still retain the variant of activities of endothelial tissue plasminogen or segment.

35. the method for any one of 1-34, the amino acid of the plasminogen is as shown in sequence 2,6,8,10 or 12.

36. the method for any one of 1-35, wherein the plasminogen is naive plasminogen.

37. the method for any one of 1-36, wherein the subject is people.

38. the method for any one of 1-37, wherein the subject lacks or missing plasminogen.

39. 38 method, wherein the shortage or missing are inborn, secondary and/or local.

40. a kind of plasminogen of method for any one of item 1-39.

41. a kind of pharmaceutical composition, it includes pharmaceutically acceptable supporting agent and for any one of the item 1-39 sides The plasminogen of method.

42. a kind of preventative or therapeutic agent box, it includes：(i) for the fibre of any one of item 1-39 the methods Lyase original and (ii) are for delivering the plasminogen to the component (means) of the subject.

43. according to the kit described in item 42, wherein the component is syringe or bottle.

44. 42 or 43 kit, also comprising label or operation instructions, the label or operation instructions instruction will The plasminogen administers the subject with any one of practical matter 1-39 the methods.

45. a kind of product, it includes：

Container containing label；With

Include the pharmaceutical composition of (i) for the plasminogen of any one of item 1-39 the methods or comprising plasminogen Object, wherein the plasminogen or composition are administered the subject with any one of practical matter 1-39 institutes by label instruction State method.

46. the kit of any one of 42-44 or the product of item 45, also comprising other one or more components or appearance Device contains other drugs in the component or container.

47. 46 kit or product, wherein the other drugs are selected from the group：Blood lipid-lowering medicine, antiplatelet drug Object, blood-pressure drug, vasodilator drug, hypoglycemic drug, anticoagulation medicine, thrombolytic agent, hepatic, the anti-rhythm of the heart lose Normal drug, cardiac drug, diuretic, anti-infectives, antiviral drugs, immunoregulation medicament, inflammation adjust class drug, resist Tumour medicine, hormone medicine, thyroxine.

48. include the slimming drugs of plasminogen.

49. include the diet products of plasminogen.

50. plasminogen is used to lower the purposes of weight.

Drug, pharmaceutical composition, product, the kit used in the above method is prepared the invention further relates to plasminogen In purposes.

On the one hand, the present invention relates to the method prevented and/or treat subject's obesity and its associated disease, including administration Subject prevents and/or the plasminogen of therapeutically effective amount.The invention further relates to plasminogens for preventing and/or treating tested The purposes of person's obesity and its associated disease.The invention further relates to plasminogens to prepare for preventing and/or treat subject Purposes in the drug of obesity and its associated disease, pharmaceutical composition, product, kit.Further, the invention further relates to For preventing and/or treating the plasminogen of subject's obesity and its associated disease.The invention further relates to for prevent and/ Or the drug comprising plasminogen, pharmaceutical composition, product, the kit for the treatment of subject's obesity and its associated disease.

In some embodiments, the obesity is obesity caused by excessive diet.In some embodiments, institute Obesity is stated as secondary obesity, such as secondary to fat metabolic disturbance disease, such as endocrine disturbance disease, glycometabolism disease Disease, liver diseases, kidney trouble, angiocardiopathy, intestines problem, thyroid disease, gall-bladder or disease of biliary tract are drunk, drug Treatment causes or adjoint fat metabolic disturbance.In some embodiments, the fat metabolic disturbance is hypertension, glycosuria Disease, chronic hepatitis, hepatic sclerosis, injury of kidney, chronic glomerulonephritis, chronic pyelonephritis, nephrotic syndrome, renal insufficiency, Kidney transplant, uremia, hypothyroidism, obstructive cholecystitis, obstructive cholangitis, drug or hormone therapy initiation or companion With fat metabolic disturbance.In some embodiments, the fat metabolic disturbance is hyperlipidemia, hyperlipoprotememia, fat Fat liver, atherosclerosis, obesity, internal organs fat deposition.

In yet another aspect, the present invention relates to prevent and/or cut down subject's fat in bodily tissue organs abnormality or mistake The method of deposition is spent, including a effective amount of plasminogen of subject is administered.The invention further relates to plasminogen for prevent and/or Cut down subject's fat in bodily tissue organs abnormality or the purposes of over-deposit.The invention further relates to plasminogens to prepare use In prevent and/or abatement subject's fat bodily tissue organs abnormality or the drug of over-deposit, pharmaceutical composition, product, Purposes in kit.Further, the invention further relates to for preventing and/or cut down subject's fat in bodily tissue organ The plasminogen of exception or over-deposit.The invention further relates to for preventing and/or cut down subject's fat in bodily tissue device The drug comprising plasminogen, pharmaceutical composition, product, the kit of official's exception or over-deposit.

In yet another aspect, the present invention relates to prevent and/or treat subject's fat in bodily tissue organs abnormality or mistake The method of illness caused by degree deposition, including a effective amount of plasminogen of subject is administered.The invention further relates to plasminogen use In the purposes for preventing and/or treating subject's fat illness caused by bodily tissue organs abnormality or over-deposit.The present invention Plasminogen is further related to prepare for preventing and/or treat subject's fat in bodily tissue organs abnormality or over-deposit Purposes in the drug of caused illness, pharmaceutical composition, product, kit.Further, the invention further relates to for preventing And/or the medicine for including plasminogen for the treatment of subject's fat illness caused by bodily tissue organs abnormality or over-deposit Object, pharmaceutical composition, product, kit.

In some embodiments, the fat refers to fat in blood in bodily tissue organs abnormality or over-deposit, Subcutaneous tissue, vascular wall, internal organs exception or over-deposit.In some embodiments, the fat is in bodily tissue Illness caused by organs abnormality or over-deposit includes obesity, and hyperlipidemia, hyperlipoprotememia, fatty liver, artery are athero- Hardening, lipid heart damage, lipid renal damage, lipid pancreas islet damage.

In yet another aspect, the present invention relates to the sides of obesity caused by preventing and/or treating subject's fat metabolic disturbance Method, including a effective amount of plasminogen of subject is administered.The invention further relates to plasminogens for preventing and/or treating subject The purposes of obesity caused by fat metabolic disturbance.It is tested for preventing and/or treating in preparation the invention further relates to plasminogen Purposes in the drug of obesity caused by person's fat metabolic disturbance, pharmaceutical composition, product, kit.Further, it is of the invention It further relates to for the plasminogen of obesity caused by preventing and/or treating subject's fat metabolic disturbance.The invention further relates to The drug comprising plasminogen of obesity, pharmaceutical composition, system caused by preventing and/or treating subject's fat metabolic disturbance Product, kit.In some embodiments, the illness includes obesity, and hyperlipidemia, fatty liver, is moved hyperlipoprotememia Pulse atherosclerosis, lipid damage to cardiac tissue, lipid injury of kidney.

In yet another aspect, the present invention relates to treat the side of subject's disease by reducing fatty exception or over-deposit Method, including a effective amount of plasminogen of subject is administered.The invention further relates to plasminogen be used for by reduce fat it is abnormal or Over-deposit treats the purposes of subject's disease.The invention further relates to plasminogen prepare be used for by reduce fat it is abnormal or Purposes in the over-deposit treatment drug of subject's disease, pharmaceutical composition, product, kit.Further, the present invention is gone back It is related to treating the plasminogen of subject's disease by reducing fatty exception or over-deposit.The invention further relates to by subtracting The drug comprising plasminogen, pharmaceutical composition, product, the reagent of few fat exception or over-deposit treatment subject's disease Box.

In some embodiments, the disease includes atherosclerosis, coronary heart diseases and angina pectoris, myocardial infarction, the rhythm of the heart Not normal, fatty liver, hepatic sclerosis, cerebral ischemia, cerebral infarction, renal insufficiency, nephrotic syndrome, renal insufficiency, obesity.

In yet another aspect, the present invention relates to the method prevented and/or treat the lipid damage of subject's histoorgan, packets Include administration a effective amount of plasminogen of subject.The invention further relates to plasminogens for preventing and/or treating subject's tissue The purposes of the lipid damage of organ.The invention further relates to plasminogens to prepare for preventing and/or treat subject's organizer Purposes in the drug of the lipid damage of official, pharmaceutical composition, product, kit.Further, the invention further relates to for pre- Anti- and/or the treatment lipid damage of subject's histoorgan plasminogen.The invention further relates to for prevent and/or treat by The drug comprising plasminogen, pharmaceutical composition, product, the kit of the lipid damage of examination person's histoorgan.

In some embodiments, the histoorgan includes ductus arteriosus wall, heart, liver, kidney, pancreas.

In yet another aspect, the present invention relates to the method for improving subject's hyperlipidemia, including subject's effective quantity is administered Plasminogen.It is used to improve the purposes of subject's hyperlipidemia the invention further relates to plasminogen.The invention further relates to fibrinolytics Proenzyme is preparing the purposes for improving in the drug of subject's hyperlipidemia, pharmaceutical composition, product, kit.Further Ground, the invention further relates to for improving the plasminogen of subject's hyperlipidemia.The invention further relates to for improving subject's height The drug comprising plasminogen, pharmaceutical composition, product, the kit of pionemia.

In some embodiments, the hyperlipidemia is chosen from the followings one or more：Hypercholesterolemia, high glycerine Three ester mass formed by blood stasis, combined hyperlipidemia familial and low hdl mass formed by blood stasis.

In yet another aspect, the present invention relates to the methods for reducing subject artery's atherosis risk, tested including being administered A effective amount of plasminogen of person.The invention further relates to plasminogen for reducing the purposes of subject artery's atherosis risk. The invention further relates to plasminogens to prepare for reducing the drug of subject artery's atherosis risk, pharmaceutical composition, system Purposes in product, kit.Further, the invention further relates to the fibrinolysins for reducing subject artery's atherosis risk It is former.The invention further relates to for reducing the drug comprising plasminogen of subject artery's atherosis risk, pharmaceutical composition, Product, kit.

In some embodiments, the subject with hypertension, obesity, diabetes, chronic hepatitis, hepatic sclerosis, Injury of kidney, chronic glomerulonephritis, chronic pyelonephritis, nephrotic syndrome, renal insufficiency, kidney transplant, uremia, thyroid gland Hypofunction, obstructive cholecystitis or obstructive cholangitis or the subject take the drug or hormone for influencing fat metabolism. In some embodiments, the plasminogen passes through one or more reduction subject artery's atherosis wind chosen from the followings Danger：It reduces total cholesterol level in blood, triglyceride levels, low-density lipoprotein white level, improve blood middle-high density lipoprotein water It is flat.

In yet another aspect, the present invention relates to the method by improving subject's hyperlipemia therapeutic disease, including administration A effective amount of plasminogen of subject.The invention further relates to plasminogens to be used for by improving subject's hyperlipemia therapeutic disease Purposes.The invention further relates to plasminogens to prepare for passing through the drug for improving subject's hyperlipemia therapeutic disease, medicine Purposes in compositions, product, kit.Further, the invention further relates to improve subject's hyperlipidemia for passing through Treat the plasminogen of disease.The invention further relates to for pass through improve subject's hyperlipemia therapeutic disease comprising fibrinolysin Drug, pharmaceutical composition, product, the kit of original.

In some embodiments, the illness includes diabetes, hypertension, atherosclerosis, coronary heart disease, heart strand Bitterly, myocardial infarction, arrhythmia cordis, chronic hepatitis, fatty liver, hepatic sclerosis, cerebral insufficiency, cerebral ischemia, cerebral infarction, chronic renal Inflammation, chronic pyelonephritis, renal insufficiency, nephrotic syndrome, uremia, obesity.

In yet another aspect, the present invention relates to the method prevented and/or treat subject's hyperlipidemia associated disease, including giving A effective amount of plasminogen of medicine subject.The invention further relates to plasminogens for preventing and/or treating subject's hyperlipidemia phase The purposes of related disorders.The invention further relates to plasminogens to prepare for preventing and/or treat subject's hyperlipidemia associated disease Drug, pharmaceutical composition, product, the purposes in kit.Further, the invention further relates to for preventing and/or treat The plasminogen of subject's hyperlipidemia associated disease.The invention further relates to related for preventing and/or treating subject's hyperlipidemia The drug comprising plasminogen, pharmaceutical composition, product, the kit of illness.In some embodiments, the illness includes Diabetes, hypertension, atherosclerosis, coronary heart diseases and angina pectoris, myocardial infarction, arrhythmia cordis, chronic hepatitis, fatty liver, liver Hardening, cerebral insufficiency, cerebral ischemia, cerebral infarction, chronic nephritis, chronic pyelonephritis, renal insufficiency, nephrotic syndrome, urine Toxication, obesity.

In any of the above-described embodiment of the present invention, the plasminogen can be with one or more other medicines or treatment Method is administered in combination.In some embodiments, one or more other medicines include hypertension therapeutic drug, diabetes Treatment drug, treatment of atherosclerosis drug, chronic glomerulonephritis medicine, chronic pyelonephritis medicine Object, Therapy of Nephrotic Syndrome drug, renal insufficiency treatment drug, uremia therapy drug, kidney transplantation treatment medication Object, fatty liver treatment drug, cirrhosis treatment drug, bariatrician drug.In some embodiments, it is described its Its drug includes：Blood lipid-lowering medicine, antiplatelet drug, blood-pressure drug, vasodilator drug, hypoglycemic drug, anticoagulation Drug, thrombolytic agent, hepatic, antiarrhythmic drug, cardiac drug, diuretic, anti-infectives, antiviral agent Object, immunoregulation medicament, inflammation adjust class drug, antitumor drug, hormone medicine, thyroxine.It is further at some In embodiment, the drug includes blood lipid-lowering medicine：Statins；Fibrates；Niacin；Cholestyramine；Clofibrate；Unsaturated fat Acid such as yishouning, lipid level and heart arteries and veins pleasure；Sodium alginate；Antiplatelet drug：Aspirin；Persantine；Clopidogrel；West Lip river he；Vasodilator drug：Hydralazine；Nitroglycerin and isosorbide dinitrate；Sodium nitroprussiate；α nitre receptor blocking pharmacon such as prazosin；α by Body blocking agent such as phentolamine；β draws receptor agonist such as albuterol；Captopril, enalapril；Nifepine, thiazolone；Willow Gamma Amino Butyric Acid, minoxidil, prostaglandin, atrial natriuretic peptide；Thrombolytic agent：Urokinase and streptokinase；Tissue-type plasminogen activator； Single-chain Urokinase-type Plasminogen Activator；TNK- tissue-type plasminogen activators；Anticoagulation medicine：Heparin；According to promise liver Element；Nagqu heparin；Bivalirudin.

In any of the above-described embodiment of the present invention, the plasminogen can have extremely with sequence 2,6,8,10 or 12 Few 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity, and still there is fibrinolytic Zymogen activity.In some embodiments, the plasminogen is on the basis of sequence 2,6,8,10 or 12, and addition is deleted And/or substitution 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 implementations 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 straight for the human plasminogen from primate or rodent To homologue or its still retain the variant of activities of endothelial tissue plasminogen or segment.In some embodiments, the plasminogen Amino acid is 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 by administered either systemically or locally, preferably by with Lower approach application：Intravenously, intramuscular, subcutaneous administration fibrinolysin was treated originally.In some embodiments of preceding method, The plasminogen is applied with appropriate peptide carrier or combination of stabilizers.It is described in some embodiments of preceding method Plasminogen is 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、1-200 mg/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

Definition

" fat metabolic disturbance " of the present invention is also known as " fat metabolism is abnormal ", " lipodystrophy ", for fatty generation Thank be abnormal, the general name of the clinic that disorderly or obstacle is caused or pathological manifestations.Herein, " fat metabolic disturbance ", " fat Fat metabolism exception ", " lipodystrophy " are used interchangeably." fat metabolism " in the present invention, " lipid metaboli ", " lipid-metabolism " can It is used interchangeably.

" fat metabolic disturbance associated disease " is the general name with the relevant illness of fat metabolic disturbance.The correlation, can To be that the cause of disease is related, pathogenesis is related, pathological manifestations are related, clinical symptoms are related and/or principle of reatment is related.

" blood fat " is the general name of triglycerides, cholesterol and phosphatide etc., and lipoprotein is made of apolipoprotein and blood fat Ball-type macromolecule complex is divided into 5 since ingredient of the lipoprotein comprising cholesterol, triglycerides is different and density is of different sizes Major class：Chylomicron (CM) very low density lipoprotein (VLDL) medium density lipoprotein (IDL) low-density lipoprotein (LDL) is highly dense Spend lipoprotein (HDL).According to blood fat danger level, clinical most common dyslipoproteinemia type：Hypercholesterolemia, Hypertriglyceridemia, combined hyperlipidemia familial and low hdl mass formed by blood stasis.Secondary dyslipidemia sees diabetes, first Shape gland hypofunction, kidney transplant, severe liver disease, obstructive disease of biliary tract, obesity, is drunk, drug therapy at nephrotic syndrome, Such as estrin treatment etc., as can excluding secondary dyslipidemia is contemplated that primary dyslipidemia.

" hyperlipidemia " refers to that the lipid compositions such as cholesterol, triglyceride, phosphatide and the not esterified resin acid in blood plasma increase Pathological condition.

" hyperlipidemia associated disease " refers to the cause of disease, pathogenesis, pathological manifestations, clinical symptoms and/or principle of reatment and height The relevant illness of blood fat.It is twisted it is preferred that the illness includes but not limited to diabetes, hypertension, atherosclerosis, coronary heart disease, the heart Bitterly, myocardial infarction, arrhythmia cordis, chronic hepatitis, fatty liver, hepatic sclerosis, cerebral insufficiency, cerebral ischemia, cerebral infarction, chronic renal Inflammation, chronic pyelonephritis, renal insufficiency, nephrotic syndrome, uremia, obesity.

Since fat metabolism or operating exception make one or more of dyslipidemias in blood plasma be known as " hyperlipidemia ", " high blood Fat disease " or " dyslipidemia " (dyslipidemia).

Since lipid is insoluble or poorly soluble in water, it is necessary to being combined into lipoprotein with protein could operate in blood circulation, Therefore hyperlipidemia is often the reflection of " hyperlipoprotememia ".

" the hyperlipidemia associated disease " of the present invention is alternatively referred to as " hyperlipidemia associated disease ", " hyperlipoprotememia correlation Illness ".

" obesity " or " obesity " refers to that body fat accumulates excessive and/or abnormal distribution.Fat or obesity is commonly used Judge index have constitutional index (BMI) and waistline (WC).The criteria for classification of the World Health Organization is with BMI >=25kgm at present² To be overweight, BMI >=30kgm²For obesity, but this index according to country, area, ethnic group difference, slightly difference up and down.Than Such as 2003《Chinese Adult is overweight and obesity prevention and control guide (on probation)》It proposes with BMI >=24kgm²For Chinese Adult Overweight boundary, BMI >=28kgm²For fat boundary.It is overweight and fertile from the point of view of the classification of above " obesity " and " overweight " Fat embodiment be degree difference.Signified obesity or obesity cover " overweight " in the claims in the present invention and specification Meaning." obesity " of the present invention, " obesity " and " overweight " can be a variety of causes, such as pass through body caused by feed merely Weight excessively increases.

Present invention experiment prove plasminogen can improve fat in vivo organ, around organ, the positions such as abdominal cavity it is different Therefore normal or over-deposit, may be used as slimming drugs, treatment is fat or overweight situation, abatement are fatty.Lower weight.

The present invention relates to the plasminogen as the slimming drugs or pharmaceutical composition comprising plasminogen, kit or systems Product.The plasminogen of the present invention is also used as food additives, in food or drink in addition to it may be used as drug.This The plasminogen of invention can also be used in cosmetic slimming product, to lose weight.Therefore, plasminogen of the invention can be with each The convenient form of kind gives the subject of weight-reducing or losing weight in need.

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^[9].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^[10,11].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, Such as hormone, growth factor and cell factor.In addition, also there are fibrinolysins and the specific physiological inhibitor of PAs.Fibrinolysin it is main Inhibitor is α 2- antiplasmins (α 2-antiplasmin).The activity of PAs is pressed down simultaneously by the plasminogen activator of uPA and tPA Preparation -1 (PAI-1) inhibits and the main plasminogen activator agent inhibitor -2 (PAI-2) for inhibiting uPA is adjusted.Certain cell tables Face has the uPA specific cell surface receptors (uPAR) of direct hydrolysis activity^[12,13]。

Plasminogen (plasminogen, plg) is a single chain glycoprotein, is made of 791 amino acid, molecular weight is about For 92kDa^[14,15].Plasminogen is mainly synthesized in liver, is largely present in extracellular fluid.Content of plasminogen is about in blood plasma 2μM.Therefore plasminogen is a huge potential source of proteolytic activity in tissue and body fluid^[16,17].Fibrinolysin It is former that there are two kinds of molecular forms：Glutamic acid-plasminogen (Glu-plasminogen) and lysine-plasminogen (Lys- plasminogen).Natural secretion and the plasminogen of uncracked form have there are one amino terminal (N- ends) glutamic acid, because This is referred to as glutamic acid-plasminogen.However, in the presence of fibrinolysin, glutamic acid-plasminogen water at Lys76- Lys77 Solution becomes lysine-plasminogen.Compared with glutamic acid-plasminogen, lysine-plasminogen has higher with fibrin Affinity, and can be activated with higher rate by PAs.The Arg560-Val561 peptide bonds of the plasminogen of both forms can It is cut by uPA or tPA, the formation of dichain proteins enzyme fibrinolysin that disulfide bond is caused to connect^[18].The amino-terminal portion of plasminogen Subpackage contains five homologous tricyclics, i.e., so-called kringles, and carboxy-terminal sections include protease domain.Some kringles The lysine-binding site to interact containing mediation plasminogen with fibrin and its inhibitor α 2-AP specificity.It is newest It was found that the segment that a plasminogen is 38kDa, is effective inhibitor of angiogenesis including kringles1- 4.This A segment is named as angiostatin, 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^[19].Fibrinolysin also has the substrate specificity of several components to ECM, including laminin, fibronectin, proteoglycans And gelatin, show fibrinolysin ECM reconstruction in also play an important role [^15,20,21].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.Cause This, it is thus proposed that, fibrinolysin may be an important upstream regulator of extracellular proteolysis^[22].In addition, fibrinolysin has There is the ability for the growth factor for activating certain potential forms^[23-25].In vitro, fibrinolysin can also hydrolyze the component of complement system simultaneously 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), encode the cDNA sequence of the sequence as shown in sequence 1, amino acid sequence is as shown in sequence 2.In vivo, it also deposits It is a kind of be at the 76-77 amino acids of Glu- plasminogens hydrolysis so as to formed Lys- plasminogens, such as 6 institute of sequence Show, encode the cDNA sequence of the amino acid sequence as shown in sequence 5.Delta- plasminogens (δ-plasminogen) are overall lengths Plasminogen has lacked the segment of Kringle2-Kringle5 structures, only containing Kringle1 and serine protease Domain^[26,27], there is the amino acid sequences (sequence 8) of document report delta- plasminogens^[27], encode the amino acid sequence CDNA sequence such as sequence 7.Miniplasminogen (Mini-plasminogen) is made of Kringle5 and serine protease domain, Having document report, it includes residue Val443-Asn791 (not contain the Glu residues of the Glu- plasminogen sequences of signal peptide For initial amino acid)^[28], 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 amino Acid)^[29], also there is patent document CN102154253A to report that its sequence includes residue Lys531-Asn791 (not contain signal peptide Glu- plasminogen sequences Glu residues be initial amino acid), this patent sequence reference patent document CN102154253A, Its amino acid sequence encodes 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, the content ratio of the meaning of the plasminogen " shortage " or activity 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 or activity of the plasminogen " missing " Content for plasminogen in subject's body is substantially less than normal person or even activity or expression is atomic, is 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 " refers in plasminogen protein, can be combined and played with the target sequence in substrate The active fragment of proteolysis function.Technical solution the present invention relates to plasminogen was covered with activities of endothelial tissue plasminogen segment generation For the technical solution of plasminogen.Activities of endothelial tissue plasminogen segment of the present invention is the serine protease comprising plasminogen The protein in domain, it is preferable that activities of endothelial tissue plasminogen segment of the present invention include sequence 14, with sequence 14 have at least 80%, 90%th, the protein of the amino acid sequence of 95%, 96%, 97%, 98%, 99% homology.Therefore, fibre of the present invention Lyase original includes containing the activities of endothelial tissue plasminogen segment and still maintains the albumen of the activities of endothelial tissue plasminogen.

At present, plasminogen in blood and its activity determination method are included：To tissue plasminogen 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 The detection of activity, 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, the latter acts on chromophoric substrate, then under the action of SK With spectrophotometric determination, absorbance increases directly proportional to activities of endothelial tissue plasminogen.In addition immuno-chemical method, gel electricity can also be used Swimming, 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 sequence Arrange the given amino acid sequence A of a certain % amino acid sequence identities of 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.The effect Fruit can be complete or partial prevention disease or its symptom and/or partially or completely cure disease and/or its symptom, and wrap It includes：(a) prevention disease occurs in subject's body, and the subject can have the procatarxis of disease, but not yet be diagnosed as having There is disease；(b) inhibit disease, that is, block its formation；(c) mitigate disease and/or its symptom, that is, cause disease and/or its disease Shape subsides.

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

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, can exist Many well known promoters, such as lactose promoter system, tryptophan (trp) promoter systems, beta- iactamase promoters System or the promoter systems from phageλ.Promoter would generally control expression, the optionally situation in operator sequence In, 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)) be 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- Ethylene glycol (PEG).It is preferred that the anti-VEGF antibodies preparaton being lyophilized is described in WO 97/04801, it includes herein as With reference to.

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)), no Degradable ethylene vinyl acetate (ethylene-vinyl acetate) (Langer, etc. ibid) or degradable Poly lactic coglycolic acid such as Lupron DepotTM (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 keys are formed for Disulfide interchange, then can by modify sulfhydryl residue, be lyophilized from acid solution, control it is wet Degree realizes stabilization 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 Range can be, for example, for example daily about 0.0001 to 2000mg/kg or about 0.001 to 500mg/kg (such as 0.02mg/kg, 0.25 mg/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 by fat and its related The plasminogen or fibrinolysin of the present invention of illness.The product preferably includes a container, label or package insert.Appropriate appearance Device has bottle, bottle, syringe etc..Container can be made of a variety of materials such as glass or plastics.The container contains composition, institute The disease of the present invention or illness and (such as the container can be intravenous solution with sterile access port can effectively be treated by stating composition Packet or bottle, containing can be by the plug that hypodermic needle penetrates).At least one of composition activating agent is fibrinolysin Original/fibrinolysin.On the container or appended label illustrates the composition and of the present invention is caused for treating by diabetes Obesity and its associated disease.The product can further include the second container containing pharmaceutically acceptable buffer solution, such as phosphate The brine of buffering, Ringer's solution and glucose solution.It can further include needed for from the point of view of business and user's angle Other materials, including other buffer solutions, diluent, filtrate, needle and syringe.In addition, the product is included with use The package insert of explanation, including for example indicating the user of the composition by plasminogen composition and treating adjoint disease The other medicines administered patient of disease.

Brief description

Fig. 1 gives the obese model mouse weight variation result of calculation of plasminogen high-calorie feed induction in 28 days.As a result It is shown as the 29th day weight and subtracts the numerical value of the 1st day weight.The results show that the change of blank control group weight is not obvious, to molten The weight that matchmaker's PBS control group mitigates, which is significantly lower than, gives plasminogen group, and statistical discrepancy significantly (* P<0.05).Illustrate fibrinolytic Proenzyme can promote the reduction of obese model mouse weight.

Fig. 2 gives the obese model mouse weight index statistical result of plasminogen high-calorie feed induction in 28 days.As a result Display is significantly lower than to plasminogen group mouse weight index and gives solvent PBS control group, and statistical discrepancy significantly (* P< 0.05, * * is P<0.01), and compared with to solvent PBS control group sky is more nearly to the body mass index of plasminogen group mouse White control group.Illustrate that plasminogen can significantly reduce the body mass index of obese model mouse, mitigate fat.

Fig. 3 gives the obese model mouse Lee's index statistical results of plasminogen high-calorie feed induction in 28 days.As a result Display is significantly lower than to plasminogen group mouse Lee's indexes and gives solvent PBS control group, and statistical discrepancy significantly (* P< 0.05), and compared with to solvent PBS control group blank control group is more nearly to the Lee's indexes of plasminogen group mouse.It says Bright plasminogen can significantly reduce the Lee's indexes of obese model mouse, mitigate fat.

The obese model lipid of mice testing result of Fig. 4 high-calorie feed induction.A is T-CHOL, B is low-density lipoprotein In vain, C is high-density lipoprotein.The results show that consolidate to plasminogen group, to total courage of solvent PBS control groups and blank control group Alcohol, low-density lipoprotein and high-density lipoprotein concentration are without significant difference.Illustrate the fertilizer that high-energy feed induces in this experiment Fat model mice blood fat, which has no, to be significantly changed.

The obese model mice serum leptin testing result of Fig. 5 high-calorie feed induction.The results show that plasminogen Group, to three groups of leptins of solvent PBS control group and blank control group without significant difference.Illustrate high-energy feed in this experiment The obese model mouse Leptin of induction, which has no, to be significantly changed.

The obese model mice serum insulin testing result of Fig. 6 high-calorie feed induction.The results show that plasminogen Group, to three groups of insulin concentrations of solvent PBS control group and blank control group without significant difference.Illustrate that high-energy is raised in this experiment The obese model mouse islets element of material induction, which has no, to be significantly changed.

Fig. 7 gives the obese model mouse peritoneal fat coefficient statistical result of plasminogen high-calorie feed induction in 28 days. Solvent PBS control group is given the results show that being significantly lower than to plasminogen group abdominal cavity fat coefficient, statistical discrepancy significantly (* P< 0.05), and compared with to solvent PBS control group blank control group is more nearly to plasminogen group mouse peritoneal fat content. Illustrate that plasminogen can significantly reduce the deposition of obese model mouse peritoneal fat.

The obese model mouse peritoneal fat HE dyeing fat that Fig. 8 gives plasminogen high-calorie feed induction in 28 days is empty Steep area statistics result.A is blank control group, and B is gives solvent PBS control group, and for C to give plasminogen group, D is quantitative analysis As a result.The results show that the area to plasminogen group average fat vacuole is significantly less than and gives solvent PBS control group, statistical discrepancy Extremely significantly (* * represent P<0.01) the more adjunction of plasminogen group fat vacuole area, and compared with to solvent PBS control group is given The white control mice of near-space.Illustrate that plasminogen can significantly decrease the size of obese model mouse adipocytes, reduce abdominal cavity Fat deposition.

Fig. 9 gives plasminogen 28 days 14-15 week old diabetic mice Serum Leptin Levels testing results.The results show that fibre Lyase original group mice serum leptin is significantly lower than solvent PBS control group is given, and extremely significantly (* * are P to statistical discrepancy<0.01), And normal mouse is more nearly to plasminogen group serum leptin level compared with to solvent PBS control group.Illustrate plasminogen Diabetes B early stage mice serum leptin level can be reduced.

Figure 10 gives plasminogen 28 days 23-25 week old diabetic mice Serum Leptin Levels testing results.The results show that fibre Lyase original group mice serum leptin is significantly lower than solvent PBS control group is given, and extremely significantly (* * are P to statistical discrepancy<0.01). Illustrate that plasminogen can reduce diabetes B late period mice serum leptin level.

16 weeks hyperlipemia model mouse of Figure 11 give plasminogen after 30 days liver oil red O stain observe result.A is Solvent PBS control group is given, for B to give plasminogen group, C is quantitative analysis results.The results show that give plasminogen group mouse liver Fat deposition, which is considerably less than, gives solvent PBS control group, and significantly (* represents P to quantitative analysis statistical discrepancy<0.05).Illustrate fibrinolytic Proenzyme can improve deposition of the fat in hyperlipemia model mouse liver.

16 weeks hyperlipemia model mouse of Figure 12 give plasminogen 30 days posterior sinus of Valsalva oil red O stain observation results. A, C is gives solvent PBS control group, and to give plasminogen group, E is quantitative analysis results by B, D.The results show that give plasminogen group Mouse aorta sinus fat deposition, which is considerably less than, gives solvent PBS control group, and significantly (* represents P to statistical discrepancy<0.05).Explanation Plasminogen can improve deposition of the fat in hyperlipemia model mouse aorta sinus.

Figure 13 gives plasminogen 3% cholesterol feed induced hyperlipidemia model mice kidney oil red O observations after 30 days As a result.A is blank control group, and B is gives solvent PBS control group, and for C to give plasminogen group, D is quantitative analysis results.As a result it shows Show, be considerably less than to plasminogen group mouse kidney fat deposition (arrow logo) and give solvent PBS control group, and quantitative analysis is united Count significant difference；It is in addition, similar to blank control group mouse to plasminogen group lipidosis level.Illustrate that plasminogen can disappear Deposition of the fat reducing fat in hyperlipemia model mouse kidney, so as to reduce the kidney injury caused by fat deposition.

3% cholesterol feed induced hyperlipidemia model mices of Figure 14 give plasminogen serum low-density fat after 20 days Protein cholesterol testing result.It is the results show that significantly low to plasminogen group mice serum concentration of low density lipoprotein cholesterol In giving solvent PBS control group, and significantly (* represents P to statistical discrepancy<0.05).Illustrate that plasminogen can reduce hyperlipemia model The content of low density lipoprotein cholesterol in mice serum has the function of to improve hyperlipidemia.

3% cholesterol feed induced hyperlipidemia model mices of Figure 15 give plasminogen after 20 days serum artery it is athero- Hardenability value testing result.The results show that it is significantly lower than to plasminogen group rat aorta atherosis index to PBS pairs of solvent According to group, and statistical difference is heteropolar that significantly (* * represent P<0.01).Illustrate that plasminogen can effectively reduce hyperlipemia model mouse hair The risk of lively pulse atherosclerosis.

Figure 16 ApoE Atherosclerosis Model mouse give plasminogen serum total cholesterol testing result after 30 days.Knot Fruit shows, is significantly lower than to plasminogen group mouse total cholesterol concentration and gives solvent PBS control group, and statistical discrepancy significantly (* Represent P<0.05).Illustrate that plasminogen can reduce the content of T-CHOL in ApoE Atherosclerosis Model mice serums, change Kind Atherosclerosis Model lipid of mice is disorderly.

Figure 17 ApoE Atherosclerosis Model mouse give plasminogen serum triglyceride testing result after 30 days.Knot Fruit shows, is significantly lower than to plasminogen group mouse triglyceride concentration and gives solvent PBS control group, and statistical discrepancy significantly (* Represent P<0.05).Illustrate that plasminogen can reduce containing for triglycerides in ApoE Atherosclerosis Model mice serums It is disorderly to improve Atherosclerosis Model lipid of mice for amount.

Figure 18 ApoE Atherosclerosis Model mouse give plasminogen serum LDL cholesterol after 30 days Testing result.Solvent PBS is given the results show that being significantly lower than to plasminogen group mice serum concentration of low density lipoprotein cholesterol Control group, and significantly (* represents P to statistical discrepancy<0.05).Illustrate that plasminogen can reduce ApoE Atherosclerosis Model mouse It is disorderly to improve Atherosclerosis Model lipid of mice for the content of low density lipoprotein cholesterol in serum.

Figure 19 gives obese model mouse hypothalamus leptin receptor immunohistochemical staining result after plasminogen.A、D For blank control group, B, E is give solvent PBS control group, and to give plasminogen group, G is quantitative analysis results by C, F.The results show that Blank control group is significantly more than to the expression of solvent PBS control group mouse hypothalamus leptin receptor；And give plasminogen group mouse The expression of hypothalamic leptin receptor is considerably less than and gives solvent PBS control group, and close to blank control group level, and statistical discrepancy is notable (P=0.01).Illustrate can to significantly reduce the expression of obesity mice hypothalamic leptin receptor to plasminogen.

Embodiment

The influence of Mice model of obesity that 1 plasminogen of embodiment induces high-calorie feed

Mouse model and grouping

The C57 male mices 14 of 8 week old are taken, are randomly divided into two groups according to weight, blank control group 4 and model group 10.The feeding of blank control group mouse is normal to maintain feed；Model group mouse feeds 45% high lipid food modeling of fat heat (TP23000, Nantong Te Luofei feed technologies Co., Ltd) 12 weeks, establishes obese model^[30].Fat heat 45% in this text High lipid food is referred to as high-calorie feed.After 12 weeks, model group mouse is weighed, and two groups is randomly divided into again according to weight, to fibre Lyase original group and solvent PBS control group is given, every group each 5.People source plasminogen is dissolved in PBS.Give plasminogen group tail vein 1mg/0.1mL/ pcs/day of people source plasminogen is injected, to the PBS of solvent PBS control group tail vein injection same volume, blank control Group is without any processing.Above-mentioned experimental animal successive administration 28 days (starting administration to be set to the 1st day), is located on the 29th day as follows Reason and detection.

Detection and result

Weight detects

Above-mentioned experimental animal weighed at the 1st and 29 day, calculated the change of weight.As a result the 29th day weight is shown as to subtract Remove the numerical value of the 1st day weight.

The results show that the change of blank control group weight is not obvious, it is considerably less than and gives to solvent PBS control group weight loss Plasminogen group, and statistical discrepancy significantly (* P<0.05) (Fig. 1).It is small to illustrate that plasminogen can significantly reduce obese model Mouse weight.

Body mass index measures

After the 29th day weighs in above-mentioned mouse, measurement Mice Body is long, calculates body mass index.Body mass index=weight (kg)/body is long²(m)。

Body mass index be at present in the world it is common weigh the fat or thin degree of human body and whether a standard of health.Weight Index equally can be as the index of the fat or thin degree of obese model animal^[43,44].The results show that refer to plasminogen group mouse weight Number, which is significantly lower than, gives solvent PBS control group, and statistical discrepancy significantly (* P<0.05) it, and compared with to solvent PBS control group gives The body mass index of plasminogen group mouse is more nearly blank control group (Fig. 2).Illustrate that plasminogen can significantly reduce obesity The body mass index of model mice mitigates fat.

Lee ' s assessment of indices

For above-mentioned mouse after the 29th day weighs in, measurement Mice Body is long, calculates Lee's indexes.

Lee's indexes are the availability indexes for reacting obese degree^[31-32].The results show that give plasminogen group mouse Lee's Index, which is significantly lower than, gives solvent PBS control group, and statistical discrepancy significantly (* P<0.05), and with giving solvent PBS control group phase Than being more nearly blank control group (Fig. 3) to the Lee's indexes of plasminogen group mouse.Illustrate that plasminogen can significantly reduce The Lee's indexes of obese model mouse mitigate fat.

Blood lipid level detects

Above-mentioned model mice plucks eyeball on the 29th day and takes blood, centrifuging and taking supernatant, using serum total cholesterol, low-density lipoprotein With high-density lipoprotein detection kit (Bioengineering Research Institute, article No. A111-1, A113-1, A112-1 are built up in Nanjing), press According to corresponding reagent box the method, detection serum total cholesterol, low-density lipoprotein and high-density lipoprotein concentration.

The results show that plasminogen group, to the T-CHOL (Fig. 4 A), low of solvent PBS control group and blank control group Density lipoprotein (Fig. 4 B) and high-density lipoprotein (Fig. 4 C) concentration are without significant difference.Illustrate that high-energy feed lures in this experiment The obese model lipid of mice led, which has no, to be significantly changed.

Serum leptin level detects

Using Serum Leptin Levels detection kit (Bioengineering Research Institute, article No. H174 are built up in Nanjing), and according to kit The method detects the leptin level in above-mentioned serum.

The results show that plasminogen group, to three groups of leptins of solvent PBS control group and blank control group without significantly Difference (Fig. 5).Illustrate that the obese model mouse Leptin of high-energy feed induction in this experiment has no to significantly change.

Leptin (Leptin, LP) is a kind of hormone secreted by adipose tissue.People generally believe that it enters blood before The adjusting of sugar, fat and energetic supersession can be participated in after cycle, body reduction is promoted to ingest, increases energy release, inhibits fat thin The synthesis of born of the same parents, and then make weight loss.But there are leptin resistance phenomenon in some obese individuals, leptin level increases in blood^[34]。 Correlative study shows db/db mouse there are leptin resistance, and serum leptin level significantly increases^[35-36]。

Serum insulin level detects

Using serum insulin detection kit (Bioengineering Research Institute, article No. H174 are built up in Nanjing), and according to reagent Box the method detects the insulin level in above-mentioned serum.

The results show that plasminogen group, to three groups of insulin concentrations of solvent PBS control group and blank control group without aobvious Write difference (Fig. 6).Illustrate that the obese model mouse islets element of high-calorie feed induction in this experiment has no to significantly change.

Abdominal cavity fat amount detects

Above-mentioned mouse puts to death materials abdominal cavity fat and weighs after the 29th day weighs in.Abdominal cavity fat coefficient (%)=(abdomen Chamber fat weight/weight) * 100.

Solvent PBS control group, and statistical difference are given the results show that being significantly lower than to plasminogen group mouse peritoneal fat coefficient Different significantly (* P<0.05), and close to blank control group mouse adipose coefficient (Fig. 7).Illustrate that plasminogen can significantly drop The deposition of low obese model mouse peritoneal fat.

The area detecting of abdominal cavity subcutaneous fat vacuole

Above-mentioned mouse was put to death at the 29th day, its abdominal cavity fat of drawing materials fixes 24-48 hours in 4% paraformaldehyde.After fixation Tissue samples carry out paraffin embedding after alcohol serial dehydration and dimethylbenzene are transparent.Histotomy thickness is 4 μm, slice dewaxing Rehydration and with haematoxylin and eosin stains (HE dyeing), ammonium hydroxide returns blue and alcohol serial dehydration mounting after the differentiation of 1% hydrochloride alcohol, Slice is in 200 times of optical microphotograph Microscopic observations.With Image-pro plus image processing softwares, the face of fat vacuole is analyzed Product.

When fat human body energy intake is more than energy expenditure, a large amount of accumulation of lipid leads to fatty ester in adipocyte Fat tissue expander, that is, adipocyte increase, the increase of fat vacuole area^[33]。

The results show that plasminogen group (Fig. 8 C) fat vacuole area be significantly less than to solvent PBS control group (figure 8B), extremely significantly (* * represent P to statistical discrepancy<0.01) (Fig. 8 D), and give plasminogen group compared with to solvent PBS control group Fat vacuole area is more nearly blank control mouse (Fig. 8 A).Illustrate that plasminogen can substantially reduce obese model mouse The size of adipocyte reduces abdominal cavity fat deposition.

2 plasminogen of embodiment reduces the concentration of diabetes early stage mice serum leptin

14-15 week old db/db male mices 12 and db/m mouse 3, db/db mouse are divided at random according to weight after weighing It is two groups, to plasminogen group and gives solvent PBS control group, every group each 6.Start administration be set to the 1st day, start within the 1st day to Plasminogen or PBS to 2mg/0.2ml/ pcs/day of plasminogen group tail vein injection people source plasminogen, give solvent PBS control The PBS of group tail vein injection same volume, successive administration 28 days.Db/m mouse are not cooked administration processing as normal control mice. Mouse fasting 16 hours in 28th day plucks eyeball on the 29th day and takes blood, centrifuging and taking supernatant, to detect the concentration of Serum Leptin Levels.Using Serum Leptin Levels detection kit (Bioengineering Research Institute, article No. H174 are built up in Nanjing), and detected according to kit the method Leptin level in above-mentioned serum.

Solvent PBS control group, and statistical difference are given the results show that being significantly lower than to plasminogen group mice serum leptin It is different that significantly (* * are P<0.01) it, and compared with to solvent PBS control group is more nearly just to plasminogen group serum leptin level Normal mouse (Fig. 9).Illustrate that plasminogen can significantly reduce diabetes B early stage mice serum leptin level.

3 plasminogen of embodiment reduces the concentration of advanced diabetes mice serum leptin

23-25 week old db/db male mices 13, mouse is randomly divided into two groups according to weight after weighing, to plasminogen Group 7 and to solvent PBS control group 6.Start to plasminogen or PBS within 1st day, give plasminogen group tail vein injection people source 2mg/0.2ml/ pcs/day of plasminogen, to the PBS of solvent PBS control group tail vein injection same volume, successive administration 28 days. Mouse fasting 16 hours in 28th day plucks eyeball on the 29th day and takes blood, centrifuging and taking supernatant, to detect the concentration of Serum Leptin Levels.Using Serum Leptin Levels detection kit (Bioengineering Research Institute, article No. H174 are built up in Nanjing), and detected according to kit the method Leptin level in above-mentioned serum.

Solvent PBS control group, statistical discrepancy are given the results show that being significantly lower than to plasminogen group mice serum leptin Extremely significantly (* * are P<0.01) (Figure 10).Illustrate that plasminogen can reduce diabetes B late period mice serum leptin water It is flat.

Deposition of 4 plasminogen of the embodiment abatement fat in 16 weeks hyperlipemia model mouse livers

6 week old male C57 mouse 11 feed high cholesterol diet (Nantong Te Luofei, article No. TP2031) high in fat 16 weeks With induced hyperlipidemia model^[37-38], this model is set to 16 weeks hyperlipemia models.Mouse after Cheng Mo, which continues to feed high courage, consolidates Alcohol feed.Before administration three days every take 50 μ l of blood to detect T-CHOL (T-CHO) content, and random according to T-CHO contents It is divided into two groups, to solvent PBS control groups 6, to plasminogen group 5.Start administration to be denoted as the 1st day, give plasminogen group tail 1mg/0.1ml/ pcs/day of intravenous injection human source plasminogen, to the PBS of solvent PBS control group tail vein injection same volume.Administration 30 days, and mouse was put to death in the 31st day, liver is taken to fix 24-48 hours in 4% paraformaldehyde, respectively at 15%, 30% sucrose In 4 DEG C sink to the bottom overnight, OCT embedding, 8 μm of frozen section thickness, oil red O stain 15min, 75% alcohol break up 5 seconds, bush uniformly dyeing Core 30 seconds, glycerin gelatine mounting.Slice is in 400 times of optical microphotograph Microscopic observations.

Oil red O stain can show lipidosis, reflect the degree of lipidosis^[39].The results show that give plasminogen group (Figure 11 B) mouse liver fat deposition is considerably less than and gives solvent PBS control group (Figure 11 A), and quantitative analysis statistical discrepancy is notable (Figure 11 C).Illustrate that plasminogen can cut down deposition of the fat in hyperlipemia model mouse liver.

5 plasminogen of embodiment reduces the deposition in 16 weeks hyperlipemia model mouse aorta sinus of lipid

6 week old male C57 mouse 11 feed high cholesterol diet (Nantong Te Luofei, article No. TP2031) high in fat 16 weeks With induced hyperlipidemia model^[37-38], this model is set to 16 weeks hyperlipemia models.Mouse after Cheng Mo, which continues to feed high courage, consolidates Alcohol feed.Before administration three days every take 50 μ l of blood to detect T-CHOL (T-CHO) content, and random according to T-CHO contents It is divided into two groups, to solvent PBS control groups 6, to plasminogen group 5.Start administration to be denoted as the 1st day, give plasminogen group tail 1mg/0.1ml/ pcs/day of intravenous injection human source plasminogen, to the PBS of solvent PBS control group tail vein injection same volume.Administration 30 days, and mouse was put to death in the 31st day, dirty 4% paraformaldehyde that is organized in of coring fixes 24-48 hours, respectively at 15%, 30% It is sunk to the bottom overnight for 4 DEG C in sucrose, OCT embeddings, 8 μm of aortic sinus frozen section thickness, oil red O stain 15min, the differentiation of 75% alcohol 5 seconds, bush uniformly dyeing core 30 seconds, glycerin gelatine mounting.Slice is in 40 times (Figure 11 A, 11B), 200 times of (Figure 11 C) times optical microphotographs Microscopic observation.

Solvent PBS is given the results show that being considerably less than to plasminogen group (Figure 12 B, 12D) mouse aorta sinus fat deposition Control group (Figure 12 A, 12C), and statistical discrepancy is significantly (Figure 12 E).Illustrate that plasminogen can reduce lipid in hyperlipemia model Deposition in mouse aorta sinus.

6 plasminogen of embodiment reduces the hyperlipemia model mouse kidney fat deposition of 3% cholesterol feed induction

9 week old male C57 mouse 16 feed 3% cholesterol high lipid food (Nantong Te Luofei) 4 weeks, induce hyperlipemia Disease^[37-38], this model is set to 3% cholesterol hyperlipemia model, and the mouse after Cheng Mo continues to feed the feeding high in fat of 3% cholesterol Material.The male C57 mouse 5 of identical week old is separately taken to be only used as blank control group, during the experiment feeding is common to maintain feed.It is being administered Every mouse of first three day takes 50 μ L of blood, detects T-CHOL, and model mice is randomly divided into two according to total cholesterol concentration and weight Group to plasminogen group and gives solvent PBS control group, every group each 8.Start administration to be denoted as the 1st day, give plasminogen group mouse 1mg/0.1ml/ pcs/day of tail vein injection people source plasminogen to the PBS of solvent PBS control group tail vein injection same volume, is given Medicine 30 days.It puts to death mouse within 31st day, kidney is taken to fix 24-48 hours in 4% paraformaldehyde, respectively at 4 in 15%, 30% sucrose It DEG C sinks to the bottom overnight, OCT embeddings, 8 μm of frozen section thickness, oil red O stain 15min, 75% alcohol breaks up 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 (Figure 13 C) mouse kidney fat deposition (arrow logo) to solvent PBS control group (Figure 13 B), and quantitative analysis statistical discrepancy is significantly (Figure 13 D)；In addition, give plasminogen group lipidosis level It is similar to blank control group mouse (Figure 13 A).Illustrate that plasminogen can cut down fat in 3% cholesterol hyperlipemia model mouse Deposition in kidney, so as to the dirty damage of kidney caused by reducing fat deposition.

7 plasminogen of embodiment reduces the hyperlipemia model mice serum low-density lipoprotein of 3% cholesterol feed induction White cholesterol levels

9 week old male C57 mouse 16 feed 3% cholesterol high lipid food (Nantong Te Luofei) 4 weeks, induce hyperlipemia Disease^[37-38], 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 be randomly divided into according to total cholesterol concentration and weight 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 16 in the 20th day Hour, it pricks within the 21st day orbital venous plexus and takes 50 μ L of blood, centrifugation obtains supernatant, using low density lipoprotein cholesterol detection kit (Bioengineering Research Institute, article No. A113-1 are built up in Nanjing) carries out low density lipoprotein cholesterol (LDL-C) and detects.

Solvent PBS control group, and statistical difference are given the results show that being significantly lower than to plasminogen group mouse (LDL-C) concentration Different notable (Figure 14).Illustrate that plasminogen can reduce containing for low density lipoprotein cholesterol in hyperlipemia model mice serum Amount.

8 plasminogen of embodiment reduces the hyperlipemia model rat aorta atherosis shape of 3% cholesterol feed induction Into risk

9 week old male C57 mouse 16 feed 3% cholesterol high lipid food (Nantong Te Luofei) 4 weeks, induce hyperlipemia Disease^[37-38], 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 (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 opens after administration in the 20th day Beginning 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 is using total Cholesterin detection reagent box (Bioengineering Research Institute, article No. A111-1 are built up in Nanjing) is detected；High-density lipoprotein courage is consolidated Using high-density lipoprotein cholesterol detection kit, (Bioengineering Research Institute, article No. are built up in Nanjing to alcohol (HDL-C) content A112-1 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^[40].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 15).Illustrate that plasminogen can reduce hyperlipemia model mouse and atherosclerosis occurs Risk.

9 plasminogen of embodiment reduces the content of ApoE atherosclerosis mice serum T-CHOLs

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^[41-42].Mouse after Cheng Mo continues to feed high cholesterol diet high in fat.Three days every before administration 50 μ l of blood are taken to detect T-CHOL (T-CHO) content, and two groups are randomly divided into according to T-CHO contents, give solvent PBS control group 7, to plasminogen group 6.Start administration to be set to the 1st day, give plasminogen group mouse tail vein injection people source plasminogen It 1mg/0.1ml/ pcs/day, to the PBS of solvent PBS control group tail vein injection same volume, is administered 30 days.Prohibit in the 30th day mouse Food 16 hours extracts eyeball on the 31st day and takes blood, and centrifugation obtains supernatant, and using T-CHOL detection kit, (biological work is built up in Nanjing Journey research institute, article No. A111-1) carry out T-CHOL detection.

Testing result is shown, is significantly lower than to plasminogen group mouse total cholesterol concentration and is given solvent PBS control groups, and Statistical discrepancy is significantly (P=0.014) (Figure 16).Illustrate that plasminogen can reduce ApoE Atherosclerosis Model mice serums The content of middle T-CHOL improves the blood fat disorder of atherosclerosis.

10 plasminogen of embodiment reduces the content of ApoE atherosclerosis mice serum triglycerides

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^[41-42].Mouse after Cheng Mo continues to feed high cholesterol diet high in fat.Three days every before administration 50 μ l of blood are taken to detect T-CHOL (T-CHO) content, and two groups are randomly divided into according to T-CHO contents, give solvent PBS control group 7, to plasminogen group 6.Start administration to be denoted as the 1st day, give plasminogen group mouse tail vein injection people source plasminogen It 1mg/0.1ml/ pcs/day, to the PBS of solvent PBS control group tail vein injection same volume, is administered 30 days.Prohibit in the 30th day mouse Food 16 hours plucks eyeball on the 31st day and takes blood, and centrifugation obtains supernatant, and using triglycerides detection kit, (bioengineering is built up in Nanjing Research institute, article No. A110-1) carry out triglycerides detection.

Testing result is shown, is significantly lower than to plasminogen group mouse triglyceride concentration and is given solvent PBS control groups, and Statistical discrepancy is significantly (P=0.013) (Figure 17).Illustrate that plasminogen can be reduced in ApoE Atherosclerosis Model mice serums The content of triglycerides improves atherosclerosis blood fat disorder.

11 plasminogen of embodiment reduces the content of ApoE atherosclerosis mice serum low density lipoprotein cholesterol

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^[41-42].Mouse after Cheng Mo continues to feed high cholesterol diet high in fat.Three days every before administration 50 μ l of blood are taken to detect T-CHOL (T-CHO) content, and two groups are randomly divided into according to T-CHO contents, give solvent PBS control group 7, to plasminogen group 6.Start administration to be denoted as the 1st day, give plasminogen group mouse tail vein injection people source plasminogen It 1mg/0.1ml/ pcs/day, to the PBS of solvent PBS control group tail vein injection same volume, is administered 30 days.Mouse fasting in 30th day It 16 hours, plucks within the 31st day eyeball and takes blood, centrifugation obtains supernatant and uses low density lipoprotein cholesterol (LDL-C) detection kit (Bioengineering Research Institute, article No. A113-1 are built up in Nanjing) carries out LDL-C detections.

Solvent PBS control group, and statistical discrepancy are given the results show that being significantly lower than to plasminogen group mouse LDL-C concentration Significantly (P=0.017) (Figure 18).Illustrate that plasminogen can reduce low density lipoprotein in ApoE Atherosclerosis Model mice serums It is disorderly to improve Atherosclerosis Model lipid of mice for protein cholesterol content.

12 plasminogen of embodiment improves the expression of obese model mouse hypothalamus leptin receptor

The C57 male mices 14 of 8 week old are taken, are randomly divided into two groups according to weight, blank control group 4 and model group 10.The feeding of blank control group mouse is normal to maintain feed；Model group mouse feeds 45% high lipid food of fat heat (TP23000, Nantong Te Luofei feed technologies Co., Ltd) modeling 12 weeks, establishes obese model^[1].After 12 weeks, model group is small Mouse is weighed, and is randomly divided into two groups again according to weight, to plasminogen group and gives solvent PBS control group, every group each 5.To fibre 1mg/0.1ml/ pcs/day of lyase original group tail vein injection people source plasminogen, gives solvent PBS control group tail vein injection same volume PBS, blank control group do not inject any liquid, and successive administration 28 days, mouse continues to feed modeling feed during administration.29th It is put to death mouse materials hypothalamus and 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 4 μm, is washed 1 time after slice dewaxing rehydration.Citric acid repairs 30 Minute, water softly rinses after ten minutes for room temperature cooling.With 3% dioxygen water incubation 15 minutes, tissue is irised out with PAP.10% Sheep blood serum (Vector laboratories, Inc., USA) is closed 1 hour；After time arrives, reject sheep blood serum liquid.Anti- leptin by 4 DEG C of overnight incubations of body antibody (Abcam), PBS are washed 2 times, every time 5 minutes.Goat anti-rabbit igg (HRP) antibody (Abcam) secondary antibody Incubation at room temperature 1 hour, PBS is washed 2 times, every time 5 minutes.It is aobvious by DAB kits (Vector laboratories, Inc., USA) Color, haematoxylin is redyed 30 seconds after washing 3 times, and flowing water returns indigo plant 5 minutes, and then PBS is washed 1 time.Serial dehydration is transparent and mounting, slice In 40 times (Figure 19 A-C), 200 times of (Figure 19 E-F) optical microphotograph Microscopic observations.

The main Physiological Function of leptin receptor is combined with leptin, is that leptin plays the internal energy balance of adjusting, fat The physiological actions such as storage, reproduction activity, and participate in the Autocrine regulation of leptin.Different types of leptin receptor is in maincenter and periphery Express to tissue selectivity^[45-47]。

The results show that the expression to solvent PBS control group (Figure 19 B, E) mouse hypothalamus leptin receptor is significantly more than blank Control group (Figure 19 A, D)；And the expression to plasminogen group (Figure 19 C, F) mouse hypothalamus leptin receptor is considerably less than to solvent PBS control group, close to blank control group level, and statistical discrepancy is significantly ((P=0.01)) (Figure 19 G).Illustrate to plasminogen The expression of obesity mice hypothalamic leptin receptor can be significantly reduced.

Bibliography

[1]http://www.ama-assn.org/ama/pub/news/news/2013/2013-06-18-new-ama- Policies-annual-meeting [EB/OL] (2013-06-18) .page.

[2]http://www.who.int/mediacentre/factsheets/fs311/en/ [EB/OL] (2015- 01- 26)

[3] HOU X, LU J, WENG J, et al.Impact of waist circumference and body mass index on risk of cardiometabolic disorder and cardiovascular disease in Chinese adults:a national diabetes and metabolic disorders survey[J].PLoS One, 2013,8 (3):e57319.

[4] LI S, XIAO J, JI L, et al.BMI and waist circumference are associated with impaired glucose metabolism and type 2diabetes in normal weight Chinese Adults [J] .J Diabet Complicat, 2014,28 (4):470-476.

[5]Christensen R,Kristensen PK,et al.Efficacy and safety of the weightloss drug rimonabant:a meta-analysis of randomised trials[J].Lancet, 2007,370(9600):1706-1713.

[6]James WP,Caterson ID,et al.Effect of sibutramine on cardiovascular outcomes in overweight and obese subjects[J].N Engl J Med,2010,363(10):905- 917.

[7]Kopelman1P,Groot Gde H,et al.Weight Loss,HbA1c Reduction,and Tolerability of Cetilistat in a Randomized,Placebo-controlled Phase 2Trial in Obese Diabetics:Comparison With Orlistat(Xenical)[J].Obesity,2010,18(1):108- 115.

[8] APOVIAN CM, ARONNE LJ, BESSESEN DH, et al.Pharmacological management of obesity:an endocrine Society clinical practice guideline[J].J Clin Endocrinol Metab, 2015,100 (2):342-362.

[9]Alexander CM and Werb,Z.(1991).Extracellular matrix degradation.In Cell Biology of Extracellular Matrix,Hay ED,ed.(New York:Plenum Press), pp.255-302.

[10]Werb,Z.,Mainardi,C.L.,Vater,C.A.,and Harris,E.D.,Jr.(1977) .Endogenous activiation of latent collagenase by rheumatoid synovial cells.Evidence for a role of plasminogen activator.N.Engl.J.Med.296,1017- 1023.

[11]He,C.S.,Wilhelm,S.M.,Pentland,A.P.,Marmer,B.L.,Grant,G.A.,Eisen, A.Z.,and Goldberg,G.I.(1989).Tissue cooperation in a proteolytic cascade activating human interstitial collagenase.Proc.Natl.Acad.Sci.U.S.A 86,2632- 2636.

[12]Stoppelli,M.P.,Corti,A.,Soffientini,A.,Cassani,G.,Blasi,F.,and Assoian,R.K.(1985). Differentiation-enhanced binding of the amino-terminal fragment of human urokinase plasminogen activator to a specific receptor on U937 monocytes.Proc.Natl.Acad.Sci.U.S.A 82,4939-4943.

[13]Vassalli,J.D.,Baccino,D.,and Belin,D.(1985).A cellular binding site for the Mr 55,000 form of the human plasminogen activator, urokinase.J.Cell Biol.100,86-92.

[14]Wiman,B.and Wallen,P.(1975).Structural relationship between" glutamic acid"and "lysine"forms of human plasminogen and their interaction with the NH2-terminal activation peptide as studied by affinity chromatography.Eur.J.Biochem.50,489-494.

[15]Saksela,O.and Rifkin,D.B.(1988).Cell-associated plasminogen activation:regulation and physiological functions.Annu.Rev.Cell Biol.4,93- 126.

[16]Raum,D.,Marcus,D.,Alper,C.A.,Levey,R.,Taylor,P.D.,and Starzl,T.E. (1980). Synthesis of human plasminogen by the liver.Science 208,1036-1037.

[17]Wallén P(1980).Biochemistry of plasminogen.In Fibrinolysis,Kline DL and Reddy KKN,eds.(Florida:CRC)

[18]Sottrup-Jensen,L.,Zajdel,M.,Claeys,H.,Petersen,T.E.,and Magnusson,S.(1975). Amino-acid sequence of activation cleavage site in plasminogen:homology with"pro"part of prothrombin.Proc.Natl.Acad.Sci.U.S.A 72,2577-2581.

[19]Collen,D.and Lijnen,H.R.(1991).Basic and clinical aspects of fibrinolysis and thrombolysis.Blood 78,3114-3124.

[20]Alexander,C.M.and Werb,Z.(1989).Proteinases and extracellular matrix remodeling. Curr.Opin.Cell Biol.1,974-982.

[21]Mignatti,P.and Rifkin,D.B.(1993).Biology and biochemistry of proteinases in tumor invasion.Physiol Rev.73,161-195.

[22]Collen,D.(2001).Ham-Wasserman lecture:role of the plasminogen system in fibrin- homeostasis and tissue remodeling.Hematology. (Am.Soc.Hematol.Educ.Program.)1-9.

[23]Rifkin,D.B.,Moscatelli,D.,Bizik,J.,Quarto,N.,Blei,F.,Dennis,P., Flaumenhaft,R., and Mignatti,P.(1990).Growth factor control of extracellular proteolysis.Cell Differ.Dev.32, 313-318.

[24]Andreasen,P.A.,Kjoller,L.,Christensen,L.,and Duffy,M.J.(1997).The urokinase-type plasminogen activator system in cancer metastasis:a review.Int.J.Cancer 72,1-22.

[25]Rifkin,D.B.,Mazzieri,R.,Munger,J.S.,Noguera,I.,and Sung,J.(1999) .Proteolytic control of growth factor availability.APMIS 107,80-85.

[26]Marder V J,Novokhatny V.Direct fibrinolytic agents:biochemical attributes,preclinical foundation and clinical potential[J].Journal of Thrombosis and Haemostasis,2010,8(3):433- 444.

[27]Hunt J A,Petteway Jr S R,Scuderi P,et al.Simplified recombinant plasmin:production and fu-nctional comparison of a novel thrombolytic molecule with plasma-derived plasmin[J]. Thromb Haemost,2008,100(3):413-419.

[28]Sottrup-Jensen L,Claeys H,Zajdel M,et al.The primary structure of human plasminogen: Isolation of two lysine-binding fragments and one“mini”- plasminogen(MW,38,000)by elastase-catalyzed-specific limited proteolysis[J] .Progress in chemical fibrinolysis and thrombolysis,1978,3:191-209.

[29]Nagai N,Demarsin E,Van Hoef B,et al.Recombinant human microplasmin:production and potential therapeutic properties[J].Journal of Thrombosis and Haemostasis,2003,1(2): 307-313.

[30]Eun Young Lee,Yeon Wook Kim,Anti-obesity effects of KR-66195,a synthetic DPP- IVinhibitor,in diet-induced obese mice and obese-diabetic ob/ ob mice,Metabolism chlinical and experimental 63(2014)793-799

[31]Yang Z,Li W,He C.Potential effect of chronic Helicobacter pylori infection on glucose metabolism of Mongolian gerbils.World J Gastroenterol.2015 Nov 28；21(44):12593-604.

[32]Hu Y,Rosa GJ,Gianola D.A GWAS assessment of the contribution of genomic imprinting to the variation of body mass index in mice.BMC Genomics.2015 Aug 5；16:576. [33]Goossens GH.The role of adipose tissue dysfunction in the pathogenesis of obesity- related insulin resistance.Physiol Behav.2008 May 23；94(2):206-18.

[34]Sahu A.Leptin signaling in the hypothalamus:emphasis on energy homeostasis and leptin resistance.Front Neuroendocrinol.2003 Dec；24(4):225- 53.

[35]Harris RB,Mitchell TD,Yan X et al.Metabolic responses to leptin in obese db/db mice are strain dependent.Am J Physiol Regul Integr Comp Physiol.2001 Jul；281(1):R115-32.

[36]Ennequin G1,Boisseau N1,Caillaud K et al.Neuregulin 1 affects leptin levels,food intake and weight gain in normal-weight,but not obese,db/ db mice.Diabetes Metab.2015 Apr；41(2):168-72.

[37]Dominika Nackiewicz,Paromita Dey,Barbara Szczerba et al.Inhibitor of differentiation 3, a transcription factor regulates hyperlipidemia associated kidney disease.Nephron Exp Nephrol.2014；126(3):141–147.

[38]Ming Gu1,Yu Zhang.,Shengjie Fan et al.Extracts of Rhizoma Polygonati Odorati Prevent High-Fat Diet-Induced Metabolic Disorders in C57BL/6 Mice.PLoS ONE 8(11): e81724.

[39]Siobhan M.Craige,PhD,Shashi Kant et al.Endothelial NADPH oxidase 4 protects ApoE-/-mice from atherosclerotic lesions.Free Radic Biol Med.2015 December；89:1–7.

[40]Sungwon Lee,Youngjoo Lee,Jiyeon Kim et al.Atorvastatin and rosuvastatin improve physiological parameters and alleviate immune dysfunction in metabolic disorders.Biochem Biophys Res Commun.2016 Sep 23；478 (3):1242-7.

[41]Yutaka Nakashima,Andrew S.Plump,Elaine W.Raines et al.Arterioscler Thromb.1994 Jan；14(1):133-40.

[42]Yvonne Nitschke,Gabriele Weissen-Plenz,Robert Terkeltaub et al.Npp1 promotes atherosclerosis in ApoE knockout mice.J.Cell.Mol.Med.Vol 15, No 11,2011 pp.2273-2283.

[43]Duan DM,Wu S,Hsu LA et al.Associations between TRPV4 genotypes and body mass index in Taiwanese subjects.Mol Genet Genomics.2015 Aug；290(4): 1357-65.

[44]Goossens GH.The role of adipose tissue dysfunction in the pathogenesis of obesity- related insulin resistance.Physiol Behav.2008 May 23；94(2):206-18.

[45]Shimizu H1,Shimomura K,Negishi M et al.Circulating concentrations of soluble leptin receptor:influence of menstrual cycle and diet therapy.Nutrition.2002 Apr；18(4):309-12.

[46]Kastin AJ1,Pan W,Maness LM et al.Decreased transport of leptin across the blood-brain barrier in rats lacking the short form of the leptin receptor.Peptides.1999 Dec；20(12):1449-53.

[47]Münzberg H1,Flier JS, C.Region-specific leptin resistance within the hypothalamus of diet-induced obese mice.Endocrinology.2004 Nov；145 (11):4880-9.Epub 2004 Jul 22。

Sequence table

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

<120>A kind of method and drug for preventing and treating obesity

<150> 2016111622375

<151> 2016-12-15

<160> 14

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2376

<212> DNA

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

<400> 1

gagcctctgg atgactatgt gaatacccag ggggcttcac tgttcagtgt cactaagaag 60

cagctgggag caggaagtat agaagaatgt gcagcaaaat gtgaggagga cgaagaattc 120

acctgcaggg cattccaata tcacagtaaa gagcaacaat gtgtgataat ggctgaaaac 180

aggaagtcct ccataatcat taggatgaga gatgtagttt tatttgaaaa gaaagtgtat 240

ctctcagagt gcaagactgg gaatggaaag aactacagag ggacgatgtc caaaacaaaa 300

aatggcatca cctgtcaaaa atggagttcc acttctcccc acagacctag attctcacct 360

gctacacacc cctcagaggg actggaggag aactactgca ggaatccaga caacgatccg 420

caggggccct ggtgctatac tactgatcca gaaaagagat atgactactg cgacattctt 480

gagtgtgaag aggaatgtat gcattgcagt ggagaaaact atgacggcaa aatttccaag 540

accatgtctg gactggaatg ccaggcctgg gactctcaga gcccacacgc tcatggatac 600

attccttcca aatttccaaa caagaacctg aagaagaatt actgtcgtaa ccccgatagg 660

gagctgcggc cttggtgttt caccaccgac cccaacaagc gctgggaact ttgtgacatc 720

ccccgctgca caacacctcc accatcttct ggtcccacct accagtgtct gaagggaaca 780

ggtgaaaact atcgcgggaa tgtggctgtt accgtgtccg ggcacacctg tcagcactgg 840

agtgcacaga cccctcacac acataacagg acaccagaaa acttcccctg caaaaatttg 900

gatgaaaact actgccgcaa tcctgacgga aaaagggccc catggtgcca tacaaccaac 960

agccaagtgc ggtgggagta ctgtaagata ccgtcctgtg actcctcccc agtatccacg 1020

gaacaattgg ctcccacagc accacctgag ctaacccctg tggtccagga ctgctaccat 1080

ggtgatggac agagctaccg aggcacatcc tccaccacca ccacaggaaa gaagtgtcag 1140

tcttggtcat ctatgacacc acaccggcac cagaagaccc cagaaaacta cccaaatgct 1200

ggcctgacaa tgaactactg caggaatcca gatgccgata aaggcccctg gtgttttacc 1260

acagacccca gcgtcaggtg ggagtactgc aacctgaaaa aatgctcagg aacagaagcg 1320

agtgttgtag cacctccgcc tgttgtcctg cttccagatg tagagactcc ttccgaagaa 1380

gactgtatgt ttgggaatgg gaaaggatac cgaggcaaga gggcgaccac tgttactggg 1440

acgccatgcc aggactgggc tgcccaggag ccccatagac acagcatttt cactccagag 1500

acaaatccac gggcgggtct ggaaaaaaat tactgccgta accctgatgg tgatgtaggt 1560

ggtccctggt gctacacgac aaatccaaga aaactttacg actactgtga tgtccctcag 1620

tgtgcggccc cttcatttga ttgtgggaag cctcaagtgg agccgaagaa atgtcctgga 1680

agggttgtag gggggtgtgt ggcccaccca cattcctggc cctggcaagt cagtcttaga 1740

acaaggtttg gaatgcactt ctgtggaggc accttgatat ccccagagtg ggtgttgact 1800

gctgcccact gcttggagaa gtccccaagg ccttcatcct acaaggtcat cctgggtgca 1860

caccaagaag tgaatctcga accgcatgtt caggaaatag aagtgtctag gctgttcttg 1920

gagcccacac gaaaagatat tgccttgcta aagctaagca gtcctgccgt catcactgac 1980

aaagtaatcc cagcttgtct gccatcccca aattatgtgg tcgctgaccg gaccgaatgt 2040

ttcatcactg gctggggaga aacccaaggt acttttggag ctggccttct caaggaagcc 2100

cagctccctg tgattgagaa taaagtgtgc aatcgctatg agtttctgaa tggaagagtc 2160

caatccaccg aactctgtgc tgggcatttg gccggaggca ctgacagttg ccagggtgac 2220

agtggaggtc ctctggtttg cttcgagaag gacaaataca ttttacaagg agtcacttct 2280

tggggtcttg gctgtgcacg ccccaataag cctggtgtct atgttcgtgt ttcaaggttt 2340

gttacttgga ttgagggagt gatgagaaat aattaa 2376

<210> 2

<211> 791

<212> PRT

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

<400> 2

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

Glu Gly Val Met Arg Asn Asn

785 790

<210> 3

<211> 2433

<212> DNA

<213>Natural plasminogen (nucleic acid sequence for deriving from swiss prot) containing signal peptide

<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 (amino acid sequence for deriving from swiss prot) containing signal peptide

<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- plasminogen amino acids 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- plasminogen amino acids 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 protokaryon 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 (the molten proenzyme amino acid sequence of fibrillin)

<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 (nucleic acid sequence of structural 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 (amino acid sequence of structural 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. purposes of the plasminogen in the drug for preparing prevention or treatment subject's obesity.

2. the purposes of claim 1, wherein the plasminogen by reduce lipid around histoorgan, histoorgan and/ Or exception or over-deposit the treatment obesity in abdominal cavity.

3. the purposes of claim 2, wherein the plasminogen reduces lipid in subcutaneous, heart, liver, lungs, kidney, blood Pipe, mesenterium, peritonaeum, body cavity, exception or over-deposit around organ.

4. the purposes of claim 1, wherein the plasminogen reduces subject's blood fat, especially triglycerides and low density lipoprotein The horizontal stretcher obesity of albumen.

5. the purposes of any one of claim 1-4, wherein the obesity causes for simple obesity or secondary to Other diseases Obesity.

6. plasminogen prepare prevention or treatment subject's obesity drug in purposes, wherein the obesity secondary to Endocrine disturbance disease, the nervous system disease, glycometabolism disease, liver diseases, kidney trouble, angiocardiopathy, intestines problem, Thyroid disease, gall-bladder or disease of biliary tract, excessive consumption of alcohol, drug effect.

7. purposes of the plasminogen in the fat drug of prevention and/or treatment subject disease concurrently is prepared, wherein described It is concurrent that the concurrent obesity of disease includes the concurrent obesity of the concurrent obesity of endocrine system disease, metabolic disease, angiocardiopathy The concurrent obesity of the concurrent obesity of obesity, disease of digestive system, degenerative disease.

8. the purposes of claim 7, wherein the fat obesity concurrent including diabetes complicated obesity, hypertension, artery The concurrent obesity of the concurrent obesity of the concurrent obesity of atherosis, liver diseases, osteoporosis.

9. the purposes in plasminogen drug of complication caused by prevention or treatment obesity is prepared, is controlled including administration subject A effective amount of plasminogen is treated, wherein the complication of the obesity includes cardiovascular and cerebrovascular disease, metabolic disease, muscle skeleton disease Disease, disease of digestive system, sleep apnea, respiratory disorder.

10. the purposes of claim 9, wherein the complication is hypertension, diabetes, coronary heart diseases and angina pectoris, myocardial infarction, Arrhythmia cordis, atherosclerosis, cerebral thrombus, cerebral hemorrhage, osteoarthritis, osteoproliferation, cholecystitis, fatty liver, hepatic sclerosis, bone Matter is loose.