CN108210892A - Prevent and treat drug of liver fibrosis and application thereof - Google Patents

Abstract

The present invention relates to a kind of method for preventing and treating liver fibrosis, including a effective amount of plasminogen of subject is administered.

Description

Prevent and treat drug of liver fibrosis and application thereof

Technical field

The present invention relates to a kind of method for preventing and treating liver fibrosis, including a effective amount of fibrinolysin of subject is administered It is former.

Background technology

Fibrosis is a kind of pathological change, shows as fibroblasts activation proliferation, fibrous connective tissue in histoorgan Increase, parenchyma is reduced, and continuing advances can cause tissue, organ structure destruction and function to lose.The fibrosis of important organ is tight Ghost image rings life in patients or even threat to life.In worldwide, tissue fibrosis is that many diseases disable, is lethal The main reason for, it is proved according to the related statistics in the U.S., in state patient lethal due to various diseases, about 45% can be attributed to Tissue fibers proliferative disease.

Fibrotic disease includes involving the disease of multisystem, such as systemic sclerosis, multifocal fibrosis, chorionitis, kidney The multisystem fibrosis of source property, also including organ-tissue specific diseases, such as skin, the heart, lung, liver, kidney fibrosis.It is different fine The cause of disease of dimensionization disease is different, such as damage, infection, immune response, chronic inflammation of histoorgan etc., but it is common It is characterized in extracellular matrix (ECM) over-deposit in the tissue and organ-tissue reconstruct^[1-3]。

Cardiac fibrosis is come across in the generation and evolution of a variety of heart diseases, such as vital myocarditis, cardiac muscle stalk Dead and hypertensive heart disease etc..Excessive cardiac fibrosis causes cardiac function to be damaged, and is that a variety of diseases move towards the main of heart failure Reason, wherein oneself with reverse myocardial fibrosis is inhibited to become the important link for the treatment of angiocardiopathy.

Liver fibrosis refers to caused by various virulence factors connective tissue paraplasm in liver, the extracellular base of diffusivity in liver The pathologic process of matter excessive deposition.Many factors can cause liver fibrosis, such as viral infection, inflammatory reaction, oxidative stress and Excessive drinking etc..The pathological characteristic of liver fibrosis is has a large amount of proliferations of fibrous tissue and deposition, but not yet shape in portal area and lobuli hepatis Be spaced in into leaflet, hepatic sclerosis then has pseudolobuli to be formed, and central vein area and portal area are spaced, the normal configuration of liver by It destroys, further development is hepatic sclerosis for liver fibrosis.In the chronic liver disease in China based on virus hepatitis, chronic viral Inflammation, necrosis, virus replication etc. are related, and are reversible in early days in the liver tissue fibrosis of hepatitis and liver.It thus will be anti- Viral therapy adjusts the therapies such as body's immunity and protection liver cell and combines with anti-fibrosis, is to prevent liver fibrosis Active measures.

Pulmonary fibrosis disease is led including idiopathic pulmonary fibrosis, sarcoidosis, hylactic pneumonia, pneumoconiosis, drug and radioactive ray Different, the in extensive range spectrum of disease of the causes of disease such as the fibrosis of cause and the fibrosis pulmonary alveolitis related with collagen vascular disease.Its Main pathological characteristic includes the pictures such as the reconstruct of lung tissue mesenchymal cell proliferation, cell extracellular matrix hyperplasia deposition and pulmonary parenchyma.Mesh It is preceding that pulmonary fibrosis is mainly treated using anti-inflammatory, anti-oxidant, anti-fibroblast proliferation and the measures such as collagen deposition and lung transplantation.

Kidney fibrosis shows as extracellular matrix and inappropriate connective tissue to be assembled in kidney, leads to Reno-colic fistula change and function Impaired pathologic process and nearly all kidney diaseases proceed to the co-channel of end-stage renal failure.Kidney fibrosis process is related to The apoptosis of inflammatory reaction, intrinsic cell and immunocyte and a variety of that modulates fibrosis factors are unbalance etc., thus can by anti-inflammatory, Anti-apoptotic and resist kidney fibrosis for approach such as fibrosis factor in treatment.

The chronic disease of histoorgan is often accompanied by fibrosis, such as the chronic inflammation of lung, chronic disease, with lung fiber Change.Liver fibrosis is also in this way, as hepatitis B, hepatitis, alcoholic liver, fatty liver, snail fever etc. are all with Early hepatic fibrosis.Liver Compensation it is very strong, fibrosis lesion is hidden always in various chronic liver diseases, General development be hepatic sclerosis when be just found, Actually hepatic sclerosis has been the severe stage of liver fibrosis.Again as chronic nephritis, glomerulitis, renal tubule inflammation etc. have kidney fibre Dimensionization；Angiocarpy, the cerebrovascular, lower limb vascular hardening narrow or congestion situations under have vascular fibrosis.

Fibrosis of skin forms cicatricial tissue.Cicatricial tissue is fibre of the granulation tissue through the reconstruction ripe ageing step formed Tie up connective tissue.When wound, fibroblast division, proliferation migrate to damaged part, generate extracellular matrix, shape Into cicatricial tissue, wound is repaired.

The formation of scar is the process of the gradual fibrosis of granulation tissue.Reticular fibre and collagenous fibres are more and more at this time, Reticular fibre collagenzation, collagenous fibres are thicker, and at the same time fibroblast is fewer and fewer, and remaining person is changed into fiber finer on a small quantity Born of the same parents；Liquid is gradually absorbed in interstitial, and neutrophil leucocyte, macrophage, lymphocyte and thick liquid cell successively disappear；Capillary It is closed, degenerates, disappears, leave seldom parteriole and veinlet.In this way, granulation tissue is to be transformed into mainly by collagenous fibres group Into blood vessel rareness cicatricial tissue, naked eyes it is white, it is strong but pliable in texture.

It since scar is tough and tensile and lacks flexibility, in addition cicatricial contraction can cause metallaxis and dysfunction, so occurring In the scar of juxtra-articular and important organ, often cause joint spasm or limitation of activity, such as in alimentary canal, the urinary tract chamber device Official then causes luminal stenosis, then causes dyskinesia in juxtra-articular.It is happened between organ or between organ and body lumen wall Cicatricial adhesion often influences its function to some extent.As extensive fibrosis, hyalinization occurs after extensive injuries in organ, Then lead to organ sclerosis.

Systemic sclerosis (SSc) is also referred to as chorionitis, be one kind with limitation or diffusivity pachyderma and fibrosis The systemic autoimmune diseases being characterized.Lesion characteristic changes for skin fiber hyperplasia and blood vessel onion-skin sample, eventually leads to skin Skin hardening, blood vessel ischemic.This disease is clinically characterized by limitation or diffusivity pachyderma and fibrosis, except skin involvement Outside, it can also influence internal organ (organs such as the heart, lung and alimentary canal).

Atherosclerosis frequently results in tissue, organ ischemia's damage, then causes the fibrosis lesion of histoorgan.It is dynamic Pulse atherosclerosis is a kind of chronic, gradual arterial disease, the fats portion that is deposited in artery during morbidity or all blocks blood Stream.When otherwise smooth, solid endarterium is roughening, thickens, and when being blocked by fat, fibrin, calcium and cell debris, Just there is atherosclerosis.Atherosclerosis is the chronic inflammation preneoplastic lesions of endarterium, leads to large and medium-sized artery Luminal stenosis or occlusion cause the damage of corresponding organ tissue ischemia, fibrosis or even necrosis.

Atherosclerosis is in close relations with diabetes, shows as diabetic and the time of atherosclerosis occurs Early, degree weight and poor prognosis, and atherosclerosis is the major causes of death of diabetic.Clinical discovery patient of diabetes Blood vessel that the characteristics of coronary artery pathological change of person, mainly lesion was involved is more, coronary artery stenosis is serious, lesion It more diffuses seriously, mechanism is considered that metabolism of blood glucose causes atherosclerosis extremely more, as further deep is ground Study carefully, it is more the result shows that, diabetes cause caused by atherosclerosis is not single factors, but through a variety of ways and More complicated mechanism is come the generation and development that induce and promote atherosclerosis, such as macrophage polarization, macrophage Migration inhibition factor approach, glycosylation end products approach, scavenger receptor up-regulation, insulin resistance, Ubiquitin-proteasome system Activation, platelet derived growth factor activated pathway etc.^[4]。

At present, fibrotic disease morbidity crowd is numerous caused by a variety of causes, and patient often has multiple organ-tissues to be involved, Still lack effective therapy, society and financial burden are heavier.Present invention research finds that plasminogen can improve organizer The fibrosis of official improves tissue organ function, so as to be opened up for the prevention and treatment of histoorgan fibrosis and its associated disease One completely new field.

Summary of the invention

The present invention includes following items：

1. a kind of side of Liver Collagen proteinosis or fibrosis caused by prevention or treatment subject liver organization damage Method, including a effective amount of plasminogen of subject is administered.

2. 1 method, wherein the damage includes Ischemia-reperfusion injury, Drug damage, immune injury, change It is damaged caused by damage, cancer caused by damage, inflammatory injury, excessive consumption of alcohol caused by learning substance.

3. 2 method, wherein the damage includes damage caused by fat deposition.

4. a kind of method prevented or treat subject's Liver Collagen proteinosis or fibrosis, has including administration subject The plasminogen of effect amount.

5. a kind of prevention or treatment sub-ject cause or the side of concurrent Liver Collagen proteinosis or fibrosis Method, including a effective amount of plasminogen of subject is administered.

6. a kind of prevention or treatment subject artery's atherosis cause or concurrent Liver Collagen proteinosis or fiber The method of change, including a effective amount of plasminogen of subject is administered.

Prevent or treatment subject's hyperlipidemia causes or concurrent Liver Collagen proteinosis or fibrosis 7. a kind of Method, including a effective amount of plasminogen of subject is administered.

8. 7 method, wherein the hyperlipidemia include it is chosen from the followings one or more：Blood triglyceride levels liter High, blood total cholesterol level raising, the raising of blood low-density lipoprotein, the raising of blood very low density lipoprotein.

9. a kind of side of Liver Collagen proteinosis for preventing or treating the initiation of subject's drug induced hepatic injury or fibrosis Method, including a effective amount of plasminogen of subject is administered.

10. 9 method, wherein the drug is hepatotoxic medication.

11. 10 method, wherein the drug includes chemotherapeutics, antibiotic medicine, blood lipid-lowering medicine, anti-hypertension Drug, hypoglycemic medicine.

12. a kind of prevention or treatment subject's alcoholic liver collagen deposition or fibrosis or nonalcoholic hepatic glue The method of former proteinosis or fibrosis, including a effective amount of plasminogen of subject is administered.

13. a kind of side of Liver Collagen proteinosis or fibrosis caused by prevention or treatment subject's chronic hepatic diseases Method, including a effective amount of plasminogen of subject is administered.

14. a kind of side of Liver Collagen proteinosis or fibrosis caused by prevention or treatment subject's virus hepatitis Method, including a effective amount of plasminogen of subject is administered.

15. a kind of method prevented or treat subject's liver fibrosis associated disease, a effective amount of including administration subject Plasminogen.

16. 15 method, wherein the liver fibrosis associated disease include the decreased liver function caused by liver fibrosis, Obstacle or forfeiture and the illness caused.

17. according to the method for any one of item 1-16, wherein the plasminogen can be with one or more other medicines or controlling Treatment means are administered in combination.

18. according to the method for item 17, wherein the other medicines include：Blood lipid-lowering medicine, antiplatelet drug, blood pressure lowering Drug, vasodilator drug, hypoglycemic drug, anticoagulation medicine, thrombolytic agent, hepatic, anti-fibrosis medicine, the anti-heart Restrain arrhythmic agents, cardiac drug, diuretic, antitumor drug, radiotherapy and chemotherapy medicine, inflammation adjust drug, immunoregulation medicament, Antiviral drugs, antibiotic.

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

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

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

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

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

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

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

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

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

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

29. the method for any one of 1-28, the shortage or missing are inborn, secondary and/or local.

30. a kind of plasminogen of method for any one of item 1-29.

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

32. a kind of preventative or therapeutic agent box, it includes：(i) for the fibre of any one of item 1-29 the methods Lyase original and (ii) are for delivering the plasminogen to the component (means) of the subject.

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

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

35. a kind of product, it includes：

Container containing label；With

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

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

37. 36 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, anti-fibrosis Drug, antiarrhythmic drug, cardiac drug, diuretic, antitumor drug, radiotherapy and chemotherapy medicine, inflammation adjust drug, are immunized Adjust drug, antiviral drugs, antibiotic.

On the one hand, the present invention relates to a kind of prevention and/or treatment subject's histoorgan collagen deposition or fibrosis And its method of associated disease, including a effective amount of plasminogen of subject is administered, wherein the subject is susceptible to suffer from histoorgan Fibrosis, in a organized way organ fibrosis are inclined to or suffer from Other diseases and accompanied by tissue organ fibrosis.The invention further relates to fibres Lyase is originally intended to prevent and/or treats the purposes of subject's histoorgan collagen deposition or fibrosis and its associated disease. The invention further relates to plasminogen be used to prepare prevention and/or treatment subject's histoorgan collagen deposition or fibrosis and The purposes of the drug of its associated disease.Moreover, it relates to for preventing and/or treating subject's histoorgan collagen egg White deposition or the plasminogen of fibrosis and its associated disease.In some embodiments, the histoorgan collagen sinks Product or fibrosis include fibrosis of skin, vascular fibrosis, cardiac fibrosis, pulmonary fibrosis, liver fibrosis, kidney fibrosis. In other embodiments, the histoorgan collagen deposition or fibrosis include infection, inflammation, hypersensitivity, tumour, It is that damage caused by tissue ischemia, histoorgan extravasated blood, chemical substance, radiation or environmental pollution causes or adjoint.Specifically, The histoorgan collagen deposition or fibrosis include bacterium, virus or histoorgan lesion is led caused by parasitic infection The histoorgan collagen deposition or fibrosis of cause, wherein, the histoorgan collagen deposition or fibrosis include knot Pulmonary fibrosis caused by core bacillus infection, hepatitis type B virus, Hepatitis C Virus or liver caused by hepatitis e virus infection Liver fibrosis caused by dirty fibrosis, infection by Schistosoma.In some embodiments, the histoorgan collagen deposition or Fiber is turned to caused by aseptic inflammation or autoimmune response.Specifically, the histoorgan collagen deposition or fiber Renal fibrosis caused by turning to chronic glomerulonephritis, pyelonephritis, nephrotic syndrome, renal insufficiency, uremia.Another In some embodiments, the histoorgan collagen deposition or fiber are turned to caused by cancer leads to injuries of tissues and organs. Specifically, liver fiber caused by pulmonary fibrosis, liver cancer caused by the histoorgan collagen deposition or fiber turn to lung cancer Renal fibrosis caused by change or cancer kidney.In other embodiments, the histoorgan collagen deposition or fibre Dimension is turned to caused by chronic ischemic tissue damage.Specifically, the histoorgan collagen deposition or fiber turn to coronal Kidney fibrous caused by heart ischemia fibrosis caused by atherosclerosis, coronary heart disease and/or chronic ischemic injury of kidney Change.In other embodiments, the histoorgan collagen deposition or fiber are organized caused by turning to angiocardiopathy Caused by organ extravasated blood.Specifically, the histoorgan collagen deposition or fiber turn to hepatic congestion or pulmonary venous pleonaemia.At some In embodiment, the histoorgan collagen deposition or fiber turn to drug induced.Specifically, the histoorgan glue Former proteinosis or fiber turn to Drug liver fibrosis or kidney fibrosis.In some embodiments, the histoorgan glue Former proteinosis or fiber turn to pulmonary fibrosis caused by imbedibility chemical substance or environmental contaminants.In the embodiment above In, the histoorgan collagen deposition or fiber turn to such as systemic loupus erythematosus, systemic sclerosis, tatanic ridge Caused by column inflammation systemic immunity disease.In some embodiments, the histoorgan fiber turns to idiopathic lung fiber Change.

In the above-described embodiment, the histoorgan fibrosis-associated disorder includes histoorgan because fibrosis lesion is led Miopragia, obstacle or the forfeiture of cause and the illness caused.Specifically, the histoorgan fibrosis-associated disorder includes artery Atherosis, coronary heart diseases and angina pectoris, myocardial infarction, arrhythmia cordis, cerebral ischemia, cerebral infarction, renal insufficiency, uremia, liver function It can obstacle, hepatic sclerosis, hepatic coma, expiratory dyspnea, pulmonary emphysema, pulmonary heart disease, pulmonary fibrosis, ankylosing spondylitis.

In the above-described embodiment, the plasminogen can be combined with one or more other medicines or therapy.Tool Body, the plasminogen can be with one or more drug combinations chosen from the followings：Blood lipid-lowering medicine, antiplatelet drug, drop Blood pressure medication, vasodilator drug, hypoglycemic drug, anticoagulation medicine, thrombolytic agent, hepatic, anti-fibrosis medicine, Antiarrhythmic drug, cardiac drug, diuretic, antitumor drug, radiotherapy and chemotherapy medicine, inflammation adjust drug, immunological regulation Drug, antiviral drugs, antibiotic.

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

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

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

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

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

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

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

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

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

In the above-described embodiment, the kit or product, should also comprising other one or more components or container Contain other drugs in component or container.In some embodiments, the other drugs are selected from the group：Blood lipid-lowering medicine resists Antiplatelet drug, blood-pressure drug, vasodilator drug, hypoglycemic drug, anticoagulation medicine, thrombolytic agent, hepatic, Anti-fibrosis medicine, antiarrhythmic drug, cardiac drug, diuretic, antitumor drug, radiotherapy and chemotherapy medicine, inflammation are adjusted Drug, immunoregulation medicament, antiviral drugs, antibiotic.

Definition

" fibrosis " is the tissues such as lung, liver, kidney, blood vessel, peritonaeum, pancreas, skin, organ by inflammation, infection, immune anti- Should, after ischemic, chemical substance, a variety of causes such as radiation lead to Continuous Damage, fibre in fibroblasts activation proliferation, histoorgan Dimension connective tissue increases, and parenchyma is reduced, tissue, the lesion that organ structure destroys and function is lost.The term can be with " fiber Change lesion " it is used interchangeably.Cardiac fibrosis, pulmonary fibrosis, liver fiber caused by the fibrosis lesion term covers a variety of causes The histoorgans fibrosis lesion such as change, kidney fibrosis, vascular fibrosis, fibrosis of skin further includes various diseases and occurs, develops Cardiac fibrosis, pulmonary fibrosis, liver fibrosis, kidney fibrosis, vascular fibrosis, fibrosis of skin for supervening in the process etc. Histoorgan fibrosis lesion.

After fibrosis lesion occurs for tissue, organ, normal configuration changes, corresponding miopragia or forfeiture, by This caused associated disease is known as " histoorgan fibrosis-associated disorder ".

" cardiac fibrosis " refers to that a variety of causes (such as inflammation, infection, immune response, ischemic, chemical substance, radiation) is led The fiber occurred in the generation and evolution of heart disease caused by damage to cardiac tissue or a variety of causes cause or adjoint Change lesion.Cardiac fibrosis lesion causes cardiac function to be damaged, and thus caused associated disease is known as " cardiac fibrosis related diseases Disease ", including but not limited to cardiac function are damaged the symptom and illness of caused each organ, tissue ischemia, such as coronary heart disease, the heart twist Bitterly, myocardial infarction, arrhythmia cordis, cerebral ischemia, expiratory dyspnea, renal insufficiency etc..

" liver fibrosis " refers to by a variety of causes (such as inflammation, infection (such as virus infection), immune response, ischemic, change Learn substance, radiation, oxidative stress and excessive drinking) caused by or adjoint liver in connective tissue paraplasm, diffusivity cell in liver Epimatrix excessive deposition, the pathological change (lesion) that is destroyed of normal configuration of liver.Further development is liver for liver fibrosis Hardening is also covered by the range of " liver fibrosis " term of the invention.Hepatic fibrosis-renal tubular ectasia syndrome lesion leads to compromised liver function, thus Caused associated disease is known as " hepatic fibrosis-renal tubular ectasia syndrome associated disease ".

" pulmonary fibrosis " refers to that a variety of causes (such as inflammation, infection, immune response, ischemic, chemical substance, radiation) causes Or adjoint lung tissue mesenchymal cell proliferation, cell extracellular matrix hyperplasia deposition and pulmonary parenchyma reconstruct and caused by pathology mistake Journey.Lungs fibrosis lesion causes lungs function to be damaged, and thus caused associated disease is known as " lungs fibrosis-associated disorder ".

" renal fibrosis " refers to that a variety of causes (such as inflammation, infection, immune response, ischemic, chemical substance, radiation) is led Cause or adjoint connective tissue causes Renal Structure to change and impaired pathologic process in the abnormal aggregation of kidney.Kidney Dirty fibrosis lesion is the co-channel that nearly all kidney trouble proceeds to the later stage.

Renal fibrosis lesion leads to Impaired kidney function, and thus caused associated disease is known as " renal fibrosis correlation Illness ", for example, renal function is not complete, renal failure, uremia etc..

The chronic disease of histoorgan is often accompanied by fibrosis, such as the chronic inflammation of lung, chronic disease, with lung fiber Change.Liver fibrosis is also in this way, all with Early hepatic fibrosis such as hepatitis B, hepatitis, alcoholic liver, fatty liver, snail fever. Again as chronic nephritis, glomerulitis, renal tubule inflammation etc. have kidney fibrosis；Angiocarpy, the cerebrovascular, lower limb vascular hardening become There is vascular fibrosis under narrow or congestion situations." fibrosis " or " fibrosis lesion " term of the invention is covered body and is respectively organized The fibrosis lesion that the chronic disease of organ is often accompanied by.

" systemic sclerosis " or " chorionitis " is that one kind turns to spy with limitation or diffusivity pachyderma and fiber The systemic autoimmune diseases of sign.Lesion characteristic changes for skin fiber hyperplasia and blood vessel onion-skin sample, and it is hard to eventually lead to skin Change, blood vessel ischemic.This disease is clinically characterized by limitation or diffusivity pachyderma and fibrosis, in addition to skin involvement, it Also internal organ (organs such as the heart, lung and alimentary canal) can be influenced.

" atherosclerosis " is a kind of chronic, gradual arterial disease, the fats portion deposited in artery during morbidity Or all block blood flow.Atherosclerosis is a progressive process.When the Lipid Concentration in blood greatly increases, Bian Huiyan It arterial wall and forms fatty streaks.These stripeds can cause metabolism of lipid and cholesterol to deposit, these precipitations are attached to originally smooth On endarterium, so as to form brief summary.Then the cicatricial tissue of fibrosis is grown below these brief summaries, leads to doped calcium.Deposition Calcium evolve as the hard film of chalk shape (be known as atheromatous plaque) that can not be removed.When with internal some tissue, device It after the artery that official is connected is blocked, is organized because caused by being obstructed the artery in the tissue, organ, the ischemic injuries of organ can be led Cause the fibrosis lesion of the tissue, organ, such as the fiber of heart, lungs, liver, kidney, blood vessel, peritonaeum, pancreas, skin Change.

Diabetes are often accompanied by the generation of atherosclerosis, and mechanism is considered the extremely caused artery congee of metabolism of blood glucose more Sample hardens, with further in-depth study, it is more the result shows that, diabetes cause atherosclerosis be not it is single because Caused by element, but through a variety of ways and more complicated mechanism induces and promote the generation of atherosclerosis and hair Exhibition^[4].Diabetes and its adjoint atherosclerosis can lead to tissue, organ damage and fibrosis, for example, heart, lungs, The fibrosis of the tissues such as liver, kidney, blood vessel, peritonaeum, pancreas, skin, organ.

Detailed description of the invention

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^[5].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^[6,7].Fibrinolysin be by The PAs that plasminogen passes through two kinds of physiologicals：Tissue-type plasminogen activator (tPA) or urokinase type plasminogen activator (uPA) proteolysis is formed.Due to plasminogen, relative level is higher in blood plasma and other body fluid, conventionally PA systems 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.The main suppression of fibrinolysin Preparation is α 2- antiplasmins (α 2-antiplasmin).The activity of PAs is inhibited simultaneously by the plasminogen activator of uPA and tPA Agent -1 (PAI-1) inhibits and the main plasminogen activator agent inhibitor -2 (PAI-2) for inhibiting uPA is adjusted.Certain cell surfaces UPA specific cell surface receptors (uPAR) with direct hydrolysis activity^[8,9]。

Plasminogen is a single chain glycoprotein, is made of 791 amino acid, and molecular weight is about 92 kDa^[10,11].Fibrinolytic Proenzyme is mainly synthesized in liver, is largely present in extracellular fluid.Content of plasminogen is about 2 μM in blood plasma.Therefore plasminogen It is a huge potential source of proteolytic activity in tissue and body fluid^[12,^13].There are two kinds of molecule shapes for plasminogen Formula：Glutamic acid-plasminogen (Glu- plasminogen) and lysine-plasminogen (Lys-plasminogen).Natural point It secretes with the plasminogen of uncracked form tool there are one amino terminal (N- ends) glutamic acid, therefore is referred to as glutamic acid-fibrinolytic Proenzyme.However, in the presence of fibrinolysin, glutamic acid-plasminogen is hydrolyzed at Lys76-Lys77 as lysine-fibrinolysin It is former.Compared with glutamic acid-plasminogen, lysine-plasminogen has higher affinity with fibrin, and can be with higher Rate activated by PAs.The Arg560-Val561 peptide bonds of the plasminogen of both forms can be cut by uPA or tPA, be caused The formation of the dichain proteins enzyme fibrinolysin of disulfide bond connection^[14].The amino terminus portion of plasminogen includes five homologous tricyclics, I.e. so-called kringles, carboxy-terminal sections include protease domain.Some kringles contain mediation plasminogen with Fibrin and its lysine-binding site of inhibitor α 2-AP specificity interaction.One plasminogen of latest find is The segment of 38kDa is effective inhibitor of angiogenesis including kringles1-4.This segment is named as blood vessel suppression Element can be generated by several protease hydrolytic plasminogens.

The main substrate of fibrinolysin is fibrin, and fibrinous dissolving is to prevent the pass that pathologic thrombus is formed Key^[15].Fibrinolysin also has the substrate specificity of several components to ECM, including laminin, fibronectin, proteoglycans And gelatin, show that fibrinolysin also plays an important role in ECM reconstructions^[11,16,17].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^[18].In addition, fibrinolysin has There is the ability for the growth factor for activating certain potential forms^[19-21].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^[22,23], there is the amino acid sequences (sequence 8) of document report delta- plasminogens^[23], 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)^[24], 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)^[25], also there is patent document CN102154253A to report that its sequence includes residue Lys531-Asn791 (not contain signal The Glu residues of the Glu- plasminogen sequences of peptide are initial amino acid), this patent sequence reference patent document CN102154253A, amino acid sequence encode cDNA sequence such as 11 institute of sequence of the amino acid sequence as shown in sequence 12 Show.

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

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

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

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

" activities of endothelial tissue plasminogen segment " 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 plasminogen live The detection (plgA) of property, 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 under the action of SK, the latter acts on chromophoric substrate, then uses and divides Light photometric determination, absorbance increase directly proportional to activities of endothelial tissue plasminogen.In addition can also be used immuno-chemical method, gel electrophoresis, Immunoturbidimetry, radioimmunodiffusion etc. are measured the activities of endothelial tissue plasminogen in blood.

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

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

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

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

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

In the case of using ALIGN-2 come comparing amino acid sequence, amino acid sequence A is given relative to given amino acid Sequence B % amino acid sequence identities (or can be expressed as having or comprising relative to, with or for given amino acid 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), sand Lei Shi Pseudomonas (Serratia) and various pseudomonas (Pseudomonas) species.In these prokaryotic hosts, it can also generate Expression vector would generally contain the expression control sequence (such as replication orgin) compatible with host cell.Permitted in addition, can exist More well known promoters, such as lactose promoter system, tryptophan (trp) promoter systems, beta- iactamase promoters system System or the promoter systems from phageλ.Promoter would generally control expression, optionally in the case of operator sequence, 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 include Chinese hamster ovary celI system, various Cos cell lines, HeLa cells, myeloma cell line and inverted B cell or Hybridoma.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- second Glycol (PEG).It is preferred that the anti-VEGF antibody formulations being lyophilized are 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 It is shorter.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 Thio Disulfide interchange and form intermolecular S -- S, 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 as caused by diabetes The plasminogen or fibrinolysin of the present invention of cardiovascular disease and its associated disease.The product preferably includes a container, label or Package insert.Appropriate container has bottle, bottle, syringe etc..Container can be made of a variety of materials such as glass or plastics.It is described Container contains composition, and the composition can effectively treat the disease of the present invention or illness and (such as described with sterile access port Container can be intravenous solution packet or bottle, and containing can be by the plug that hypodermic needle penetrates).In the composition at least A kind of activating agent is plasminogen/fibrinolysin.On the container or appended label illustrates the composition for treating this hair The bright cardiovascular disease as caused by diabetes and its associated disease.The product can further include containing pharmaceutically acceptable buffer solution Second container, the brine of such as phosphate-buffered, Ringer's solution and glucose solution.It can further include from business With other materials required from the point of view of user's angle, including other buffer solutions, diluent, filtrate, needle and syringe.In addition, The product includes the package insert with operation instruction, including for example indicating the user of the composition by plasminogen group It closes object and treats the other medicines administered patient of adjoint disease.

Brief description

The induced liver fibrosis model mouse liver sirius red stains that Fig. 1 gives 28 days carbon tetrachloride of plasminogen are seen Examine 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 Display is considerably less than to plasminogen group collagen deposition (arrow logo) and gives solvent PBS control group, and statistical discrepancy is shown Write (* expressions P<0.05)；Compared with to solvent PBS control group, blank pair is more nearly to plasminogen group collagen deposition level According to mouse.Illustrate that plasminogen can reduce the deposition of Liver Fibrosis Model mouse liver collagen, improve hepatic fibrosis-renal tubular ectasia syndrome.

Fig. 2 ApoE Atherosclerosis Model mouse give plasminogen 30 days posterior sinus of Valsalva sirius red stains generations Table picture.A, C is gives solvent PBS control group, and B, D is give plasminogen group.As a result it is shown to plasminogen group collagen The area of deposition (arrow logo), which is significantly less than, gives solvent PBS control group, illustrates that plasminogen can cut down artery congee hardening mould Type mouse aorta sinus Fibrosis levels.

Fig. 3 gives the Liver Fibrosis Model mouse liver sirius red stains generation of plasminogen tetrachloro-methane induction after 14 days Table picture.A is blank control group, and B is gives solvent PBS control group, and C is gives plasminogen group.The results show that plasminogen Group collagen deposition, which is considerably less than, gives solvent PBS control groups, and is on close level with blank control mouse collagen deposition. Illustrate that plasminogen can reduce the deposition of Liver Collagen albumen, improve Liver Fibrosis Model mouse liver fibrosis.

It is representative that 16 weeks hyperlipemia model mouse of Fig. 4 give 30 days posterior sinus of Valsalva sirius red stains of plasminogen Picture.A, C is gives solvent PBS control group, and B, D is give plasminogen group.The results show that give plasminogen group aortic sinus tube wall The area of inner membrance collagen deposition (arrow logo), which is significantly less than, gives solvent PBS control group, illustrates that plasminogen can be cut down The intimal fibrosis of hyperlipemia model mouse aorta sinus tube wall is horizontal.

The systemic sclerosis model mice of Fig. 5 bleomycin induceds gives plasminogen skin sirius red stains after 21 days Representative picture.A is blank control group, and B is gives solvent PBS control group, and for C to give plasminogen group, D is damaged group for PLG activity. The results show that in the systemic sclerosis disease mouse model of bleomycin induced, group is damaged to solvent PBS groups and PLG activity, very Skin top collagenous fiber bundle showed increased, collagenous fibres are coarse, and arrangement is fine and close, and skin corium thickens；Give plasminogen group skin corium Middle fibroblast is considerably less than and gives solvent PBS groups, and dermis of skin layer thickness is close to normal level.

The systemic sclerosis model mice of Fig. 6 bleomycin induceds gives plasminogen lung sirius red stains generation after 21 days Table picture.A is gives solvent PBS control group, and for B to give plasminogen group, C is quantitative analysis results.It is the results show that mould in rich Lay In the systemic sclerosis disease mouse model of element induction, to solvent PBS groups mouse pulmonary fibrosis (arrow logo) degree ratio to fibrinolytic Proenzyme group is high；To plasminogen group mouse lung alveolar wall form close to normally, level of inflammation cell significantly reduces, fibrosis journey Degree, which is significantly lower than, gives solvent PBS groups, and significantly (* represents P to statistical discrepancy<0.05).

The systemic sclerosis model mice of Fig. 7 bleomycin induceds gives plasminogen heart sirius red stains after 21 days Representative picture.A is gives solvent PBS control group, and B is gives plasminogen group.The study found that in the systematicness of bleomycin induced It hardens in disease mouse model, plasminogen group is given to solvent PBS control group myocardial collagen deposition (arrow logo) degree ratio It is high.Illustrate that plasminogen can effectively reduce the cardiac fibrosis of bleomycin induced.

The systemic sclerosis model mice of Fig. 8 bleomycin induceds gives plasminogen kidney sirius red stains after 21 days Representative picture.A is gives solvent PBS control group, and B is gives plasminogen group.The results show that in the systematicness of bleomycin induced It hardens in disease mouse model, it is high to plasminogen group to solvent PBS control group renal collagen fibrosis (arrow logo) degree ratio. Illustrate that plasminogen can effectively reduce the renal fibrosis of bleomycin induced.

Fig. 9 24-25 week old diabetic mices give plasminogen after 31 days kidney IV Collagen Type VIs immunostaining observation knot Fruit.A is gives solvent PBS control group, and B is gives plasminogen group.The results show that colour (arrow to the plasminogen group IV collagens positive Leader is known) it is significantly more than and gives solvent PBS control group, illustrate that plasminogen can improve the fibrosis of diabetic mice kidney.

26 week old diabetic mices of Figure 10 give plasminogen after 35 days kidney masson dyeing observation result.A is to molten Matchmaker's PBS control group, B is gives plasminogen group.The results show that solvent PBS control groups, glomerular mesangial matrixes, extracellular matrix Increase, renal interstitial mild fibrosis (arrow logo), the fibrosis of hyperplasia is in blue.Give plasminogen group mesangial cell And matrix is significantly fewer than control group, kidney region fibrosis significantly reduces.Illustrate that plasminogen can improve diabetic mice kidney Fibrosis lesion.

Figure 11 24-25 week old diabetic mices give plasminogen after 31 days heart masson dyeing observation result.A is Solvent PBS control group is given, B is gives plasminogen group.The results show that visible blue between solvent PBS control group cardiac muscle fibre The collagenous fibres (arrow logo) of the hyperplasia of color, in slight myocardial fibrosis；To visible few between plasminogen group cardiac muscle fibre The collagenous fibres of the hyperplasia azury of amount, compared to control group, myocardial fibrosis is substantially reduced.Illustrate that plasminogen can change The fibrosis of kind diabetic mice heart.

Figure 12 17-18 week old diabetic mices give plasminogen heart sirius red stains representativeness picture after 35 days. A is gives solvent PBS control group, and B is gives plasminogen group.The results show that deposition (the arrow to plasminogen group mouse collagen fiber Leader is known) it is considerably less than and gives solvent PBS control group.Illustrate that plasminogen can reduce versus young (17-18 week old) diabetes The fibrosis of mouse heart.

Figure 13 26-27 week old diabetic mices give plasminogen heart sirius red stains representativeness picture after 35 days. A is gives solvent PBS control group, and B is gives plasminogen group.The results show that deposition (the arrow to plasminogen group mouse collagen Leader is known) it is considerably less than and gives solvent PBS control group.It is small to illustrate that plasminogen can weaken relatively old (26-27 week old) diabetes The fibrosis of rat heart.

Figure 14 cis-platinum kidney fibrosis model mices give plasminogen after 7 days kidney IV Collagen Type VIs immunostaining observation knot Fruit.A is gives solvent PBS control group, and B is gives plasminogen group.The results show that give solvent PBS control group kidney IV Collagen Type VIs sun Property expression (arrow logo) be apparently higher than and give plasminogen group.Illustrate that plasminogen can improve cis-platinum kidney fibrosis model mice kidney Dirty fibrosis.

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

Figure 16 C57 high blood lipid model mouse give plasminogen heart sirius red stains representativeness picture after 30 days.A To give solvent PBS control group, B is gives plasminogen group.The results show that the deposition (arrow logo) to plasminogen group collagen is bright Aobvious be less than gives solvent PBS control group, illustrates that plasminogen can mitigate high blood lipid model mouse heart fibrosis.

The Chronic Renal Impairment model mice of Figure 17 purine induction gives plasminogen kidney sirius red stains sight after 10 days Examine result.A is gives solvent PBS control group, and B is gives plasminogen group, and C is damaged group for PLG activity, and D is quantitative analysis results. It is considerably less than to the deposition (arrow logo) of plasminogen group collagen and is damaged group to solvent PBS control group and PLG activity, and to fibre Lyase original group is damaged group quantitative analysis statistical discrepancy significantly (* expressions P with PLG activity<0.05).Illustrate that plasminogen can mitigate Renal fibrosis caused by Chronic Renal Impairment promotes the reparation of injury of kidney.

Figure 18 24-25 week old diabetic mices give plasminogen after 31 days pancreas islet sirius red stains observe result.A To give solvent PBS control group, for B to give plasminogen group, C is quantitative analysis results.The results show that give plasminogen group mouse pancreas Island collagen deposition (arrow logo), which is considerably less than, gives solvent PBS control group, and significantly (* represents P to statistical discrepancy<0.05).Explanation Plasminogen can improve the islet damage and fibrosis caused by diabetes.

Figure 19 give plasminogen after 30 days 3% cholesterol hyperlipemia model mouse kidney sirius red stains observation knot Fruit.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.The results show that It is considerably less than to plasminogen group renal collagen proteinosis (arrow logo) and gives solvent PBS control group, and statistical discrepancy is shown It writes；Normal level is substantially returned to plasminogen group fibrosis.It is high in fat to illustrate that plasminogen can effectively reduce by 3% cholesterol Blood stasis model mouse kidney fibrosis.

Embodiment

1 plasminogen of embodiment reduces tetrachloro-methane induction hepatic fibrosis-renal tubular ectasia syndrome process Liver Collagen proteinosis

7-8 week old C57 female mices 20, are randomly divided into three groups, blank control group 5, to solvent PBS control group 7 With to plasminogen group 8.Tetrachloro is injected intraperitoneally to solvent PBS control group and to plasminogen group mouse according to 1mL/kg weight Change carbon, three-times-weekly, continuously inject surrounding, establish Liver Fibrosis Model^[36,37], blank control mouse peritoneal injection respective volume Corn oil.Carbon tetrachloride need to be diluted with corn oil, and carbon tetrachloride is 1 with corn oil dilution ratio:3.Start on the day of modeling to Medicine is denoted as the 1st day, to 1mg/0.1mL/ pcs/day of plasminogen group mouse tail vein injection people source plasminogen, gives solvent PBS The PBS of control group tail vein injection same volume, blank control group do not do injection treatment, successive administration 28 days.It puts to death within 29th day small Mouse takes liver to fix 24 hours in 4% paraformaldehyde.Liver after fixation is transparent laggard through alcohol serial dehydration and dimethylbenzene Row paraffin embedding.Histotomy thickness is 3 μm, and slice dewaxing was to washing 1 time after water, with 0.1% sirius red stains 60 minutes Afterwards, flowing water rinses, haematoxylin dyeing 1 minute, and flowing water rinses, and indigo plant is returned in 1% hydrochloride alcohol and ammonium hydroxide differentiation, and flowing water rinses, drying Mounting afterwards is sliced in 200 times of optical microphotograph Microscopic observations.

Solvent PBS control groups (figure is given the results show that being considerably less than to plasminogen group (Fig. 1 C) collagen deposition 1B), and statistical discrepancy is notable (Fig. 1 D)；Compared with to solvent PBS control group, to plasminogen group collagen deposition (arrow logo) Level is more nearly blank control mouse (Figure 1A).Illustrate that plasminogen can reduce Liver Fibrosis Model mouse liver collagen egg White deposition improves hepatic fibrosis-renal tubular ectasia syndrome.

2 plasminogen of embodiment improves ApoE atherosclerosis mouse aorta sinus fibrosis

6 week old male ApoE mouse 13 feed high cholesterol diet (Nantong Te Luofei, TP2031) high in fat 16 weeks to lure Lead Atherosclerosis Model^[31,32].Mouse after Cheng Mo before administration three days every 50 μ l of blood is taken to detect T-CHOL (T- CHO) content, and two groups are randomly divided into according to T-CHO contents, to solvent PBS control group 7, to plasminogen group 6.Start Administration is set to the 1st day, to 1mg/0.1mL/ pcs/day of plasminogen group mouse tail vein injection people source plasminogen, to solvent The PBS of PBS control group tail vein injection same volume.Administration 30 days, and mouse was put to death in the 31st day, it cores dirty in 4% poly first Aldehyde fixes 24-48 hours, is sunk to the bottom overnight respectively at 4 DEG C in 15%, 30% sucrose, OCT embeddings, 8 μm of frozen section thickness, with After 0.1% Picro-Sirius red saturation picric acid dyes 30 minutes, flowing water rinses 2min, haematoxylin dyeing 1 minute, and flowing water rinses, and 1% Hydrochloride alcohol breaks up, and ammonium hydroxide returns indigo plant, and flowing water rinses, neutral gum mounting after drying, be sliced 40 (2A, 2B), 200 times (2C, 2D) optical microphotograph Microscopic observation.

The results show that plasminogen group (Fig. 2 B, D) aortic sinus tube wall inner membrance collagen deposition (arrow logo) Area is significantly less than to solvent PBS control group (Fig. 2A, C), illustrates that plasminogen can cut down artery congee hardening model mouse master Artery sinus Fibrosis levels.

3 plasminogen of embodiment improves the hepatic fibrosis-renal tubular ectasia syndrome of tetrachloro-methane induction

9 week old C57 female mices 15, are randomly divided into three groups, blank control group, to solvent PBS control groups and to fibrinolytic Proenzyme group, every group each 5.Tetrachloro is injected intraperitoneally to solvent PBS control group and to plasminogen group mouse according to 1mL/kg weight Change carbon, three-times-weekly, Liver Fibrosis Model is established in continuous injection two weeks^[36,37], blank control mouse injects according to model mice Mode injects the corn oil of respective volume.Carbon tetrachloride need to be diluted with corn oil, and carbon tetrachloride is 1 with corn oil dilution ratio: 3.Start to be administered after model foundation, start administration the same day be denoted as the 1st day, to plasminogen group mouse according to 1mg/0.1mL/ only/ Its tail vein injection people source plasminogen, to the PBS of solvent PBS control group tail vein injection same volume, blank control group is not done Injection treatment, successive administration 14 days.It puts to death mouse within 15th day and takes liver, 24 hours are fixed in 4% paraformaldehyde.After fixation Liver carries out paraffin embedding after alcohol serial dehydration and dimethylbenzene are transparent.Histotomy thickness is 3 μm, after slice dewaxing to water Washing 1 time, with 0.1% sirius red stains after sixty minutes, flowing water are rinsed, haematoxylin dyeing 1 minute, and flowing water rinses, 1% hydrochloric acid Indigo plant is returned in alcohol and ammonium hydroxide differentiation, and flowing water rinses, and mounting after drying is sliced in 200 times of optical microphotograph Microscopic observations.

Solvent PBS control groups (figure is given the results show that being considerably less than to plasminogen group (Fig. 3 C) collagen deposition 3B), and to plasminogen group ratio to PBS group collagen deposition levels closer to blank control mouse (Fig. 3 A).Illustrate fibrinolytic Proenzyme can reduce the deposition of Liver Collagen albumen, improve Liver Fibrosis Model mouse liver fibrosis.

4 plasminogen of embodiment reduces by 16 weeks hyperlipemia model mouse aorta sinus fibrosis

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^[30,31], 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, mouse was put to death in the 31st day, materials heart fixes 24-48 hours in 4% paraformaldehyde.Tissue after fixation is through alcohol ladder Paraffin embedding is carried out after degree dehydration and dimethylbenzene are transparent.Aortic sinus slice thickness is 3 μm, is washed 1 time after slice dewaxing rehydration, After being dyed 30 minutes with 0.1% Picro-Sirius red saturation picric acid, flowing water rinses 2min, haematoxylin dyeing 1 minute, and flowing water rinses, 1% hydrochloride alcohol breaks up, and ammonium hydroxide returns indigo plant, and flowing water rinses, neutral gum mounting after drying, 40 (4A, 4B), 200 times (4C, 4D) optical microphotograph Microscopic observation.

The results show that give plasminogen group (Fig. 4 B, 4D) aortic sinus tube wall inner membrance collagen deposition (arrow logo) Area be significantly less than and give solvent PBS control group (Fig. 4 A, 4C), illustrate that plasminogen can cut down hyperlipemia model mouse master Artery sinus Fibrosis levels.

5 plasminogen of embodiment reduces systemic sclerosis mouse skin fibrosis

12 week old C57 heros mouse 15 are taken, are randomly divided into three groups, blank control group, (PBS is phosphoric acid buffer to solvent PBS Salting liquid (Phosphate Buffer Saline) is the solvent of plasminogen herein.) control group and plasminogen group is given, Every group each 5, and 13 week old PLG activity is taken to be damaged mouse 5.Experiment is denoted as grouping of weighing in the 0th day on the day of starting, and opens within second day Beginning modeling is administered, and mouse is damaged by 0.1mg/0.1ml/ pcs/day to solvent PBS control group, to plasminogen group and PLG activity Bleomycin induced systemic sclerosis is subcutaneously injected^[26].Physiological saline is subcutaneously injected by 0.1ml/ pcs/day in blank control group, It starts simultaneously to plasminogen or PBS and is denoted as the 1st day, continuous modeling is administered 21 days.1mg/ is pressed to plasminogen group mouse 0.1ml/ pcs/day of tail vein injection plasminogen, gives the PBS of same volume to solvent PBS control group, normal mouse group and PLG activity is damaged mouse and does not do administration processing.Mouse was put to death at the 22nd day and back skin tissues is taken to be fixed in 4% paraformaldehyde 24 hours are fixed in liquid.Fixed skin histology carries out paraffin embedding after alcohol serial dehydration and dimethylbenzene are transparent.Tissue is cut Piece thickness is 3 μm, is washed 1 time after slice dewaxing to water, after being dyed 30 minutes with 0.1% Picro-Sirius red saturation picric acid, flowing water Rinse 2min, haematoxylin dyeing 1 minute, flowing water rinses, and 1% hydrochloride alcohol differentiation, ammonium hydroxide returns indigo plant, and flowing water rinses, after drying in Property natural gum mounting, in 100 times of optical microphotograph Microscopic observations.

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

The results show that in the systemic sclerosis disease mouse model of bleomycin induced, observed under microscope to solvent PBS groups (Fig. 5 B) and PLG activity are damaged a group mouse (Fig. 5 D) upper part of dermis collagenous fiber bundle showed increased, and collagenous fibres are coarse, Arrangement is fine and close, and skin corium thickens；Give plasminogen group (Fig. 5 C), fibroblast is considerably less than to PBS pairs of solvent in skin corium According to group, dermis of skin layer thickness is substantially close to normal level (Fig. 5 A).Illustrate that plasminogen can effectively reduce bleomycin induced Fibrosis of skin.

6 plasminogen of embodiment reduces systemic sclerosis mouse pulmonary fibrosis

12 week old C57 heros mouse 17 are taken, two groups are randomly divided into, to solvent PBS control group 11 and to plasminogen group 6 Only.Experiment is denoted as the 0th day and grouping of weighing on the day of starting, start within the 1st day modeling administration, and two groups of mouse press 0.1mg/0.1ml/ Pcs/day be subcutaneously injected bleomycin induced systemic sclerosis^[26], and start to plasminogen or PBS, continuous modeling administration 21 My god.To plasminogen group mouse by 1mg/0.1ml/ pcs/day of tail vein injection plasminogen, to solvent PBS control group with identical Mode gives the PBS of same volume.It puts to death within 22nd day mouse and that lung tissue is taken to fix 24 in 4% paraformaldehyde fixer is small When.Lung tissue after fixation carries out paraffin embedding after alcohol serial dehydration and dimethylbenzene are transparent.Histotomy thickness is 3 μm, It is washed 1 time after slice dewaxing to water, after being dyed 30 minutes with 0.1% Picro-Sirius red saturation picric acid, flowing water rinses 2min, bush Uniformly dyeing color 1 minute, flowing water rinse, and 1% hydrochloride alcohol differentiation, ammonium hydroxide returns indigo plant, and flowing water rinses, neutral gum mounting after drying, 200 times of optical microphotograph Microscopic observations.

The study found that in the systemic sclerosis disease mouse model of bleomycin induced, Microscopic observation gives solvent PBS groups (Fig. 6 A) collagenous fibrosis (arrow logo) degree ratio gives plasminogen group (Fig. 6 B) height；Give plasminogen group mouse lung lung Steep that wall form is significantly reduced close to normal level, inflammatory cell, and fibrosis is significantly lower than and gives solvent PBS groups, and statistical difference Different notable (Fig. 6 C).Illustrate that plasminogen can effectively reduce the systemic sclerosis mouse lung tissue fiber of bleomycin induced Change.

7 plasminogen of embodiment reduces systemic sclerosis mouse heart fibrosis

12 week old C57 heros mouse 10 are taken, are randomly divided into two groups, to solvent PBS control group and each 5 to plasminogen group. Experiment is denoted as the 0th day and grouping of weighing on the day of starting, start within the 1st day modeling administration, by 0.1mg/0.1ml/ pcs/day of hypodermic injection Bleomycin induced systemic sclerosis^[26], and start to plasminogen or PBS, successive administration 21 days.It is small to plasminogen group Mouse presses 1mg/0.1ml/ pcs/day of tail vein injection plasminogen, to solvent PBS control group tail vein injection same volume PBS put to death mouse at the 22nd day and cores and dirty fixes 24 hours in 4% paraformaldehyde fixer.Heart after fixation is through wine Paraffin embedding is carried out after smart serial dehydration and dimethylbenzene are transparent.Histotomy thickness is 3 μm, and 1 is washed after slice dewaxing to water Secondary, after being dyed 30 minutes with 0.1% Picro-Sirius red saturation picric acid, flowing water rinses 2min, haematoxylin dyeing 1 minute, flowing water punching It washes, 1% hydrochloride alcohol differentiation, ammonium hydroxide returns indigo plant, and flowing water rinses, and neutral gum mounting after drying is seen under 200 times of light microscopes It examines.

The study found that in the systemic sclerosis disease mouse model of bleomycin induced, Microscopic observation is to PBS pairs of solvent Plasminogen group (Fig. 7 B) height is given according to group (Fig. 7 A) myocardial collagen deposition rate.Illustrate that plasminogen can effectively reduce rich Lay The cardiac fibrosis of mycin induction.

8 plasminogen of embodiment reduces systemic sclerosis mouse kidney fibrosis

12 week old C57 heros mouse 10 are taken only two groups are randomly divided into, to solvent PBS control group and to plasminogen group each 5 Only.Experiment is denoted as the 0th day and grouping of weighing on the day of starting, start within the 1st day modeling administration, and all mouse press 0.1mg/0.1ml/ Pcs/day bleomycin induced systemic sclerosis is subcutaneously injected, and start to plasminogen or PBS, continuous modeling is administered 21 days. To plasminogen group mouse by 1mg/0.1ml/ pcs/day of tail vein injection plasminogen, noted to solvent PBS control group tail vein Penetrate the PBS of same volume.Mouse was put to death at the 22nd day and kidney is taken to fix 24 hours in 4% paraformaldehyde fixer.It is fixed Kidney afterwards carries out paraffin embedding after alcohol serial dehydration and dimethylbenzene are transparent.Histotomy thickness is 3 μm, and slice dewaxing is extremely It is washed 1 time after water, after being dyed 30 minutes with 0.1% Picro-Sirius red saturation picric acid, flowing water rinses 2min, and haematoxylin dyeing 1 divides Clock, flowing water rinse, and 1% hydrochloride alcohol differentiation, ammonium hydroxide returns indigo plant, and flowing water rinses, neutral gum mounting after drying, in 200 times of optics Micro- Microscopic observation.

The results show that in the systemic sclerosis disease mouse model of bleomycin induced, solvent PBS control groups (figure is given 8A) renal collagen fibrosis (arrow logo) degree ratio gives plasminogen group (Fig. 8 B) height.Illustrate that plasminogen can be reduced effectively The renal fibrosis of bleomycin induced.

9 plasminogen of embodiment reduces diabetic mice renal collagen proteinosis

24-25 week old db/db heros mouse 10, is randomly divided into two groups, to solvent PBS control group and gives plasminogen group, often Each 5 of group.Experiment is denoted as the 0th day and grouping of weighing on the day of starting, and the 1st day starts to plasminogen or PBS, successive administration 31 My god.To plasminogen group mouse by 2mg/0.2ml/ pcs/day of tail vein injection plasminogen, phase is given to solvent PBS control group The PBS of same volume.To putting to death mouse after plasminogen 31 days and that renal tissue is taken to fix 24 in 4% paraformaldehyde fixer is small When.Renal tissue after fixation carries out paraffin embedding after alcohol serial dehydration and dimethylbenzene are transparent.Histotomy thickness is 4 μ M is washed 1 time after being sliced dewaxing rehydration.With 3% dioxygen water incubation 15 minutes, 0.01MPBS was washed 2 times, every time 5 minutes.10% just Normal sheep blood serum (Vector laboratories, Inc., USA) is closed 1 hour, and the time gets rid of serum deprivation after, is irised out with PAP Tissue.For 4 DEG C of overnight incubations of rabbit anti-mouse polyclonal antibody (Abcam) of IV collagens, TBS is washed 2 times, every time 5 minutes.Mountain Goat anti-rabbit igg (HRP) antibody (Abcam) secondary antibody is incubated at room temperature 1 hour, and TBS is washed 2 times.By DAB kits (Vector Laboratories, Inc., USA) colour developing, haematoxylin is redyed 30 seconds after washing 3 times, and flowing water rinses 5 minutes, and graded ethanol takes off Water, dimethylbenzene is transparent, neutral gum mounting, is sliced in 200 times of optical microphotograph Microscopic observations.

Diabetic nephropathy is chronic complicating diseases of diabetes, and glomerulosclerosis and kidney region fibrosis are its typical pathology Change^[27]。

Solvent PBS control group (figure is given the results show that being significantly more than to the coloring of plasminogen group (Fig. 9 B) the IV collagens positive 9A), illustrate that plasminogen can reduce the deposition (arrow logo) of renal tissue collagen, plasminogen is prompted to be expected to pass through drop The deposition of low renal tissue collagen prevents the renal tissue fibrosis caused by diabetes.

10 plasminogen of embodiment improves diabetic mice renal fibrosis

26 week old db/db heros mouse 10, are randomly divided into two groups, to solvent PBS control group and give plasminogen group, every group each 5.Experiment is denoted as the 0th day and grouping of weighing on the day of starting, and starts within the 1st day to plasminogen or PBS, successive administration 35 days.It gives Plasminogen group mouse presses 2mg/0.2ml/ pcs/day of tail vein injection plasminogen, and same volume is given to solvent PBS control group PBS.It puts to death within 36th day mouse and renal tissue is taken to fix 24 hours in 4% paraformaldehyde fixer.Kidney after fixation Tissue carries out paraffin embedding after alcohol serial dehydration and dimethylbenzene are transparent.Histotomy thickness is 4 μm, slice dewaxing rehydration After be put into potassium bichromate solution and stay overnight.Garapa uniformly dyeing 3 to 5 minutes, flowing water is slightly washed.1% hydrochloride alcohol breaks up, AMMONIA TREATMENT 1 Second, washing.Ponceaux acid fuchsin liquid contaminates 8 minutes, short rinse in water.1% phosphomolybdic acid aqueous solution is handled about 2 minutes, aniline Blue liquid is redyed 6 minutes.1% glacial acetic acid rinses 1 minute or so.Mounting after absolute ethyl alcohol dehydration dimethylbenzene is transparent, is sliced 200 Times optical microphotograph Microscopic observation.

Masson dyes the fibrosis that can show tissue.The results show that solvent PBS control group (Figure 10 A) glomerulus Mesangial matrix *, extracellular matrix increase, renal interstitial mild fibrosis (arrow logo), and the fibrosis of hyperplasia is in blue.To fibrinolysin Former group of (Figure 10 B) mesangial cell and matrix are significantly fewer than control group, and kidney region fibrosis significantly reduces.Illustrate fibrinolysin Proper energy improves the fibrosis of diabetic mice kidney.

11 plasminogen of embodiment improves 24-25 week old diabetic mice cardiac fibrosis

24-25 week old db/db heros mouse 10, is randomly divided into two groups, to solvent PBS control group and gives plasminogen group, often Each 5 of group.Experiment is denoted as the 0th day and grouping of weighing on the day of starting, and the 1st day starts to plasminogen or PBS, successive administration 31 My god.To plasminogen group mouse by 2mg/0.2ml/ pcs/day of tail vein injection plasminogen, phase is given to solvent PBS control group The PBS of same volume.To putting to death mouse after plasminogen 31 days and that heart tissue is taken to fix 24 in 4% paraformaldehyde fixer is small When.Heart tissue after fixation carries out paraffin embedding after alcohol serial dehydration and dimethylbenzene are transparent.Histotomy thickness is 4 μ M is put into potassium bichromate solution and stays overnight after slice dewaxing rehydration.Garapa uniformly dyeing 3 to 5 minutes, flowing water is slightly washed.1% hydrochloride alcohol Differentiation, AMMONIA TREATMENT 1 second, washing.Ponceaux acid fuchsin liquid contaminates 8 minutes, short rinse in water.At 1% phosphomolybdic acid aqueous solution Reason about 2 minutes, aniline blue liquid is redyed 6 minutes.1% glacial acetic acid rinses general 1 minute or so.Absolute ethyl alcohol dehydration dimethylbenzene is transparent Mounting afterwards is sliced in 200 times of optical microphotograph Microscopic observations.

The most common complication of diabetes is connective tissue excessive accumulation (pathologic fibrosis), and Myocardial Interstitial Fibrosis can It can be the characteristic pathology change of Leonurus heterophyllus sweet^[28-29]。

Masson dyes the fibrosis that can show tissue.It is the results show that fine to solvent PBS control group (Figure 11 A) cardiac muscle The collagenous fibres (arrow logo) of the hyperplasia of visible blue between dimension, in slight myocardial fibrosis；Give plasminogen group (figure 11B) between cardiac muscle fibre visible a small amount of hyperplasia azury collagenous fibres, compared to control group, myocardial fibrosis is apparent Mitigate.Illustrate that plasminogen can improve the fibrosis of diabetic mice heart.

12 plasminogen of embodiment reduces 17-18 week old diabetic mices myocardial collagen deposition

17-18 week old db/db heros mouse 8, is randomly divided into two groups, to solvent PBS control group and gives plasminogen group, every group Each 4.Experiment is denoted as the 0th day and grouping of weighing on the day of starting, and starts within the 1st day to plasminogen or PBS, successive administration 35 days. To plasminogen group mouse by 2mg/0.2ml/ pcs/day of tail vein injection plasminogen, given to solvent PBS control group identical The PBS of volume.To putting to death mouse after plasminogen 35 days and that heart tissue is taken to fix 24 in 4% paraformaldehyde fixer is small When.Heart after fixation carries out paraffin embedding after alcohol serial dehydration and dimethylbenzene are transparent.Histotomy thickness is 3 μm, is cut Piece is washed 1 time after dewaxing to water, and after being dyed 30 minutes with 0.1% Picro-Sirius red saturation picric acid, flowing water rinses 2min, bush Uniformly dyeing color 1 minute, flowing water rinse, and 1% hydrochloride alcohol differentiation, ammonium hydroxide returns indigo plant, and flowing water rinses, neutral gum mounting after drying, 200 times of optical microphotograph Microscopic observations.

The results show that the deposition (arrow logo) to plasminogen group (Figure 12 B) mouse collagen fiber is considerably less than to molten Matchmaker's PBS control group (Figure 12 A).Illustrate that plasminogen can reduce the deposition of heart tissue collagen, plasminogen is prompted to be expected to By reducing the deposition of heart tissue collagen, versus young (17-18 week old) diabetic mice heart tissue fiber is reduced Change.

13 plasminogen of embodiment reduces 26-27 week old diabetic mices myocardial collagen deposition

26-27 week old db/db heros mouse 9, is randomly divided into two groups, to solvent PBS control group 5 and to plasminogen group 4 Only.Experiment is denoted as the 0th day and grouping of weighing on the day of starting, and starts within the 1st day to plasminogen or PBS, successive administration 35 days.To fibre Lyase original group mouse presses 2mg/0.2ml/ pcs/day of tail vein injection plasminogen, and same volume is given to solvent PBS control group PBS.To putting to death mouse after plasminogen 35 days and heart tissue taken to fix 24 hours in 4% paraformaldehyde fixer.It is fixed Heart afterwards carries out paraffin embedding after alcohol serial dehydration and dimethylbenzene are transparent.Histotomy thickness is 3 μm, slice dewaxing It is washed after to water 1 time, with 0.1% sirius red stains after sixty minutes, flowing water rinses, haematoxylin dyeing 1 minute, and flowing water rinses, Indigo plant is returned in 1% hydrochloride alcohol and ammonium hydroxide differentiation, and flowing water rinses, mounting after drying, in 200 times of optical microphotograph Microscopic observations.

The results show that the deposition (arrow logo) to plasminogen group (Figure 13 B) mouse collagen fiber is considerably less than to molten Matchmaker's PBS control group (Figure 13 A).Illustrate that plasminogen can reduce the deposition of heart tissue collagen, plasminogen is prompted to be expected to By reducing the deposition of heart tissue collagen, relatively old (26-27 week old) diabetic mice heart tissue fiber is reduced Change.

14 plasminogen of embodiment reduces the fibrosis of cis-platinum renal fibrosis model mice kidney

The male C57 mouse 10 of 8-9 week old health, are randomly divided into two groups, to solvent PBS control group and to plasminogen Group, every group each 5.After the completion of grouping, by 10mg/Kg weight single intraperitoneal injection cis-platinums, renal fibrosis model is established^[30]。 After establishing model, give plasminogen through tail vein injection for 1mg/0.1ml/ pcs/day to plasminogen group, give solvent PBS control Group gives the PBS of same volume.Experiment is denoted as the 0th day on the day of starting and weighs and be grouped, and starts within the 1st day intraperitoneal injection cis-platinum and makes Mould gives plasminogen or solvent PBS, dosage period 7 days in 3 hours after modeling.It puts to death mouse within 8th day, takes kidney more than 4% 24 hours are fixed in polyformaldehyde fixer.Renal tissue after fixation carries out paraffin after alcohol serial dehydration and dimethylbenzene are transparent Embedding.Histotomy thickness is 5 μm, is washed 1 time after slice dewaxing rehydration.Citric acid is repaired 30 minutes, and room temperature cools down 10 minutes Water softly rinses afterwards.With 3% dioxygen water incubation 15 minutes, tissue is irised out with PAP.10% sheep blood serum (Vector Laboratories, Inc., USA) it closes 1 hour；After time arrives, reject sheep blood serum liquid.Rabbit anti-mouse IV collagen antibodies (Abcam) 4 DEG C of overnight incubations, TBS are washed 2 times, every time 5 minutes.Goat anti-rabbit igg (HRP) antibody (Abcam) secondary antibody room temperature is incubated It educates 1 hour, TBS is washed 2 times, every time 5 minutes.It develops the color by DAB kits (Vector laboratories, Inc., USA), water Haematoxylin is redyed 30 seconds after washing 3 times, and flowing water returns indigo plant 5 minutes, and then TBS is washed 1 time.Serial dehydration is transparent and mounting, is sliced 200 Times optical microphotograph Microscopic observation.

Cis-platinum be clinically be widely used, the reliable broad-spectrum anti-cancer drug of curative effect, but with serious renal toxicity, mainly It is renal tubule and interregnal damage, finally develops into kidney fibrosis^[30].The experimental result is shown, gives solvent PBS control group (figure 14A) kidney IV Collagen Type VIs positive expression (arrow logo), which is apparently higher than, gives molten plasminogen group (Figure 14 B).Illustrate plasminogen The fibrosis of cis-platinum renal fibrosis model mice kidney can be improved.

15 plasminogen of embodiment improves ApoE atherosclerosis mouse heart Fibrosis levels

6 week old ApoE male mices 13 feed high cholesterol diet (Nantong Te Luofei, TP2031) high in fat 16 weeks, lure Lead atherosclerosis^[31,32].Three days every mouse take 50 μ L of blood before administration, detect total cholesterol concentration, and according to detection As a result mouse is randomly divided into two groups, to solvent PBS control group 7 and to plasminogen group 6.Start administration and be set to the 1st My god, it is quiet to solvent PBS control group tail to 1mg/0.1ml/ pcs/day of plasminogen group mouse tail vein injection people source plasminogen Arteries and veins injects the PBS of same volume.Administration 30 days, during which feeds high cholesterol diet high in fat always.Mouse was put to death in the 31st day, is cored It is dirty to fix 24-48 hours in 4% paraformaldehyde.Tissue after fixation carries out paraffin after alcohol serial dehydration and dimethylbenzene are transparent Embedding.Histotomy thickness is 3 μm, is washed 1 time after slice dewaxing rehydration, 30 are dyed with 0.1% Picro-Sirius red saturation picric acid After minute, flowing water rinses 2min, haematoxylin dyeing 1 minute, and flowing water rinses, the differentiation of 1% hydrochloride alcohol, and ammonium hydroxide returns indigo plant, flowing water punching It washes, neutral gum mounting after drying, in 200 times of optical microphotograph Microscopic observations.

The results show that the deposition (arrow logo) to plasminogen group (Figure 15 B) collagen considerably less than gives solvent PBS control Group (Figure 15 A) prompts plasminogen that can sink by reducing the collagen of ApoE Atherosclerosis Model mouse heart tissues Product prevents and reduces the cardiac fibrosis caused by atherosclerosis.

16 plasminogen of embodiment reduces high blood lipid model mouse heart fibrosis

6 week old C57 male mices 11 feed high cholesterol diet (Nantong Te Luofei, TP2031) high in fat 16 weeks, induction Hyperlipidemia^[33,34].Three days every mouse take 50 μ L of blood before administration, detect total cholesterol concentration, and divide mouse at random according to it It it is two groups, to solvent PBS control group 6 and to plasminogen group 5.Start administration to be denoted as the 1st day, it is small to plasminogen group 1mg/0.1ml/ pcs/day of tail vein injection people source plasminogen, to solvent PBS control group tail vein injection same volume PBS.Administration 30 days, during which feeds high cholesterol diet high in fat always.Mouse, dirty be organized in more than 4% of coring were put to death in the 31st day Polyformaldehyde fixes 24-48 hours.Tissue after fixation carries out paraffin embedding after alcohol serial dehydration and dimethylbenzene are transparent.Tissue Slice thickness is 3 μm, is washed 1 time after slice dewaxing rehydration, after being dyed 30 minutes with 0.1% Picro-Sirius red saturation picric acid, stream Water rinses 2min, haematoxylin dyeing 1 minute, and flowing water rinses, and 1% hydrochloride alcohol differentiation, ammonium hydroxide returns indigo plant, and flowing water rinses, after drying Neutral gum mounting, in 200 times of optical microphotograph Microscopic observations.

Solvent PBS control group is given the results show that being considerably less than to plasminogen group (Figure 16 B) collagen deposition (arrow logo) (Figure 16 A) prompts plasminogen can be by reducing high blood lipid model mouse heart tissue collagen deposition, so as to prevent and drop Cardiac fibrosis caused by low hyperlipidemia.

17 plasminogen of embodiment repairs the fibrosis of chronic renal failure model kidney

The PLG activity Normal male mice 12 and PLG activity of 8-9 week old are damaged male mice 6, and PLG activity is just Normal mouse is randomly divided into two groups, to plasminogen group and each 6 to solvent PBS control group.The feeding 0.25% daily of three groups of mouse Purine feed (Nantong Te Luofei) establishes chronic renal failure model^[35].It is denoted as the 1st day on the day of modeling, starts simultaneously at administration.It gives Plasminogen group gives plasminogen by 1mg/0.1ml/ pcs/day, and same volume is given in the same manner to solvent PBS control group Long-pending PBS, continuous modeling are administered 10 days, and PLG activity is damaged mouse and does not process.It puts to death mouse within 11st day and takes kidney in more than 4% 24 hours are fixed in polyformaldehyde.Kidney after fixation carries out paraffin embedding after alcohol serial dehydration and dimethylbenzene are transparent.Tissue Slice thickness is 3 μm, is washed after slice dewaxing to water 1 time, with 0.1% sirius red stains after sixty minutes, flowing water rinses, bush Uniformly dyeing color 1 minute, flowing water rinse, and indigo plant is returned in 1% hydrochloride alcohol and ammonium hydroxide differentiation, and flowing water rinses, and mounting after drying is sliced 200 Times optical microphotograph Microscopic observation.

The results show that the deposition (arrow logo) to plasminogen group (Figure 17 B) collagen will be considerably less than to PBS pairs of solvent Group (Figure 17 C) is damaged, and group statistical discrepancy significantly (P is damaged to plasminogen group and PLG activity according to group (Figure 17 A) and PLG activity =0.018) (Figure 17 D).Illustrate that plasminogen can significantly mitigate the deposition of collagen in Chronic Renal Impairment animal kidney tissue, It prevents and mitigates the renal fibrosis caused by Chronic Renal Impairment.

18 plasminogen of embodiment reduces diabetic mice pancreas islet collagen deposition

24-25 week old db/db male mices 16, are randomly divided into two groups, to plasminogen group 10, to PBS pairs of solvent According to group 6.To 2mg/0.2ml/ pcs/day of plasminogen group tail vein injection people source plasminogen, solvent PBS control group tail is given It is injected intravenously the PBS of same volume.Experiment is denoted as grouping of weighing in the 0th day on the day of starting, and starts within the 1st day, to plasminogen or PBS, to connect Continuous administration 31 days.Mouse was put to death at the 32nd day, takes pancreas fixed in 4% paraformaldehyde.Pancreatic tissues after fixation are through alcohol Paraffin embedding is carried out after serial dehydration and dimethylbenzene are transparent.Histotomy thickness is 3 μm, is washed 1 time after slice dewaxing to water, with After sixty minutes, flowing water rinses 0.1% sirius red stains, haematoxylin dyeing 1 minute, and flowing water rinses, 1% hydrochloride alcohol and ammonium hydroxide Indigo plant is returned in differentiation, and flowing water rinses, and mounting after drying is sliced in 200 times of optical microphotograph Microscopic observations.

The results show that plasminogen group mouse (Figure 18 B) pancreas islet collagen deposition (arrow logo) significantly lower than to solvent PBS control group (Figure 18 A), and statistical discrepancy is significantly (Figure 18 C).Illustrate that plasminogen can significantly mitigate diabetic mice pancreas group The deposition of middle collagen is knitted, prevents and mitigate damage and the fibrosis of pancreas.

19 plasminogen of embodiment reduces by 3% cholesterol hyperlipemia model mouse kidney fibrosis

9 week old male C57 mouse 16 feed 3% cholesterol high lipid food (Nantong Te Luofei) 4 weeks, induce hyperlipemia Disease^[30,31], 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. Mouse is administered 30 days after administration in 30th day, and mouse was put to death in the 31st day, and materials kidney fixes 24-48 hours in 4% paraformaldehyde. Tissue after fixation carries out paraffin embedding after alcohol serial dehydration and dimethylbenzene are transparent.Slice thickness is 3 μm, and slice dewaxing is multiple It is washed 1 time after water, after being dyed 30 minutes with 0.1% Picro-Sirius red saturation picric acid, flowing water rinses 2min, and haematoxylin dyeing 1 divides Clock, flowing water rinse, and 1% hydrochloride alcohol differentiation, ammonium hydroxide returns indigo plant, and flowing water rinses, neutral gum mounting after drying, in 200 times of optics Micro- Microscopic observation.

The results show that it is considerably less than to plasminogen group (Figure 19 C) renal collagen proteinosis (arrow logo) to solvent PBS control group (Figure 19 B), and statistical discrepancy is significantly (Figure 19 D)；Normal level is substantially returned to plasminogen group fibrosis (Figure 19 A).Illustrate that plasminogen can effectively reduce by 3% cholesterol hyperlipemia model mouse kidney fibrosis.

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Sequence table

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

<120>A kind of method for preventing and treating liver fibrosis

<130> PDK03581

<160> 14

<170> PatentIn version 3.5

<210> 1

<211> 2376

<212> DNA

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

<400> 1

gagcctctgg atgactatgt gaatacccag ggggcttcac tgttcagtgt cactaagaag 60

cagctgggag caggaagtat agaagaatgt gcagcaaaat gtgaggagga cgaagaattc 120

acctgcaggg cattccaata tcacagtaaa gagcaacaat gtgtgataat ggctgaaaac 180

aggaagtcct ccataatcat taggatgaga gatgtagttt tatttgaaaa gaaagtgtat 240

ctctcagagt gcaagactgg gaatggaaag aactacagag ggacgatgtc caaaacaaaa 300

aatggcatca cctgtcaaaa atggagttcc acttctcccc acagacctag attctcacct 360

gctacacacc cctcagaggg actggaggag aactactgca ggaatccaga caacgatccg 420

caggggccct ggtgctatac tactgatcca gaaaagagat atgactactg cgacattctt 480

gagtgtgaag aggaatgtat gcattgcagt ggagaaaact atgacggcaa aatttccaag 540

accatgtctg gactggaatg ccaggcctgg gactctcaga gcccacacgc tcatggatac 600

attccttcca aatttccaaa caagaacctg aagaagaatt actgtcgtaa ccccgatagg 660

gagctgcggc cttggtgttt caccaccgac cccaacaagc gctgggaact ttgtgacatc 720

ccccgctgca caacacctcc accatcttct ggtcccacct accagtgtct gaagggaaca 780

ggtgaaaact atcgcgggaa tgtggctgtt accgtgtccg ggcacacctg tcagcactgg 840

agtgcacaga cccctcacac acataacagg acaccagaaa acttcccctg caaaaatttg 900

gatgaaaact actgccgcaa tcctgacgga aaaagggccc catggtgcca tacaaccaac 960

agccaagtgc ggtgggagta ctgtaagata ccgtcctgtg actcctcccc agtatccacg 1020

gaacaattgg ctcccacagc accacctgag ctaacccctg tggtccagga ctgctaccat 1080

ggtgatggac agagctaccg aggcacatcc tccaccacca ccacaggaaa gaagtgtcag 1140

tcttggtcat ctatgacacc acaccggcac cagaagaccc cagaaaacta cccaaatgct 1200

ggcctgacaa tgaactactg caggaatcca gatgccgata aaggcccctg gtgttttacc 1260

acagacccca gcgtcaggtg ggagtactgc aacctgaaaa aatgctcagg aacagaagcg 1320

agtgttgtag cacctccgcc tgttgtcctg cttccagatg tagagactcc ttccgaagaa 1380

gactgtatgt ttgggaatgg gaaaggatac cgaggcaaga gggcgaccac tgttactggg 1440

acgccatgcc aggactgggc tgcccaggag ccccatagac acagcatttt cactccagag 1500

acaaatccac gggcgggtct ggaaaaaaat tactgccgta accctgatgg tgatgtaggt 1560

ggtccctggt gctacacgac aaatccaaga aaactttacg actactgtga tgtccctcag 1620

tgtgcggccc cttcatttga ttgtgggaag cctcaagtgg agccgaagaa atgtcctgga 1680

agggttgtag gggggtgtgt ggcccaccca cattcctggc cctggcaagt cagtcttaga 1740

acaaggtttg gaatgcactt ctgtggaggc accttgatat ccccagagtg ggtgttgact 1800

gctgcccact gcttggagaa gtccccaagg ccttcatcct acaaggtcat cctgggtgca 1860

caccaagaag tgaatctcga accgcatgtt caggaaatag aagtgtctag gctgttcttg 1920

gagcccacac gaaaagatat tgccttgcta aagctaagca gtcctgccgt catcactgac 1980

aaagtaatcc cagcttgtct gccatcccca aattatgtgg tcgctgaccg gaccgaatgt 2040

ttcatcactg gctggggaga aacccaaggt acttttggag ctggccttct caaggaagcc 2100

cagctccctg tgattgagaa taaagtgtgc aatcgctatg agtttctgaa tggaagagtc 2160

caatccaccg aactctgtgc tgggcatttg gccggaggca ctgacagttg ccagggtgac 2220

agtggaggtc ctctggtttg cttcgagaag gacaaataca ttttacaagg agtcacttct 2280

tggggtcttg gctgtgcacg ccccaataag cctggtgtct atgttcgtgt ttcaaggttt 2340

gttacttgga ttgagggagt gatgagaaat aattaa 2376

<210> 2

<211> 791

<212> PRT

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

<400> 2

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

Glu Gly Val Met Arg Asn Asn

785 790

<210> 3

<211> 2433

<212> DNA

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

<400> 3

atggaacata aggaagtggt tcttctactt cttttatttc tgaaatcagg tcaaggagag 60

cctctggatg actatgtgaa tacccagggg gcttcactgt tcagtgtcac taagaagcag 120

ctgggagcag gaagtataga agaatgtgca gcaaaatgtg aggaggacga agaattcacc 180

tgcagggcat tccaatatca cagtaaagag caacaatgtg tgataatggc tgaaaacagg 240

aagtcctcca taatcattag gatgagagat gtagttttat ttgaaaagaa agtgtatctc 300

tcagagtgca agactgggaa tggaaagaac tacagaggga cgatgtccaa aacaaaaaat 360

ggcatcacct gtcaaaaatg gagttccact tctccccaca gacctagatt ctcacctgct 420

acacacccct cagagggact ggaggagaac tactgcagga atccagacaa cgatccgcag 480

gggccctggt gctatactac tgatccagaa aagagatatg actactgcga cattcttgag 540

tgtgaagagg aatgtatgca ttgcagtgga gaaaactatg acggcaaaat ttccaagacc 600

atgtctggac tggaatgcca ggcctgggac tctcagagcc cacacgctca tggatacatt 660

ccttccaaat ttccaaacaa gaacctgaag aagaattact gtcgtaaccc cgatagggag 720

ctgcggcctt ggtgtttcac caccgacccc aacaagcgct gggaactttg tgacatcccc 780

cgctgcacaa cacctccacc atcttctggt cccacctacc agtgtctgaa gggaacaggt 840

gaaaactatc gcgggaatgt ggctgttacc gtgtccgggc acacctgtca gcactggagt 900

gcacagaccc ctcacacaca taacaggaca ccagaaaact tcccctgcaa aaatttggat 960

gaaaactact gccgcaatcc tgacggaaaa agggccccat ggtgccatac aaccaacagc 1020

caagtgcggt gggagtactg taagataccg tcctgtgact cctccccagt atccacggaa 1080

caattggctc ccacagcacc acctgagcta acccctgtgg tccaggactg ctaccatggt 1140

gatggacaga gctaccgagg cacatcctcc accaccacca caggaaagaa gtgtcagtct 1200

tggtcatcta tgacaccaca ccggcaccag aagaccccag aaaactaccc aaatgctggc 1260

ctgacaatga actactgcag gaatccagat gccgataaag gcccctggtg ttttaccaca 1320

gaccccagcg tcaggtggga gtactgcaac ctgaaaaaat gctcaggaac agaagcgagt 1380

gttgtagcac ctccgcctgt tgtcctgctt ccagatgtag agactccttc cgaagaagac 1440

tgtatgtttg ggaatgggaa aggataccga ggcaagaggg cgaccactgt tactgggacg 1500

ccatgccagg actgggctgc ccaggagccc catagacaca gcattttcac tccagagaca 1560

aatccacggg cgggtctgga aaaaaattac tgccgtaacc ctgatggtga tgtaggtggt 1620

ccctggtgct acacgacaaa tccaagaaaa ctttacgact actgtgatgt ccctcagtgt 1680

gcggcccctt catttgattg tgggaagcct caagtggagc cgaagaaatg tcctggaagg 1740

gttgtagggg ggtgtgtggc ccacccacat tcctggccct ggcaagtcag tcttagaaca 1800

aggtttggaa tgcacttctg tggaggcacc ttgatatccc cagagtgggt gttgactgct 1860

gcccactgct tggagaagtc cccaaggcct tcatcctaca aggtcatcct gggtgcacac 1920

caagaagtga atctcgaacc gcatgttcag gaaatagaag tgtctaggct gttcttggag 1980

cccacacgaa aagatattgc cttgctaaag ctaagcagtc ctgccgtcat cactgacaaa 2040

gtaatcccag cttgtctgcc atccccaaat tatgtggtcg ctgaccggac cgaatgtttc 2100

atcactggct ggggagaaac ccaaggtact tttggagctg gccttctcaa ggaagcccag 2160

ctccctgtga ttgagaataa agtgtgcaat cgctatgagt ttctgaatgg aagagtccaa 2220

tccaccgaac tctgtgctgg gcatttggcc ggaggcactg acagttgcca gggtgacagt 2280

ggaggtcctc tggtttgctt cgagaaggac aaatacattt tacaaggagt cacttcttgg 2340

ggtcttggct gtgcacgccc caataagcct ggtgtctatg ttcgtgtttc aaggtttgtt 2400

acttggattg agggagtgat gagaaataat taa 2433

<210> 4

<211> 810

<212> PRT

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

<400> 4

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

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

785 790 795 800

Thr Trp Ile Glu Gly Val Met Arg Asn Asn

805 810

<210> 5

<211> 2145

<212> DNA

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

<400> 5

aaagtgtatc tctcagagtg caagactggg aatggaaaga actacagagg gacgatgtcc 60

aaaacaaaaa atggcatcac ctgtcaaaaa tggagttcca cttctcccca cagacctaga 120

ttctcacctg ctacacaccc ctcagaggga ctggaggaga actactgcag gaatccagac 180

aacgatccgc aggggccctg gtgctatact actgatccag aaaagagata tgactactgc 240

gacattcttg agtgtgaaga ggaatgtatg cattgcagtg gagaaaacta tgacggcaaa 300

atttccaaga ccatgtctgg actggaatgc caggcctggg actctcagag cccacacgct 360

catggataca ttccttccaa atttccaaac aagaacctga agaagaatta ctgtcgtaac 420

cccgataggg agctgcggcc ttggtgtttc accaccgacc ccaacaagcg ctgggaactt 480

tgtgacatcc cccgctgcac aacacctcca ccatcttctg gtcccaccta ccagtgtctg 540

aagggaacag gtgaaaacta tcgcgggaat gtggctgtta ccgtgtccgg gcacacctgt 600

cagcactgga gtgcacagac ccctcacaca cataacagga caccagaaaa cttcccctgc 660

aaaaatttgg atgaaaacta ctgccgcaat cctgacggaa aaagggcccc atggtgccat 720

acaaccaaca gccaagtgcg gtgggagtac tgtaagatac cgtcctgtga ctcctcccca 780

gtatccacgg aacaattggc tcccacagca ccacctgagc taacccctgt ggtccaggac 840

tgctaccatg gtgatggaca gagctaccga ggcacatcct ccaccaccac cacaggaaag 900

aagtgtcagt cttggtcatc tatgacacca caccggcacc agaagacccc agaaaactac 960

ccaaatgctg gcctgacaat gaactactgc aggaatccag atgccgataa aggcccctgg 1020

tgttttacca cagaccccag cgtcaggtgg gagtactgca acctgaaaaa atgctcagga 1080

acagaagcga gtgttgtagc acctccgcct gttgtcctgc ttccagatgt agagactcct 1140

tccgaagaag actgtatgtt tgggaatggg aaaggatacc gaggcaagag ggcgaccact 1200

gttactggga cgccatgcca ggactgggct gcccaggagc cccatagaca cagcattttc 1260

actccagaga caaatccacg ggcgggtctg gaaaaaaatt actgccgtaa ccctgatggt 1320

gatgtaggtg gtccctggtg ctacacgaca aatccaagaa aactttacga ctactgtgat 1380

gtccctcagt gtgcggcccc ttcatttgat tgtgggaagc ctcaagtgga gccgaagaaa 1440

tgtcctggaa gggttgtagg ggggtgtgtg gcccacccac attcctggcc ctggcaagtc 1500

agtcttagaa caaggtttgg aatgcacttc tgtggaggca ccttgatatc cccagagtgg 1560

gtgttgactg ctgcccactg cttggagaag tccccaaggc cttcatccta caaggtcatc 1620

ctgggtgcac accaagaagt gaatctcgaa ccgcatgttc aggaaataga agtgtctagg 1680

ctgttcttgg agcccacacg aaaagatatt gccttgctaa agctaagcag tcctgccgtc 1740

atcactgaca aagtaatccc agcttgtctg ccatccccaa attatgtggt cgctgaccgg 1800

accgaatgtt tcatcactgg ctggggagaa acccaaggta cttttggagc tggccttctc 1860

aaggaagccc agctccctgt gattgagaat aaagtgtgca atcgctatga gtttctgaat 1920

ggaagagtcc aatccaccga actctgtgct gggcatttgg ccggaggcac tgacagttgc 1980

cagggtgaca gtggaggtcc tctggtttgc ttcgagaagg acaaatacat tttacaagga 2040

gtcacttctt ggggtcttgg ctgtgcacgc cccaataagc ctggtgtcta tgttcgtgtt 2100

tcaaggtttg ttacttggat tgagggagtg atgagaaata attaa 2145

<210> 6

<211> 714

<212> PRT

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

<400> 6

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

Thr Trp Ile Glu Gly Val Met Arg Asn Asn

705 710

<210> 7

<211> 1245

<212> DNA

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

<400> 7

gagcctctgg atgactatgt gaatacccag ggggcttcac tgttcagtgt cactaagaag 60

cagctgggag caggaagtat agaagaatgt gcagcaaaat gtgaggagga cgaagaattc 120

acctgcaggg cattccaata tcacagtaaa gagcaacaat gtgtgataat ggctgaaaac 180

aggaagtcct ccataatcat taggatgaga gatgtagttt tatttgaaaa gaaagtgtat 240

ctctcagagt gcaagactgg gaatggaaag aactacagag ggacgatgtc caaaacaaaa 300

aatggcatca cctgtcaaaa atggagttcc acttctcccc acagacctag attctcacct 360

gctacacacc cctcagaggg actggaggag aactactgca ggaatccaga caacgatccg 420

caggggccct ggtgctatac tactgatcca gaaaagagat atgactactg cgacattctt 480

gagtgtgaag aggcggcccc ttcatttgat tgtgggaagc ctcaagtgga gccgaagaaa 540

tgtcctggaa gggttgtagg ggggtgtgtg gcccacccac attcctggcc ctggcaagtc 600

agtcttagaa caaggtttgg aatgcacttc tgtggaggca ccttgatatc cccagagtgg 660

gtgttgactg ctgcccactg cttggagaag tccccaaggc cttcatccta caaggtcatc 720

ctgggtgcac accaagaagt gaatctcgaa ccgcatgttc aggaaataga agtgtctagg 780

ctgttcttgg agcccacacg aaaagatatt gccttgctaa agctaagcag tcctgccgtc 840

atcactgaca aagtaatccc agcttgtctg ccatccccaa attatgtggt cgctgaccgg 900

accgaatgtt tcatcactgg ctggggagaa acccaaggta cttttggagc tggccttctc 960

aaggaagccc agctccctgt gattgagaat aaagtgtgca atcgctatga gtttctgaat 1020

ggaagagtcc aatccaccga actctgtgct gggcatttgg ccggaggcac tgacagttgc 1080

cagggtgaca gtggaggtcc tctggtttgc ttcgagaagg acaaatacat tttacaagga 1140

gtcacttctt ggggtcttgg ctgtgcacgc cccaataagc ctggtgtcta tgttcgtgtt 1200

tcaaggtttg ttacttggat tgagggagtg atgagaaata attaa 1245

<210> 8

<211> 414

<212> PRT

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

<400> 8

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410

<210> 9

<211> 1104

<212> DNA

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

<400> 9

gtcaggtggg agtactgcaa cctgaaaaaa tgctcaggaa cagaagcgag tgttgtagca 60

cctccgcctg ttgtcctgct tccagatgta gagactcctt ccgaagaaga ctgtatgttt 120

gggaatggga aaggataccg aggcaagagg gcgaccactg ttactgggac gccatgccag 180

gactgggctg cccaggagcc ccatagacac agcattttca ctccagagac aaatccacgg 240

gcgggtctgg aaaaaaatta ctgccgtaac cctgatggtg atgtaggtgg tccctggtgc 300

tacacgacaa atccaagaaa actttacgac tactgtgatg tccctcagtg tgcggcccct 360

tcatttgatt gtgggaagcc tcaagtggag ccgaagaaat gtcctggaag ggttgtaggg 420

gggtgtgtgg cccacccaca ttcctggccc tggcaagtca gtcttagaac aaggtttgga 480

atgcacttct gtggaggcac cttgatatcc ccagagtggg tgttgactgc tgcccactgc 540

ttggagaagt ccccaaggcc ttcatcctac aaggtcatcc tgggtgcaca ccaagaagtg 600

aatctcgaac cgcatgttca ggaaatagaa gtgtctaggc tgttcttgga gcccacacga 660

aaagatattg ccttgctaaa gctaagcagt cctgccgtca tcactgacaa agtaatccca 720

gcttgtctgc catccccaaa ttatgtggtc gctgaccgga ccgaatgttt catcactggc 780

tggggagaaa cccaaggtac ttttggagct ggccttctca aggaagccca gctccctgtg 840

attgagaata aagtgtgcaa tcgctatgag tttctgaatg gaagagtcca atccaccgaa 900

ctctgtgctg ggcatttggc cggaggcact gacagttgcc agggtgacag tggaggtcct 960

ctggtttgct tcgagaagga caaatacatt ttacaaggag tcacttcttg gggtcttggc 1020

tgtgcacgcc ccaataagcc tggtgtctat gttcgtgttt caaggtttgt tacttggatt 1080

gagggagtga tgagaaataa ttaa 1104

<210> 10

<211> 367

<212> PRT

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

<400> 10

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

<210> 11

<211> 750

<212> DNA

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

<400> 11

gccccttcat ttgattgtgg gaagcctcaa gtggagccga agaaatgtcc tggaagggtt 60

gtaggggggt gtgtggccca cccacattcc tggccctggc aagtcagtct tagaacaagg 120

tttggaatgc acttctgtgg aggcaccttg atatccccag agtgggtgtt gactgctgcc 180

cactgcttgg agaagtcccc aaggccttca tcctacaagg tcatcctggg tgcacaccaa 240

gaagtgaatc tcgaaccgca tgttcaggaa atagaagtgt ctaggctgtt cttggagccc 300

acacgaaaag atattgcctt gctaaagcta agcagtcctg ccgtcatcac tgacaaagta 360

atcccagctt gtctgccatc cccaaattat gtggtcgctg accggaccga atgtttcatc 420

actggctggg gagaaaccca aggtactttt ggagctggcc ttctcaagga agcccagctc 480

cctgtgattg agaataaagt gtgcaatcgc tatgagtttc tgaatggaag agtccaatcc 540

accgaactct gtgctgggca tttggccgga ggcactgaca gttgccaggg tgacagtgga 600

ggtcctctgg tttgcttcga gaaggacaaa tacattttac aaggagtcac ttcttggggt 660

cttggctgtg cacgccccaa taagcctggt gtctatgttc gtgtttcaag gtttgttact 720

tggattgagg gagtgatgag aaataattaa 750

<210> 12

<211> 249

<212> PRT

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

<400> 12

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Trp Ile Glu Gly Val Met Arg Asn Asn

245

<210> 13

<211> 684

<212> DNA

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

<400> 13

gttgtagggg ggtgtgtggc ccacccacat tcctggccct ggcaagtcag tcttagaaca 60

aggtttggaa tgcacttctg tggaggcacc ttgatatccc cagagtgggt gttgactgct 120

gcccactgct tggagaagtc cccaaggcct tcatcctaca aggtcatcct gggtgcacac 180

caagaagtga atctcgaacc gcatgttcag gaaatagaag tgtctaggct gttcttggag 240

cccacacgaa aagatattgc cttgctaaag ctaagcagtc ctgccgtcat cactgacaaa 300

gtaatcccag cttgtctgcc atccccaaat tatgtggtcg ctgaccggac cgaatgtttc 360

atcactggct ggggagaaac ccaaggtact tttggagctg gccttctcaa ggaagcccag 420

ctccctgtga ttgagaataa agtgtgcaat cgctatgagt ttctgaatgg aagagtccaa 480

tccaccgaac tctgtgctgg gcatttggcc ggaggcactg acagttgcca gggtgacagt 540

ggaggtcctc tggtttgctt cgagaaggac aaatacattt tacaaggagt cacttcttgg 600

ggtcttggct gtgcacgccc caataagcct ggtgtctatg ttcgtgtttc aaggtttgtt 660

acttggattg agggagtgat gaga 684

<210> 14

<211> 228

<212> PRT

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

<400> 14

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Gly Val Met Arg

225

Claims (10)

1. a kind of method of Liver Collagen proteinosis or fibrosis caused by prevention or treatment subject liver organization damage, packet Include administration a effective amount of plasminogen of subject.

2. the method for claim 1 wherein it is described damage include Ischemia-reperfusion injury, Drug damage, immune injury, It is damaged caused by damage, cancer caused by damage, inflammatory injury, excessive consumption of alcohol caused by chemical substance.

3. the method for claim 2, wherein the damage includes damaging caused by fat deposition.

4. a kind of method prevented or treat subject's Liver Collagen proteinosis or fibrosis, including subject's effective quantity is administered Plasminogen.

5. a kind of prevention or treatment sub-ject cause or the method for concurrent Liver Collagen proteinosis or fibrosis, packet Include administration a effective amount of plasminogen of subject.

Prevent or treatment subject artery's atherosis causes or concurrent Liver Collagen proteinosis or fibrosis 6. a kind of Method, including a effective amount of plasminogen of subject is administered.

7. a kind of prevention or treatment subject's hyperlipidemia cause or the method for concurrent Liver Collagen proteinosis or fibrosis, Including a effective amount of plasminogen of subject is administered.

8. the method for claim 7, wherein the hyperlipidemia is including chosen from the followings one or more：Blood triglyceride levels Raising, the raising of blood total cholesterol level, the raising of blood low-density lipoprotein, the raising of blood very low density lipoprotein.

9. a kind of Liver Collagen proteinosis or the method for fibrosis prevented or treatment subject drug induced hepatic injury causes, packet Include administration a effective amount of plasminogen of subject.

10. the method for claim 9, wherein the drug is hepatotoxic medication.