CN108210896A - Prevent and treat drug of hyperlipidemia and application thereof - Google Patents

Abstract

The present invention relates to for preventing and/or treating the method for subject's hyperlipidemia and its associated disease, it is susceptible to suffer from hyperlipidemia including administration, with hyperlipidemia or suffers from Other diseases and with subject's prevention of hyperlipidemia and/or the plasminogen of therapeutically effective amount.The invention further relates to for being used to prevent and/or treat subject's hyperlipidemia and its drug comprising plasminogen, pharmaceutical composition, product, the kit of associated disease.

Description

Prevent and treat drug of hyperlipidemia and application thereof

Technical field

It is easy including being administered the present invention relates to a kind of method prevented and/or treat fat metabolic disturbance and its associated disease A effective amount of plasminogen of subject of fat metabolic disturbance and its associated disease is suffered from, with abatement fat in bodily tissue The abnormal deposition of organ, so as to fulfill preventing and/or treating fat metabolic disturbance and its associated disease, the purpose of complication.

Background technology

Fat metabolic disturbance is also known as lipodystrophy, is one kind in metabolic disease, is by primary or acquisition The exception of lipid (lipid) and its metabolite quality and quantity in blood caused by sexual factor and its hetero-organization organ.The metabolism of lipid Including lipid small intestine it is digested, absorb, blood circulation (being transported by lipoprotein) is entered by lymphatic system, is converted through liver, store up Adipose tissue is stored in, is utilized when needing by tissue.The main function of lipid in vivo is oxidation energy supply, and adipose tissue is body Energy warehouse, fat can also cooperate with skin, bone, muscle protection internal organ, prevent body temperature from distributing and helping fat-soluble dimension in food The absorption of raw element.Phosphatide is the important feature ingredient of all cell membranes, and cholesterol is cholic acid and steroid hormone (adrenal cortex Hormone and gonadal hormone) precursor.Lipid metabolism is adjusted by histoorgans such as heredity, neurohumor, hormone, enzyme and livers Section.When these are because being known as abnormal, disorders of lipid metabolism and the pathophysiological change in relation to organ, such as hyperlipoprotememia can be caused Clinical syndrome, obesity, fatty liver etc. caused by and its.

Hyperlipoprotememia (Hyperlipoproteinemia) is caused by the lipoprotein in blood is excessively high.Fat in blood Matter such as triglyceride (TG), free cholesterol (FC), cholesterol ester (CE) and phosphatide etc. are seldom dissolved in water, only with apolipoprotein (APO) composition giant molecule compound (lipoprotein), could dissolve in blood, operates and be metabolized.When blood fat is higher than normal person's upper limit As hyperlipidemia (Hyperlipemia).Since blood fat is transported in blood with lipoprotein form, therefore hyperlipidemia is also referred to as height Lipoproteinemia.Generally with empty stomach blood triglyceride of being grown up more than 160mg/dl, cholesterol is more than 260mg/dl, children's cholesterol It is standard more than 160mg/dl^[1]。

Hyperlipoprotememia (hyperlipidemia) is the one of the major reasons of atherosclerotic lesion, is HypercholesterolemicRats Abnormal performance.Due to the type of blood fat or lipoprotein difference, blood fat or lipoprotein beyond normal range (NR) also can be different, So hyperlipoprotememia is divided into five types by the World Health Organization (WHO)：I type, mainly chylomicron increase, and serum muddiness is in Milky, wherein containing a large amount of triglycerides (TG)；II type is divided into two hypotype of II a types and II b, the former is mainly low density lipoprotein Egg (LDL) significantly increases, and the latter's very low density lipoprotein (VLDL) also has increase；III type, often muddiness LDL and VLDL is all for serum There is an increase, fusion both on electrophoresis；IV type, predominantly VLDL increase, and serum or have muddiness；V type, chylomicron and VLDL are all There is increase, serum muddiness is creamy white.It is wherein most common with II type familial combined hyperlipidemia^[1]。

Hyperlipidemia can be divided into primary and secondary two major class according to the cause of disease.Primary is mostly due to lipid and lipoprotein generation Birth defects (or hereditary detect) and certain environmental factors (including diet, nutrition and drug etc.) are thanked by unknown machine It manages and causes.It is secondary mainly secondary to certain disease, as diabetes, liver diseases, kidney trouble, thyroid disease and It drinks, is fat.The environmental factors such as diet and life style are also the cause of disease of the disease.

Diabetes often merge disorders of lipid metabolism, and therefore diabetes are also known as " glycolipid disease "^[2].The pathogenesis of diabetes with B cell function damage and insulin resistance are related, show as chronic hyperglycemia, and the disorder of glycometabolism often merges lipid-metabolism Disorder.Lipids metabolic disturbance in diabetes mellitus has become the risk factor of angiocardiopathy independence, is mainly shown as high glycerine three Pionemia, low-level HDL and LDL concentration increase.

The mechanism of diabetes disorders of lipid metabolism is unclear, but numerous evidences show that insulin resistance is its generation Key link.Research in recent years also found that intestines insulin resistance also assists in wherein.Diabetes animal model and crowd are ground Study carefully discovery and further result in insulin resistance with the relevant certain abnormal gene expressions of lipid metaboli.Diabetic's Atherosclerosis The generation of change is related to many factors, but the exception of plasma lipid level is most important factor.Research shows that diabetic The morbidity and mortality of angiocardiopathy are apparently higher than non-diabetic patients, and diabetes have become the independence of angiocardiopathy Risk factor^[3]。

In recent years, nephrosis and the relationship of disorders of lipid metabolism are more and more noticeable, in chronic progressive injury of kidney companion often With Anomalous lipid metablism, and hyperlipidemia can promote and aggravate the damage of kidney, other than mediating glomerular injury, between tubule It also works in matter damage.Munk first describes the dyslipidemia of nephrotic syndrome within 1913.There are scholar's report, 70%- 10% Patients With Nephrotic Symdrome may occur in which hyperlipidemia.It is mainly shown as that blood T-CHOL (TC) significantly increases, and with low Based on density lipoprotein-cholesterol raising, the raising of the slight increase of triglycerides (TG), wherein low-density lipoprotein (LDL) There is certain correlation with Urine proteins^[4].Chronic renal insufficiency patient based on moderate triglyceride, consolidate by the total courage of blood plasma Normally, cholesterol increases alcohol level in VLDLC, intermediate density lipoprotein cholesterol (IDLC).High-density lipoprotein courage is consolidated Alcohol (HDLC) is reduced, and the content of triglyceride in various lipoprotein increases.Its basic reason is exactly uremia environment to glycerine The synthesis of three esters and the adverse effect of catabolism and the inhibiting effect to reverse cholesterol transport^[5]。

It is wide with the popularity of kidney transplant therapy and various neotype immunosuppressants (particularly CsA, prednisone) General application, the life cycle of chronic renal failure (CRF) patient significantly extends, however the incidence of hyperlipidemia is non-after kidney transplant Chang Gao.Hyperlipemia of patients after underwent kidney transplantation is mainly shown as total plasma cholesterol (TC), triglycerides (TG), low-density lipoprotein courage Sterol (LDLC), C-VLDL (VLDLC) level increase^[6]。

Clinical research confirmation has certain correlation between disorders of lipid metabolism and diabetic nephropathy.Diabetic's fat Metabolic disorder, raised lipid deposition is in glomerular basement membrane, stimulation basilar memebrane cell Proliferation and extracellular matrix generation.Early in 1936, Kimmelstiel and Wilson just had found have in the renal arterioles of Diabetic Nephropathy patients, glomerulus and renal tubule A large amount of lipidosis^[7].Anomalous lipid metablism makes glomerulus and renal interstitial fibrosis that renal function progressive be caused to damage One of the major reasons^[8]。

Disorders of lipid metabolism may also lead to the generation of obesity (obesity syndrome).Obesity is divided to pure and secondary two Class.Simple obesity refers to the obesity without apparent incretion metabolism disease.Constitutional obesity and fat two kinds acquired can be divided into again. Constitutional obesity has a household heredity factors, and patient feeds abundant since the childhood, enters amount surplus, from small obesity, adipocyte is in hypertrophy. It is acquired fat mostly caused by overnutrition and/or physical exertion are reduced, as physical conditions for life changes after people in middle age Kind, disease recovery and abundant, postpartum of recuperating stop physical training or manual labor etc..Adipocyte is in hypertrophy change, is not increased Raw phenomenon, therapeutic effect are preferable.Caused by Secondary Obesity is mainly neuroendocrine disorder.Neuroendocrine has metabolism important Adjustment effect：1. hypothalamus has the maincenter of modulation of appetite, inflammation of the central nervous system sequelae, wound, tumour etc. that can cause Hypothalamic function is abnormal, makes honey stomach and causes obesity.2. insulin secretion increases, such as early stage non-insulin-depending type glycosuria Patient injects excessive insulin, causes hyperinsulinemia；The insulin of B cell knurl hypersecretion, this all makes fatty conjunction Into increase, cause obesity.Subtract 3. pituitary function is low, particularly promoting sexual gland hormone and thyrotropic hormone reduction causes sexual gland and first During shape gland hypofunction, obesity can occur.4. obesity easily occurs for multipara or oral contraceptive for female person, this prompting estrogen Play the role of promoting Fatty synthesis.5. hypercortisolism is often accompanied by central obesity.6. hypothyroidism, due to metabolism Rate is low, fat accumulation, and with myxoedema.7. sexual gland lowly also can be fat, such as dystrophia adiposogenitalis (cerebral adiposity Disease, Fu Luoli klinefelter syndromes caused by wound, encephalitis, hypophysoma, craniopharyngioma equivalent damage hypothalamus, show as centrality fertilizer It is fat, slow with diabetes insipidus and sexual development).

Disorders of lipid metabolism often results in fatty liver.Fatty liver refers to fat accumulation mistake in caused liver cell due to various reasons More lesions.Liver plays especially important effect in lipid-metabolism, it can synthesize lipoprotein, is conducive to lipid transport, It is fatty acid oxidation and the main place of ketoboidies formation.Liver amount containing lipid is few when normal, and about 4%, wherein mainly phosphatide. If liver cannot in time transport fat, fat is accumulated in liver cell, that is, forms fatty liver.

Fatty liver can be that an independent disease can also be caused by other reasons, such as obesity fatty liver, alcohol Property fatty liver, quick weight-lossing fatty liver, dystrophic fatty liver, diabetic fatty liver, Drug fatty liver etc..

Some drugs or chemical toxicant cause fatty liver by inhibiting the synthesis of protein, such as tetracycline, adrenal cortex Hormone, puromycin, ring amine, ipecine and arsenic, lead, silver, mercury etc..Lipid-lowering medicine also can by interfere the metabolism of lipoprotein and Form fatty liver.

One of harm of fatty liver is one of the formation that it promotes atherosclerosis, the reason of leading to atherosclerosis It is that Patients with Fatty Liver is often accompanied by hyperlipemia, blood viscosity increases, and low-density lipoprotein (LDL) therein is because of its molecular weight pole It is small, it is easy to it is calm in vascular wall across arterial intima, reduce arterial elasticity, caliber narrows, and flexibility weakens, finally Lead to blood circulation disorder.The harm of fatty liver second is that induce or aggravate hypertension, coronary heart disease, be easy to cause myocardial infarction and Sudden death.The three of the harm of fatty liver are encephalopathy fatty liver syndrome (Reye syndromes).The four of the harm of fatty liver are to lead to liver Hardening, liver failure, liver cancer.

Fatty liver is the product of liver lipid metabolism imbalance, while is the pathogenic factor for aggravating hepar damnification again, this is a kind of Reciprocal causation, the development of vicious circle.Fat drips increase in liver cell, make hepatic cell fattydegeneration, enlargement, and nucleus is extruded partially From center.The metabolism work of fat will carry out in mitochondria, and to extracellular transport mainly by smooth surfaced endoplasmic reticulum, fat exists fat Accumulation in liver cell, which further aggravates mitochondria and the burden of endoplasmic reticulum, reduces its function, and then influences other nutrients, swashs The metabolism of element, vitamin.Long-term degeneration of liver cells can lead to aregeneratory and the necrosis of liver cell, and then form liver fiber Change, hepatic sclerosis.The probability of the secondary hepatocellular carcinoma of hepatic sclerosis is higher.

The five of the harm of fatty liver are acute gestational fatty livers, and case fatality rate is high.This disease is also known as obstetrics acute yellow liver and withers Contracting, is a kind of pregnancy complication more rare, prognosis is dangerous.Last three months to mostly occur in pregnancy, clinical manifestation often with urgency Weight liver is similar, may occur in which acute liver failure, pancreatitis, renal failure, whole body disturbance of blood coagulation and lead to quick death, it is first The pregnant woman of secondary gestation is in the majority.

The six of the harm of fatty liver are induction or aggravate diabetes.If obesity fatty liver patient blood sugar concentration is more than normal Level, though the not up to diagnostic criteria of diabetes, it is considered that it is prediabetes.Fatty liver and diabetes usually together and It is raw, and interact, this brings the difficulty of bigger to clinical treatment.

Present invention research finds that plasminogen can prevent and/or cut down abnormal deposition of the fat in bodily tissue organ, Such as can prevent and cut down abnormal deposition of the lipid in the tissue between blood, vascular wall, internal organs and organ, improve The function of these histoorgans, so as to be carried for fat metabolic disturbance and its associated disease and its adjoint disease or complication Completely new prevention and treatment scheme is supplied

Summary of the invention

The present invention relates to following items：

On the one hand, the present invention relates to：Item 1. is a kind of to be used to prevent and/or treat subject's hyperlipidemia and its related diseases The method of disease is susceptible to suffer from hyperlipidemia including administration, with hyperlipidemia or suffers from Other diseases and with the tested of hyperlipidemia Person prevents and/or the plasminogen of therapeutically effective amount.

The method that item is 2. 1, wherein the hyperlipidemia is endocrine disturbance disease, glycometabolism disease, liver diseases, kidney Dirty disease, angiocardiopathy, intestines problem, thyroid disease, gall-bladder or disease of biliary tract obesity, are drunk, drug therapy initiation It is or adjoint.

The method of 3. 2, wherein the hyperlipidemia is hypertension, diabetes, chronic hepatitis, hepatic sclerosis, injury of kidney, Chronic glomerulonephritis, chronic pyelonephritis, nephrotic syndrome, renal insufficiency, kidney transplant, uremia, thyroid function are low Under, obstructive cholecystitis, obstructive cholangitis, estrin treatment cause or it is adjoint.

The method of any one of 4. 1-3 of item, wherein the hyperlipidemia is I types, IIa types, IIb types, type III or IV types Hyperlipidemia.

The method of any one of 5. 1-4 of item, wherein the hyperlipidemia is chosen from the followings one or more：Serum glycerine Three esters (TG) raising, serum low-density fat egg (LDL) raising, very low density lipoprotein (VLDL) raising.

On the other hand, the present invention relates to：Item 6. is a kind of for athero- by improving hyperlipidemia prevention subject artery The method of hardening, including applying a effective amount of plasminogen to subject.

On the other hand, the present invention relates to：Item 7. is a kind of to be used for by improving hyperlipidemia prevention and/or abatement subject The method that fat is deposited in bodily tissue organs abnormality, including applying a effective amount of plasminogen to subject.

On the other hand, the present invention relates to：Item 8. is a kind of to be used for by improving hyperlipidemia prevention and/or treatment subject The method of fat disease caused by bodily tissue organs abnormality deposition, including applying a effective amount of fibrinolysin to subject It is former.

The method that item is 9. 7 or 8, wherein the fat refers to fat at subcutaneous group in bodily tissue organs abnormality deposition It knits, the abnormal deposition of vascular wall, internal organs.

The method that item is 10. 9, wherein fat disease caused by bodily tissue organs abnormality deposition includes obesity Disease, fatty liver, the lipid mechanism harm of heart, the lipid mechanism harm of kidney, atherosclerosis.

On the other hand, the present invention relates to：Item 11. is a kind of for improving the methods of subject's hyperlipidemia, described high in fat Mass formed by blood stasis includes chosen from the followings one or more：Hypercholesterolemia, hypertriglyceridemia, combined hyperlipidemia familial and low height Density lipoprotein mass formed by blood stasis, including applying a effective amount of plasminogen to subject.

On the other hand, the present invention relates to：A kind of 12. methods for reducing subject artery's atherosis risk of item, It includes applying a effective amount of plasminogen to subject.

The method that item is 13. 12, wherein the plasminogen passes through following one or more reduction subject artery congee Sample hardens risk：T-CHOL in blood, triglycerides, low-density lipoprotein white level is reduced to realize.

The method that item is 14. 13, wherein the plasminogen is by improving the reduction of blood middle-high density lipoprotein levels Subject artery's atherosis risk.

The method of any one of 15. 12-14 of item, wherein the plasminogen is heavy in the exception of vascular wall by reducing lipid Product reduces subject artery's atherosis risk.

The method that item is 16. 12, wherein the plasminogen moves the removing reduction subject of lipid by enhancing liver Pulse atherosclerosis risk.

17. according to the method for any one of item 1-16, wherein the plasminogen can with one or more other medicines or Therapy is administered in combination.

The method that item is 18. 17, wherein one or more other medicines include treating diabetes drug, artery congee Sample hardening treating drug, Therapy of Nephrotic Syndrome drug, renal insufficiency treatment drug, uremia therapy drug, Kidney transplantation treatment drug, fatty liver treatment drug, cirrhosis treatment drug, bariatrician drug.

19. according to the method for item 18, wherein the other medicines include：Blood lipid-lowering medicine, antiplatelet drug, drop blood Pressing object, vasodilator drug, hypoglycemic drug, anticoagulation medicine, thrombolytic agent, hepatic, antiarrhythmic drug, Cardiac drug, diuretic, anti-infectives, antiviral drugs, immunoregulation medicament, inflammation adjust class drug, antineoplastic Object, hormone medicine, thyroxine.

The method of any one of 20. 1-19 of item, 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.

The method of any one of 21. 1-20 of item, the plasminogen is on the basis of sequence 2,6,8,10 or 12, is added Add, delete and/or replace 1-100,1-90,1-80,1-70,1-60,1-50,1-45,1-40,1-35,1-30,1-25,1- 20th, 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.

The method of any one of 22. 1-21 of item, the plasminogen are comprising activities of endothelial tissue plasminogen segment and still have There is the protein of activities of endothelial tissue plasminogen.

The method of any one of 23. 1-22, the plasminogen are selected from Glu- plasminogens, Lys- plasminogens, small Plasminogen, Microplasminogen, delta- plasminogens or their variant for retaining activities of endothelial tissue plasminogen.

The method of any one of 24. 1-23, the plasminogen for natural or synthetic human plasminogen or its still Retain the variant or segment of activities of endothelial tissue plasminogen.

The method of any one of 25. 1-23 of item, the plasminogen are the people from primate or rodent Plasminogen directly to homologue or its still retain the variant of activities of endothelial tissue plasminogen or segment.

The method of any one of 26. 1-25 of item, the amino acid of the plasminogen is as shown in sequence 2,6,8,10 or 12.

The method of any one of 27. 1-26 of item, wherein the plasminogen is naive plasminogen.

The method of any one of 28. 1-27 of item, wherein the subject is people.

The method of any one of 29. 1-28 of item, wherein the subject lacks or missing plasminogen.

The method that item is 30. 29, wherein the shortage or missing are inborn, secondary and/or local.

On the other hand, the present invention relates to：A kind of plasminogens of the methods for any one of item 1-29 of item 31..

On the other hand, the present invention relates to：A kind of 32. pharmaceutical compositions, it includes pharmaceutically acceptable supporting agent and For the plasminogen of any one of item 1-29 the methods.

On the other hand, the present invention relates to：A kind of preventative or therapeutic agent boxes of item 33., it includes：(i) for item The plasminogen of any one of 1-29 the methods and (ii) are for delivering the plasminogen to the component of the subject (means)。

34. kit according to item 33 of item, wherein the component is syringe or bottle.

The kit that item is 35. 33 or 34 is also indicated comprising label or operation instructions, the label or operation instructions The plasminogen is administered into the subject with any one of practical matter 1-29 the methods.

On the other hand, the present invention relates to：A kind of 36. products of item, 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.

The kit of any one of 37. 33-35 or the product of item 36, also comprising other one or more components or Container contains other drugs in the component or container.

The kit or product that item is 38. 37, wherein the other drugs are selected from the group：Blood lipid-lowering medicine, antiplatelet Drug, blood-pressure drug, vasodilator drug, hypoglycemic drug, anticoagulation medicine, thrombolytic agent, hepatic, the anti-rhythm of the heart Arrhythmic agents, cardiac drug, diuretic, anti-infectives, antiviral drugs, immunoregulation medicament, inflammation adjust class drug, Antitumor drug, hormone medicine, thyroxine.

The invention further relates to plasminogen for the purposes of the method for any one of practical matter 1-29.

The invention further relates to plasminogen in preparation for the drug of the method for any one of item 1-29, pharmaceutical composition, system Purposes in product, kit.

The invention further relates to prevent and/or treat subject's fat metabolic disturbance and its associated disease.

On the one hand, the present invention relates to the method prevented and/or treat subject's fat metabolic disturbance and its associated disease, packets The plasminogen of administration subject prevention and/or therapeutically effective amount is included, wherein the subject is susceptible to suffer from fat metabolic disturbance, suffers from Fat metabolic disturbance suffers from Other diseases and with fat metabolic disturbance.The invention further relates to plasminogen for prevent and/ Or the purposes for the treatment of subject's fat metabolic disturbance and its associated disease.The invention further relates to plasminogens to prepare for preventing And/or the purposes in treatment subject's fat metabolic disturbance and its drug of associated disease, pharmaceutical composition, product, kit. Further, the invention further relates to for preventing and/or treat the fibrinolysin of subject's fat metabolic disturbance and its associated disease It is former.The invention further relates to for prevent and/or treat subject's fat metabolic disturbance and its associated disease comprising plasminogen Drug, pharmaceutical composition, product, kit.

In some embodiments, the fat metabolic disturbance is endocrine disturbance disease, glycometabolism disease, liver disease Disease, kidney trouble, angiocardiopathy, intestines problem, thyroid disease, gall-bladder or disease of biliary tract, obesity, drink, drug is controlled Treat initiation or adjoint fat metabolic disturbance.In some embodiments, the fat metabolic disturbance for hypertension, diabetes, Chronic hepatitis, hepatic sclerosis, injury of kidney, chronic glomerulonephritis, chronic pyelonephritis, nephrotic syndrome, renal insufficiency, kidney move Plant, uremia, hypothyroidism, obstructive cholecystitis, obstructive cholangitis, drug or hormone therapy initiation or adjoint Fat metabolic disturbance.In some embodiments, the fat metabolic disturbance for hyperlipidemia, hyperlipoprotememia, fatty liver, Atherosclerosis, obesity, internal organs fat deposition.In other embodiment, the atherosclerosis includes Atherosclerosis of aorta, coronary atherosclerosis, cerebral atherosclerosis, renal artery atherosclerosis, arteria hepatica atherosis, Superior mesenteric atherosclerosis, lower limb atherosclerosis.

In yet another aspect, the present invention relates to prevent and/or cut down subject's fat to deposit in bodily tissue organs abnormality Method, including be administered a effective amount of plasminogen of subject.The invention further relates to plasminogen for prevent and/or cut down by The purposes that examination person's fat is deposited in bodily tissue organs abnormality.The invention further relates to plasminogen prepare for prevent and/or Drug that abatement subject's fat is deposited in bodily tissue organs abnormality, pharmaceutical composition, product, the purposes in kit.Into One step, the invention further relates to for preventing and/or cut down the fibrinolysin that subject's fat is deposited in bodily tissue organs abnormality It is former.The invention further relates to include fibrinolysin what bodily tissue organs abnormality deposited for preventing and/or cutting down subject's fat Drug, pharmaceutical composition, product, the kit of original.

In yet another aspect, the present invention relates to prevent and/or treat subject's fat to deposit in bodily tissue organs abnormality The method of caused illness, including a effective amount of plasminogen of subject is administered.The invention further relates to plasminogens for preventing And/or the purposes for the treatment of subject's fat illness caused by bodily tissue organs abnormality deposition.The invention further relates to fibrinolysins Original is preparing drug, medicine for preventing and/or treating subject's fat illness caused by bodily tissue organs abnormality deposition Purposes in compositions, product, kit.Further, the invention further relates to fatty for preventing and/or treating subject The drug comprising plasminogen, pharmaceutical composition, product, the kit of illness caused by bodily tissue organs abnormality deposition.

In some embodiments, the fat refers to fat in blood in bodily tissue organs abnormality deposition, subcutaneous group It knits, the abnormal deposition of vascular wall, internal organs.In some embodiments, the fat is deposited in bodily tissue organs abnormality Caused illness includes obesity, hyperlipidemia, hyperlipoprotememia, fatty liver, atherosclerosis, lipid heart damage Harmful, lipid renal damage, lipid pancreas islet damage.

In yet another aspect, the present invention relates to prevent and/or treatment subject's fat metabolic disturbance caused by illness method, Including a effective amount of plasminogen of subject is administered.The invention further relates to plasminogens for preventing and/or treating subject's fat The purposes of illness caused by fat metabolism disorder.The invention further relates to plasminogens to prepare for preventing and/or treat subject's fat Purposes in the drug of illness caused by fat metabolism disorder, pharmaceutical composition, product, kit.Further, the invention further relates to For the plasminogen of illness caused by preventing and/or treating subject's fat metabolic disturbance.The invention further relates to for prevent and/ Or the drug comprising plasminogen, pharmaceutical composition, product, the kit of illness caused by treatment subject's fat metabolic disturbance. In some embodiments, the illness includes obesity, hyperlipidemia, hyperlipoprotememia, fatty liver, Atherosclerosis Change, lipid damage to cardiac tissue, lipid injury of kidney.

In yet another aspect, the present invention relates to the method for treating subject's disease by reducing fatty abnormal deposition, including A effective amount of plasminogen of subject is administered.The invention further relates to plasminogen be used for by reduce fatty abnormal deposition treatment by The purposes of examination person's disease.It is used for the invention further relates to plasminogen in preparation by reducing fatty abnormal deposition treatment subject's disease The drug of disease, pharmaceutical composition, product, the purposes in kit.Further, the invention further relates to reduce fat for passing through Abnormal deposition treats the plasminogen of subject's disease.The invention further relates to treat subject's disease by reducing fatty abnormal deposition The drug comprising plasminogen, pharmaceutical composition, product, the kit of disease.

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

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

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

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

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

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

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

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

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

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

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

In any of the above-described embodiment of the present invention, the plasminogen can have extremely with sequence 2,6,8,10 or 12 Few 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity, and still there is fibrinolytic Zymogen activity.In some embodiments, the plasminogen is on the basis of sequence 2,6,8,10 or 12, and addition is deleted And/or substitution 1-100,1-90,1-80,1-70,1-60,1-50,1-45,1-40,1-35,1-30,1-25,1-20,1- 15, 1-10,1-5,1-4,1-3,1-2,1 amino acid, and still there is the protein of activities of endothelial tissue plasminogen.

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

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

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

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

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

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

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

Definition

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

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

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

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

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

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

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

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

" fatty liver " refers to the lesion that fat accumulation is excessive in caused liver cell due to various reasons, can be one Independent disease can also be caused by other reasons, such as obesity fatty liver, alcoholic fatty liver, quick weight-lossing are fatty Liver, dystrophic fatty liver, diabetic fatty liver, Drug fatty liver etc..

Fat drips increase in fatty liver, liver cell, make hepatic cell fattydegeneration, enlargement, and nucleus is extruded partially From center.The metabolism work of fat will carry out in mitochondria, and to extracellular transport mainly by smooth surfaced endoplasmic reticulum, fat exists fat Accumulation in liver cell, which further aggravates mitochondria and the burden of endoplasmic reticulum, reduces its function, and then influences other nutrients, swashs The metabolism of element, vitamin.Long-term degeneration of liver cells can lead to aregeneratory and the necrosis of liver cell, and then form liver fiber Change, hepatic sclerosis.

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

People not yet determine the exact cause of atherosclerosis, it has been found that important pathogenic factor： Hyperlipidemia, hypertension have smoking history, have atherosclerosis family history (suffering from the disease before 60 years old) or diabetes.It is high in fat Mass formed by blood stasis can promote the formation of fatty streaks.Because hypertension applies artery certain constant force, accelerate obstruction of artery and hardened Journey, therefore the illness rate of atherosclerosis can be increased.Smoking can be led to arterial contraction, limit blood flowing, thus be Arteries and veins obstruction creates condition.Diabetes can also promote the generation of atherosclerosis, especially for the artery of very little.

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

The main reason for atherosclerosis is coronary heart disease, cerebral infarction, peripheral vascular disease.Lipidosis is artery congee The lesion basis of sample hardening, its main feature is that involvement arterial disease since inner membrance, generally first has lipid and the accumulation of compound carbohydrate, goes out Blood and thrombosis, and then proliferation of fibrous tissue and calcinosis, and have the gradual transformation and calcification of arterial media, lead to artery Wall thickening is hardened, lumen of vessels is narrow.Lesion often involves big-and-middle muscular artery, is enough to block lumen of artery once developing to, then this is dynamic The tissue or organ that arteries and veins is supplied are by ischemic or necrosis.

Atherosclerosis is a kind of systemic disease, an organ angiogenesis atherosclerotic lesion, it is meant that Blood vessel elsewhere may also have existed same lesion；Equally, vascular events occur for an organ, it is meant that other ground The dangerous of Fang Fasheng vascular events increases.

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^[9].In addition, fibrinolysin, which can activate some metalloprotein enzyme precursors (pro-MMPs), forms active metalloproteinases (MMPs).Therefore fibrinolysin is considered as an important upstream regulation object of extracellular proteolysis effect^[10,11].Fibrinolysin is The PAs of two kinds of physiologicals is passed through by plasminogen：Tissue-type plasminogen activator (tPA) or urokinase type plasminogen activation What agent (uPA) proteolysis was formed.Due to plasminogen, relative level is higher in blood plasma and other body fluid, conventionally PA The adjusting of system is mainly realized by the synthesis of PAs and activity level.The synthesis of PA system components is strictly adjusted by different factors, Such as hormone, growth factor and cell factor.In addition, also there are fibrinolysins and the specific physiological inhibitor of PAs.Fibrinolysin it is main Inhibitor is α 2- antiplasmins (α 2-antiplasmin).The activity of PAs is pressed down simultaneously by the plasminogen activator of uPA and tPA Preparation -1 (PAI-1) inhibits and the main plasminogen activator agent inhibitor -2 (PAI-2) for inhibiting uPA is adjusted.Certain cell tables Face has the uPA specific cell surface receptors (uPAR) of direct hydrolysis activity^[12,13]。

Plasminogen is a single chain glycoprotein, is made of 791 amino acid, and molecular weight is about 92 kDa^[14,15].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^[16,17].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^[18].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^[19].Fibrinolysin also has the substrate specificity of several components to ECM, including laminin, fibronectin, proteoglycans And gelatin, show fibrinolysin ECM reconstruction in also play an important role [^15,20,21].Indirectly, fibrinolysin can also pass through conversion Certain protease precursors are active protease come the other components for the ECM that degrades, including MMP-1, MMP-2, MMP-3 and MMP-9.Cause This, it is thus proposed that, fibrinolysin may be an important upstream regulator of extracellular proteolysis^[22].In addition, fibrinolysin has There is the ability for the growth factor for activating certain potential forms^[23-25].In vitro, fibrinolysin can also hydrolyze the component of complement system simultaneously Discharge chemotactic complement fragment.

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

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

Glu- plasminogens are the plasminogens of Native full-length, are made of 791 amino acid and (do not contain 19 amino acid Signal peptide), encode the cDNA sequence of the sequence as shown in sequence 1, amino acid sequence is as shown in sequence 2.In vivo, it also deposits It is a kind of be at the 76-77 amino acids of Glu- plasminogens hydrolysis so as to formed Lys- plasminogens, such as 6 institute of sequence Show, encode the cDNA sequence of the amino acid sequence as shown in sequence 5.Delta- plasminogens (δ-plasminogen) are overall lengths Plasminogen has lacked the segment of Kringle2-Kringle5 structures, only containing Kringle1 and serine protease Domain^[26,27], there is the amino acid sequences (sequence 8) of document report delta- plasminogens^[27], encode the amino acid sequence CDNA sequence such as sequence 7.Miniplasminogen (Mini-plasminogen) is made of Kringle5 and serine protease domain, Having document report, it includes residue Val443-Asn791 (not contain the Glu residues of the Glu- plasminogen sequences of signal peptide For initial amino acid)^[28], amino acid sequence encodes cDNA sequence such as 9 institute of sequence of the amino acid sequence as shown in sequence 10 Show.And Microplasminogen (Micro-plasminogen) only contains serine protease domain, there is its amino acid of document report Sequence include residue A la543-Asn791 (using do not contain signal peptide Glu- plasminogen sequences Glu residues for originate amino Acid)^[29], also there is patent document CN102154253A to report that its sequence includes residue Lys531-Asn791 (not contain signal peptide Glu- plasminogen sequences Glu residues be initial amino acid), this patent sequence reference patent document CN102154253A, Its amino acid sequence encodes the cDNA sequence of the amino acid sequence as shown in sequence 11 as shown in sequence 12.

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

In this application, 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 activities of endothelial tissue plasminogen Detection (plgA), the detection (plgAg) of plasma tissue plasminogen antigen, Plasma Tissue-Type Plasminogen Activitor mortifier live The detection of property, the detection of Plasma Tissue-Type Plasminogen Activitor mortifier antigen, plasma fibrin lyase-antifibrinolysin Multienzyme complex detects (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

16 weeks hyperlipemia model mouse of Fig. 1 give plasminogen after 30 days liver oil red O stain observe result.A be to Solvent PBS control group, for B to give plasminogen group, C is quantitative analysis results.The results show that give plasminogen group mouse liver fat Fat deposition, which is considerably less than, gives solvent PBS control group, and significantly (* represents P to quantitative analysis statistical discrepancy<0.05).Illustrate fibrinolysin Proper energy improves deposition of the fat in hyperlipemia model mouse liver.

16 weeks hyperlipemia model mouse of Fig. 2 give plasminogen 30 days posterior sinus of Valsalva oil red O stain observation results. A, C is gives solvent PBS control group, and to give plasminogen group, E is quantitative analysis results by B, D.The results show that give plasminogen group Mouse aorta sinus fat deposition, which is considerably less than, gives solvent PBS control group, and significantly (* represents P to statistical discrepancy<0.05).Explanation Plasminogen can improve deposition of the fat in hyperlipemia model mouse aorta sinus.

16 weeks hyperlipemia model mouse of Fig. 3 give plasminogen 30 days representative pictures of posterior sinus of Valsalva HE dyeing.A、 C is gives solvent PBS control group, and B, D is give plasminogen group.The results show that the visible bubble of solvent PBS control group active blood vessel wall Foam cell deposition (arrow is signified), plaque deposition is serious；Give plasminogen group active blood vessel wall only visible slight foam cells Deposition, and apparent atheromatous plaque deposition is had no under inner membrance, it is lighter to plasminogen group Aortic injury.Illustrate plasminogen energy Improve damage of the hyperlipemia model mouse aorta sinus inner wall caused by lipidosis.

16 weeks hyperlipemia model mouse of Fig. 4 give plasminogen cardiac fibers protein immunization histochemical staining figure after 30 days Piece.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 small to plasminogen group The fibrinous positive expression of rat heart, which is considerably less than, gives solvent PBS control group, and significantly (* represents P to statistical discrepancy<0.05). Illustrate that plasminogen can reduce the heart injury caused by hyperlipidemia.

16 weeks hyperlipemia model mouse of Fig. 5 give plasminogen heart IgM immunostaining representativeness pictures after 30 days.A To give solvent PBS control group, B is gives plasminogen group.The results show that the positive expression to plasminogen group mouse heart IgM Solvent PBS control group is considerably less than given, illustrates that plasminogen can mitigate heart injury caused by hyperlipidemia.

16 weeks hyperlipemia model mouse of Fig. 6 give plasminogen heart sirius red stains representativeness picture after 30 days. A is gives solvent PBS control group, and B is gives plasminogen group.The results show that the deposition to plasminogen group collagen is considerably less than given Solvent PBS control group illustrates that plasminogen can mitigate hyperlipemia model mouse heart fibrosis.

16 weeks hyperlipemia model mouse of Fig. 7 give plasminogen serum cardiac troponin I testing result after 30 days.As a result it shows Show, be apparently higher than to solvent PBS control group serum cardiac troponin concentration and give plasminogen group, and statistical discrepancy significantly (* tables Show P<0.05).Illustrate that plasminogen can significantly repair the damage of hyperlipidemia heart.

3% cholesterol hyperlipemia model mouse of Fig. 8 give plasminogen 10 days and serum high-density LP after 20 days Cholesterol detection result.The results show that after giving plasminogen to plasminogen group mice serum HDL-C concentration be apparently higher than to Solvent PBS control group, and heteropolar significantly (the * * tables of the two equal statistical difference of high-density lipoprotein concentration after being administered 10 days and 20 days Show P<0.01).Illustrate that plasminogen can effectively improve containing for hyperlipemia model mice serum middle-high density lipoprotein cholesterol It is disorderly to improve hyperlipemia model lipid of mice for amount.

3% cholesterol hyperlipemia model mouse of Fig. 9 give plasminogen serum total cholesterol testing result after 20 days. Solvent PBS control groups are given, and statistical discrepancy is notable the results show that being significantly lower than to plasminogen group mouse total cholesterol concentration (* represents P<0.05).Illustrate that plasminogen can reduce the content of T-CHOL in hyperlipemia model mice serum, having reduces The function of blood fat.

3% cholesterol hyperlipemia model mouse of Figure 10 give plasminogen after 20 days serum low-density LP courage consolidate Alcohol testing result.Solvent PBS control group, and statistical difference are given the results show that being significantly lower than to plasminogen group mouse LDL-C concentration It is different that significantly (* represents P<0.05).Illustrate that plasminogen can reduce low-density lipoprotein courage in hyperlipemia model mice serum and consolidate The content of alcohol has the function of to improve hyperlipidemia.

3% cholesterol hyperlipemia model mouse of Figure 11 give plasminogen serum atherosclerosis index after 20 days Testing result.Solvent PBS control group is given the results show that being significantly lower than to plasminogen group rat aorta atherosis index, and Statistical difference is heteropolar, and significantly (* * represent P<0.01).Illustrate that plasminogen can effectively reduce hyperlipemia model mouse and artery congee occurs The risk of sample hardening.

3% cholesterol hyperlipemia model mouse of Figure 12 give plasminogen serum cardiac risk danger index knot after 20 days Fruit.Solvent PBS control group is given the results show that being significantly less than to plasminogen group CRI, and extremely significantly (* * represent P to statistical discrepancy <0.01).Illustrate that plasminogen can effectively reduce the risk that heart disease occurs for hyperlipemia model mouse.

Figure 13 24-25 week diabetic mices give plasminogen liver oil red O stain picture after 35 days.The results show that it gives The lipidosis area of plasminogen group mouse liver, which is significantly smaller than, gives solvent PBS control groups, and statistical discrepancy significantly (* tables Show P<0.05).Illustrate that plasminogen can reduce deposition of the fat in diabetic mice liver.

Figure 14 24-25 week old diabetic mices give plasminogen 31 days aorta posterior HE dyeing pictures.A, C is to molten Matchmaker's PBS control group, B, D is give plasminogen group.The results show that there is foam cells deposition to solvent PBS control group vascular wall (arrow logo), middle elastic film is disorganized, vessel wall thickening, and tube wall convex-concave is uneven；Give plasminogen group middle elastic film Tactical rule, undulate, vascular wall thickness are uniform.Show that injecting plasminogen has the Aortic injury caused by diabetes There is certain repair.

26 week old diabetic mices of Figure 15 give plasminogen ventricle oil red O stain representativeness picture after 35 days.A be to Solvent PBS control group, B is gives plasminogen group.The results show that plasminogen group mouse ventricle lipidosis (arrow logo) Considerably less than give solvent PBS control group.Illustrate that plasminogen can reduce diabetic mice ventricle lipidosis, promote ventricle damage Reparation.

26 week old diabetic mices of Figure 16 give the content of plasminogen serum middle-high density lipoprotein cholesterol after 35 days Testing result.The results show that after people source fibrinolysin source is continuously injected to diabetic mice 35 days, plasminogen group mouse is given The content of HDL-C, which is higher than, in serum gives solvent PBS control group, and statistical discrepancy is notable.Illustrate that blood can be promoted by injecting plasminogen The content raising of aloof from politics and material pursuits density lipoprotein-cholesterol, improves diabetic mice blood fat disorder.

Figure 17 24-25 week old diabetic mices give plasminogen low density lipoprotein cholesterol in serum after 31 days (LDL-C) content detection result.The results show that diabetic mice continuously injected people source fibrinolysin source after 31 days, to fibre LDL-C contents in lyase original group mice serum are less than solvent PBS control group is given, and statistical discrepancy is close to significantly (P=0.1).It says Bright plasminogen can reduce the content of low density lipoprotein cholesterol in diabetic mice serum.

Figure 18 ApoE Atherosclerosis Model mouse give plasminogen serum total cholesterol testing result after 30 days. Solvent PBS control group is given, and statistical discrepancy is notable the results show that being significantly lower than to plasminogen group mouse total cholesterol concentration (* represents P<0.05).Illustrate that plasminogen can reduce containing for T-CHOL in ApoE Atherosclerosis Model mice serums It is disorderly to improve Atherosclerosis Model lipid of mice for amount.

Figure 19 ApoE Atherosclerosis Model mouse give plasminogen serum triglyceride testing result after 30 days. Solvent PBS control group is given, and statistical discrepancy is notable the results show that being significantly lower than to plasminogen group mouse triglyceride concentration (* represents P<0.05).Illustrate that plasminogen can reduce containing for triglycerides in ApoE Atherosclerosis Model mice serums It is disorderly to improve Atherosclerosis Model lipid of mice for amount.

Figure 20 ApoE Atherosclerosis Model mouse give plasminogen serum LDL cholesterol after 30 days Testing result.Solvent PBS control group, and statistical discrepancy are given the results show that being significantly lower than to plasminogen group mouse LDL-C concentration Significantly (* represents P<0.05).Illustrate that plasminogen can reduce low-density lipoprotein in ApoE Atherosclerosis Model mice serums The content of white cholesterol, it is disorderly to improve Atherosclerosis Model lipid of mice.

Figure 21 ApoE Atherosclerosis Model mouse give plasminogen liver oil red O stain representative diagram after 30 days Piece.A is gives solvent PBS control group, and for B to give plasminogen group, C is quantitative analysis results.It is the results show that small to plasminogen group Mouse Liver fatty deposition, which is considerably less than, gives solvent PBS control groups, and significantly (* represents P to quantitative analysis statistical discrepancy<0.05).It says Bright plasminogen can reduce deposition of the fat in Atherosclerosis Model mouse liver.

Figure 22 ApoE Atherosclerosis Model mouse give 30 days posterior sinus of Valsalva oil red O stains of plasminogen and represent Property picture.A is gives solvent PBS control group, and B is gives plasminogen group.The results show that give plasminogen group mouse aorta sinus fat Fat deposition, which is considerably less than, gives solvent PBS control group.Illustrate that plasminogen can improve fat in Atherosclerosis Model mouse master Deposition in artery sinus.

16 week old hyperlipemia model mouse of Figure 23 give 30 days posterior sinus of Valsalva sirius red stains of plasminogen and represent Property 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 blood The area of inside pipe wall collagen deposition (arrow logo), which is significantly less than, gives solvent PBS control group, illustrates that plasminogen can be cut down Hyperlipemia model mouse aorta sinus Fibrosis levels.

Figure 24 ApoE Atherosclerosis Model mouse give plasminogen heart coefficients statistics result after 30 days.As a result Display is significantly lower than to plasminogen group mouse heart coefficient and gives solvent PBS control group.Illustrate that plasminogen can improve ApoE and move Cardiac compensation caused by pulse atherosclerosis model mice heart injury is loose.

Figure 25 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.Illustrate that plasminogen can effectively reduce 3% cholesterol hyperlipemia Disease model mice renal fibrosis.

Figure 26 give plasminogen after 30 days 3% cholesterol hyperlipemia model mouse kidney oil red O observe result.A is Blank control group, B is gives solvent PBS control group, and for C to give plasminogen group, D is quantitative analysis results.The results show that fibre Lyase original group mouse kidney fat deposition (arrow logo) is considerably less than and gives solvent PBS control group, and quantitative analysis statistical discrepancy Significantly；It is in addition, similar to blank control group mouse to plasminogen group lipidosis level.Illustrate that plasminogen can cut down fat Deposition in hyperlipemia model mouse kidney, so as to reduce the kidney injury caused by fat deposition.

Embodiment

Deposition of 1 plasminogen of the embodiment abatement fat in 16 weeks hyperlipemia model mouse livers

6 week old male C57 mouse 11 feed high cholesterol diet (Nantong Te Luofei, article No. TP2031) high in fat 16 weeks With induced hyperlipidemia model^[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, and mouse was put to death in the 31st day, liver is taken to fix 24-48 hours in 4% paraformaldehyde, respectively at 15%, 30% sucrose In 4 DEG C sink to the bottom overnight, OCT embedding, 8 μm of frozen section thickness, oil red O stain 15min, 75% alcohol break up 5 seconds, bush uniformly dyeing Core 30s, glycerin gelatine mounting.Slice is in 400 times of optical microphotograph Microscopic observations.

Oil red O stain can show lipidosis, reflect the degree of lipidosis^[32].The results show that give plasminogen group (Figure 1B) mouse liver fat deposition is considerably less than to solvent PBS control group (Figure 1A), and quantitative analysis statistical discrepancy is notable (Fig. 1 C).Illustrate that plasminogen can cut down deposition of the fat in hyperlipemia model mouse liver.

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

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, and mouse was put to death in the 31st day, dirty 4% paraformaldehyde that is organized in of coring fixes 24-48 hours, respectively at 15%, 30% It is sunk to the bottom overnight for 4 DEG C in sucrose, OCT embeddings, 8 μm of aortic sinus frozen section thickness, oil red O stain 15min, the differentiation of 75% alcohol 5 seconds, bush uniformly dyeing core 30s, glycerin gelatine mounting.Slice is in 40 (Fig. 2A, 2B), 200 times of (Fig. 2 C, 2D) times light microscopes Lower observation.

The results show that it is considerably less than to plasminogen group (Fig. 2 B, 2D) mouse aorta sinus fat deposition to PBS pairs of solvent According to group (Fig. 2A, 2C), and statistical discrepancy is significantly (Fig. 2 E).Illustrate that plasminogen can reduce lipid in hyperlipemia model mouse master Deposition in artery sinus.

3 plasminogen of embodiment improves hyperlipemia model mouse aorta sinus damage in 16 weeks

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, and mouse was put to death in the 31st day, dirty 4% paraformaldehyde that is organized in of coring fixes 24-48 hours.Tissue after fixation is through wine Paraffin embedding is carried out after smart serial dehydration and dimethylbenzene are transparent.Tissue samples after fixation are saturating through alcohol serial dehydration and dimethylbenzene Paraffin embedding is carried out after bright.Aortic sinus histotomy thickness is 3 μm, slice dewaxing rehydration and with haematoxylin and eosin stains (HE dyeing), ammonium hydroxide returns blue and alcohol serial dehydration mounting after the differentiation of 1% hydrochloride alcohol, be sliced 40 (Fig. 3 A, B), 200 times (Fig. 3 C, D) optical microphotograph Microscopic observation.

The results show that solvent PBS control group (Fig. 3 A, 3C) aortic sinus inner wall foam cells deposition (arrow is signified) is given, Plaque deposition weight；It is deposited, and under inner membrance to the only visible slight foam cells of plasminogen group (Fig. 3 B, 3D) aortic sinus inner wall It has no apparent atheromatous plaque deposition, is damaged to plasminogen group aortic sinus inner wall lighter.Illustrate that plasminogen can improve height Pionemia model mice artery sinus inner wall damages.

4 plasminogen of embodiment reduces the fibrinous expression of hyperlipemia model mouse heart in 16 weeks

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, and mouse was put to death in the 31st day, dirty 4% paraformaldehyde that is organized in of coring fixes 24-48 hours.Tissue after fixation is through wine Paraffin embedding is carried out after smart serial dehydration and dimethylbenzene are transparent.Histotomy thickness is 3 μm, is washed 1 time after slice dewaxing rehydration. With 3% dioxygen water incubation 15 minutes, wash 2 times, every time 5 minutes.5% normal sheep serum liquid (Vector Laboratories, Inc., USA) it closes 30 minutes；After time arrives, reject sheep blood serum liquid irises out tissue with PAP.With 3% Dioxygen water incubation 15 minutes is washed 2 times, every time 5 minutes.4 DEG C of overnight incubations of rabbit anti-mouse fibrin antibody (Abcam), 0.01MPBS is washed 2 times, every time 5 minutes.Goat anti-rabbit igg (HRP) antibody (Abcam) secondary antibody is incubated at room temperature 1 hour, and PBS washes 2 It is secondary, 5 minutes every time.It develops the color by DAB kits (Vector laboratories, Inc., USA), haematoxylin is answered after washing 3 times Dye 30 seconds, flowing water rinse 5 minutes.Gradient alcohol dehydration, dimethylbenzene is transparent and neutral gum mounting, is sliced and is shown in 200 times of optics Micro- Microscopic observation.

Fibrinogen is fibrinous precursor, is deposited in the case of a fracture in tissue, as body to the one of damage Kind stress reaction, fibrinogen are hydrolyzed into fibrin deposition in damage location^[33,34].Therefore, local fiber will can be damaged A mark of the protein level as degree of injury.

Immunohistochemical staining is the results show that apparent to the positive expression of plasminogen group mouse (Fig. 4 B) cardiac fibers albumen Less than solvent PBS control group (Fig. 4 A) is given, and statistical discrepancy is significantly (Fig. 4 C), caused by illustrating that plasminogen can reduce hyperlipidemia Myocardial damage.

The hyperlipemia model mouse cardiac muscle damage in 16 weeks of 5 plasminogen effective protection of embodiment

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, and mouse was put to death in the 31st day, dirty 4% paraformaldehyde that is organized in of coring fixes 24-48 hours.Tissue after fixation is through wine Paraffin embedding is carried out after smart serial dehydration and dimethylbenzene are transparent.Histotomy thickness is 3 μm, is washed 1 time after slice dewaxing rehydration. With 3% dioxygen water incubation 15 minutes, wash 2 times, every time 5 minutes.5% normal sheep serum liquid (Vector Laboratories, Inc., USA) it closes 30 minutes；After time arrives, reject sheep blood serum liquid irises out tissue with PAP.With 3% Dioxygen water incubation 15 minutes is washed 2 times, every time 5 minutes.Mountain sheep anti mouse IgM (HRP) antibody (Abcam) is incubated at room temperature 1 hour, PBS is washed 2 times, every time 5 minutes.It develops the color by DAB kits (Vector laboratories, Inc., USA), revives after washing 3 times Lignin dye core 30 seconds, flowing water rinses 5 minutes.Gradient alcohol dehydration, dimethylbenzene is transparent, and neutral gum mounting is sliced at 200 times Optical microphotograph Microscopic observation.

IgM antibody plays an important role during apoptosis and non-viable non-apoptotic cell is removed, the histoorgan local I gM of damage The level of antibody is proportionate with degree of injury^[35,^36].Therefore, the level of detection histoorgan local I gM antibody can reflect The degree of injury of the histoorgan.

Immunostaining results are shown, are considerably less than to the positive expression of plasminogen group mouse (Fig. 5 B) heart IgM to molten Matchmaker's PBS control group (Fig. 5 A), illustrates that plasminogen can reduce the damage of hyperlipemia model animal hearts.

6 plasminogen of embodiment mitigates 16 weeks hyperlipemia model mouse heart 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, and mouse was put to death in the 31st day, dirty 4% paraformaldehyde that is organized in of coring fixes 24-48 hours.Tissue after fixation is through wine Paraffin embedding is carried out after smart serial dehydration and dimethylbenzene are transparent.Histotomy 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, in 200 times of optical microphotograph Microscopic observations.

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

Coloration result is shown, is considerably less than to the deposition of plasminogen group (Fig. 6 B) collagen and is given solvent PBS control group (figure 6A), illustrate that plasminogen can mitigate the deposition of hyperlipemia model mouse heart tissue collagen, mitigate myocardial fibrosis.

7 plasminogen of embodiment repairs hyperlipemia model mouse cardiac muscle damage in 16 weeks

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 fasted after administration in the 30th day, fasting 16 hours, plucks within the 31st day eyeball and takes blood, centrifugation obtains supernatant, uses Cardiac troponin (Cardiac troponin I, CTNI) detection kit (Nanjing is built up) detection serum cTnT Concentration.

Cardiac muscle troponin I is the important symbol object of myocardial damage, and serum-concentration is capable of the journey of reflecting myocardium damage Degree^[37]。

Testing result is shown, is apparently higher than to solvent PBS control group serum cardiac troponin concentration and is given plasminogen group, And statistical discrepancy is significantly (Fig. 7).Illustrate that plasminogen can significantly improve the damage of hyperlipemia model mouse heart.

It is dense that 8 plasminogen of embodiment improves 3% cholesterol hyperlipemia model mice serum high-density lipoprotein cholesterol Degree

9 week old male C57 mouse 16 feed 3% cholesterol high lipid food (Nantong Te Luofei) 4 weeks, induce hyperlipemia Disease^[30,31], this model is set to 3% cholesterol hyperlipemia model.Mouse after Cheng Mo continues to feed the feeding high in fat of 3% cholesterol Material.Three days every mouse take 50 μ L of blood before administration, detect T-CHOL, and be randomly divided into according to total cholesterol concentration and weight Two groups, every group each 8.Start administration to be denoted as the 1st day, give plasminogen group mouse tail vein injection people source plasminogen 1mg/ It 0.1ml/ pcs/day, to the PBS of solvent PBS control group tail vein injection same volume, is administered 20 days.In mouse fasting in the 10th, 20 day It 16 hours, pricks within the 11st, 21 day orbital venous plexus and takes 50 μ l of blood, centrifugation obtains supernatant, to detect serum high-density LP courage Sterol (HDL-C).By detection kit, (bio-engineering research is built up in Nanjing to high-density lipoprotein cholesterol content herein Institute, article No. A112-1) the method detection.

High-density lipoprotein is a kind of plasma lipoprotein of antiatherosclerosis, is the protective factors of coronary heart disease, is commonly called as " blood vessel scavenger ".

Testing result is shown, is apparently higher than to plasminogen group mice serum HDL-C concentration and is given solvent PBS control group, and The two equal statistical discrepancy of HDL-C concentration after being administered 10,20 days is notable (Fig. 8).Illustrate that plasminogen can improve hyperlipidemia mould The content of type mice serum middle-high density lipoprotein cholesterol improves hyperlipemia in mice blood fat disorder.

9 plasminogen of embodiment reduces by 3% cholesterol hyperlipemia model mice serum total cholesterol level

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.Mouse after Cheng Mo continues to feed the feeding high in fat of 3% cholesterol Material.Three days every mouse take 50 μ L of blood before administration, detect T-CHOL, and be randomly divided into according to total cholesterol concentration and weight Two groups, every group each 8.Start administration to be denoted as the 1st day, give plasminogen group mouse tail vein injection people source plasminogen 1mg/ It 0.1ml/ pcs/day, to the PBS of solvent PBS control group tail vein injection same volume, is administered 20 days.In mouse fasting 16 in the 20th day Hour, it pricks within the 21st day orbital venous plexus and takes 50 μ L of blood, centrifugation obtains supernatant, and (Nanjing is built up using T-CHOL detection kit Bioengineering Research Institute, article No. A111-1) carry out T-CHOL detection.

Testing result is shown, is significantly lower than to plasminogen group mouse total cholesterol concentration and is given solvent PBS control groups, and Statistical discrepancy is significantly (Fig. 9).Illustrate that plasminogen can reduce the content of T-CHOL in hyperlipemia model mice serum.

10 plasminogen of embodiment reduces by 3% cholesterol hyperlipemia model mice serum low density lipoprotein cholesterol water It is flat

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.Mouse after Cheng Mo continues to feed the feeding high in fat of 3% cholesterol Material.Three days every mouse take 50 μ l of blood before administration, detect T-CHOL, and be randomly divided into according to total cholesterol concentration and weight Two groups, every group each 8.Start administration to be denoted as the 1st day, give plasminogen group mouse tail vein injection people source plasminogen 1mg/ It 0.1ml/ pcs/day, to the PBS of solvent PBS control group tail vein injection same volume, is administered 20 days.In mouse fasting 16 in the 20th day Hour, it pricks within the 21st day orbital venous plexus and takes 50 μ L of blood, centrifugation obtains supernatant, using low density lipoprotein cholesterol detection kit (Bioengineering Research Institute, article No. A113-1 are built up in Nanjing) carries out low density lipoprotein cholesterol (LDL-C) and detects.

Low-density lipoprotein is a kind of hdl particle for delivering cholesterol and entering peripheral tissues' cell, can be oxidized to oxygen Change low-density lipoprotein, when working as low-density lipoprotein (OX-LDL) excess of low-density lipoprotein, especially oxidative modification, it takes The cholesterol of band is just accumulated on arterial wall, causes artery sclerosis.Therefore low density lipoprotein cholesterol is referred to as that " bad courage is consolidated Alcohol ".

Solvent PBS control group, and statistical difference are given the results show that being significantly lower than to plasminogen group mouse (LDL-C) concentration Different notable (Figure 10).Illustrate that plasminogen can reduce containing for low density lipoprotein cholesterol in hyperlipemia model mice serum Amount improves hyperlipemia in mice blood fat disorder.

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

9 week old male C57 mouse 16 feed 3% cholesterol high lipid food (Nantong Te Luofei) 4 weeks, induce hyperlipemia Disease^[30,31], this model is set to 3% cholesterol hyperlipemia model.Mouse after Cheng Mo continues to feed the feeding high in fat of 3% cholesterol Material.Three days every mouse take 50 μ l of blood before administration, detect T-CHOL (T-CHO), and according to total cholesterol concentration and weight It is randomly divided into two groups, every group each 8.Start administration to be denoted as the 1st day, give plasminogen group mouse tail vein injection people source fibrinolytic 1mg/0.1ml/ pcs/day of proenzyme, to the PBS of solvent PBS control group tail vein injection same volume.The mouse after administration in the 20th day It is fasted, fasting 16 hours, pricks within the 21st day orbital venous plexus and take 50 μ L of blood, centrifugation obtains supernatant, and total cholesterol level uses T-CHOL detection kit (Bioengineering Research Institute, article No. A111-1 are built up in Nanjing) is detected；High-density lipoprotein courage Using high-density lipoprotein cholesterol detection kit, (Bioengineering Research Institute, article No. are built up in Nanjing to sterol (HDL-C) content A112-1 it) is detected.

Atherosclerosis index is the overall target for clinically predicting atherosclerosis, it is believed that it is as to hat Clinical meaning is than the T-CHOL of individual event, triglycerides, high-density lipoprotein and low-density in terms of the estimation of worry degree of risk Lipoprotein bigger^[38].Atherosclerosis index=(T-CHO- HDL-C)/HDL-C.

Result of calculation is shown, is significantly lower than to plasminogen group rat aorta atherosis index and is given solvent PBS control Group, and statistical discrepancy is significantly (Figure 11).Illustrate that plasminogen can reduce hyperlipemia model mouse and atherosclerosis occurs Risk.

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

9 week old male C57 mouse 16 feed 3% cholesterol high lipid food (Nantong Te Luofei) 4 weeks, induce hyperlipemia Disease^[30,31], this model is set to 3% cholesterol hyperlipemia model.Mouse after Cheng Mo continues to feed the feeding high in fat of 3% cholesterol Material.Three days every mouse take 50 μ l of blood, detection T-CHOL (T-CHO) before administration, and are divided at random according to total cholesterol concentration It is two groups, every group each 8.Start administration to be denoted as the 1st day, give plasminogen group mouse tail vein injection people source plasminogen 1mg/0.1ml/ pcs/day, to the PBS of solvent PBS control group tail vein injection same volume.Mouse starts to prohibit after administration in the 20th day Food, fasting 16 hours prick orbital venous plexus on the 21st day and take 50 μ L of blood, and centrifugation obtains supernatant, and total cholesterol level is consolidated using total courage Alcohol detection kit (Bioengineering Research Institute, article No. A111-1 are built up in Nanjing) is detected；High-density lipoprotein cholesterol (HDL-C) using high-density lipoprotein cholesterol detection kit, (Bioengineering Research Institute, article No. A112- are built up in Nanjing to content 1) it is detected.Cardiac risk index=T-CHO/HDL-C.

Cardiac risk index (cardiac risk index, CRI) to assess blood fat disorder induce heart disease wind Danger^[38]。

Solvent PBS control group is given the results show that being significantly less than to plasminogen group CRI, and statistical discrepancy extremely significantly (figure 12).Illustrate that plasminogen can effectively reduce the risk that heart disease occurs for hyperlipemia model mouse.

13 plasminogen of embodiment improves diabetic mice liver lipids deposition

24-25 week old male db/db mouse 10, are randomly divided into two groups, to solvent PBS control group and give plasminogen group Each 5.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.It is small to plasminogen group Mouse presses 2mg/0.2ml/ pcs/day of tail vein injection plasminogen, and same volume is given to solvent PBS control group tail vein injection PBS, successive administration 35 days.Putting to death mouse at the 36th day takes 4% paraformaldehyde of liver organization to fix 24-48 hours, respectively at 15%th, in 30% sucrose 4 DEG C sink to the bottom overnight, OCT embeddings, 8 μm of frozen section thickness, oil red O dyeing 15min, 75% alcohol point Change 5 seconds, bush uniformly dyeing core 30s, glycerin gelatine mounting.Slice is in 400 times of optical microphotograph Microscopic observations.

Coloration result shows, to plasminogen group (Figure 13 B) mouse liver lipidosis area significantly less than to solvent PBS control group (Figure 13 A), and statistical discrepancy is significantly (P=0.02) (Figure 13 C).Illustrate that plasminogen can reduce fat in glycosuria Deposition in sick mouse liver.

14 plasminogen of embodiment mitigates the damage of diabetic mice active blood vessel wall

24-25 week old male db/db mouse 10, are randomly divided into two groups, to solvent PBS control group and give plasminogen group Each 5.Experiment is denoted as grouping of weighing in the 0th day on the day of starting, and starts within the 1st day to PBS or plasminogen, successive administration 31 days.It gives Plasminogen group mouse presses 2mg/0.2ml/ pcs/day of tail vein injection plasminogen, gives solvent PBS control group tail vein injection Give the PBS of same volume.Mouse was put to death at the 32nd day and aorta is taken to fix 24 in 10% neutral formalin fixer Hour.The transparent rear progress paraffin embedding of active pulse classic alcohol serial dehydration and dimethylbenzene after fixation.Histotomy thickness is 5 μ M, slice dewaxing rehydration simultaneously use haematoxylin and eosin stains (HE dyeing), and it is terraced to return blue simultaneously alcohol for ammonium hydroxide after the differentiation of 1% hydrochloride alcohol Degree dehydration mounting is sliced in 400 times of (Figure 14 A, B) light microscopes and 1000 times (Figure 14 C, D) oily Microscopic observation.

Diabetes and hyperlipidemia complications are the relatively conventional complication of diabetes, are the significant risks of Diabetic Macrovascular Complications The factor^[39]。

Coloration result shows have foam cells to deposit (arrow mark to solvent PBS control group (Figure 14 A, 14C) vascular wall Know), middle elastic film is disorganized, vessel wall thickening, and tube wall convex-concave is uneven；Give plasminogen group (Figure 14 B, 14D) middle level bullet Property membrane structure rule, undulate, vascular wall thickness is uniform.Show that diabetic mice aorta can be mitigated by injecting plasminogen The lipidosis of tube wall has certain protective effect to damage caused by ductus arteriosus wall lipidosis.

15 plasminogen of embodiment reduces diabetic mice ventricle lipidosis

26 week old male db/db mouse 9 are grouped at random, to plasminogen group 4, to solvent PBS control group 5.It gives 2mg/0.2ml/ pcs/day of plasminogen group tail vein injection people source plasminogen gives solvent PBS control group tail vein injection consubstantiality Long-pending PBS is administered 35 days.In the 36th day put to death mouse, core it is dirty in 4% paraformaldehyde fix 24-48 hours, respectively at 15%th, in 30% sucrose 4 DEG C sink to the bottom overnight, OCT embeddings, 8 μm of frozen section thickness, oil red O stain 15min, 75% alcohol point Change 5 seconds, bush uniformly dyeing core 30s, glycerin gelatine mounting.Slice is in 200 times of optical microphotograph Microscopic observations.

The results show that it is considerably less than to plasminogen group mouse (Figure 15 B) ventricle lipidosis (arrow logo) to solvent PBS control group (Figure 15 A).Illustrate that plasminogen can reduce fat and be deposited in diabetic mice ventricle, promote repairing for ventricle damage It is multiple.

16 plasminogen of embodiment improves the High-density Lipoprotein-cholesterol in diabetic mice serum

26 week old male db/db mouse 20, experiment are denoted as the 0th day and weigh, be randomly divided into according to weight on the day of starting Two groups, to plasminogen group 11, each 9 to solvent PBS control group., the 1st day starts to plasminogen or PBS, successive administration 35 days.To 2mg/0.2ml/ pcs/day of plasminogen group tail vein injection people source plasminogen, control group tail vein injection same volume PBS.36th day mouse plucks eyeball and adopts whole blood, and 4 DEG C of 3500r/min are centrifuged 10 minutes, take supernatant and using high density lipoprotein level White detection kit (Bioengineering Research Institute, article No. A112-1 are built up in Nanjing) detection serum middle-high density lipoprotein cholesterol (HDL-C) concentration.

Testing result is shown, is higher than to the content of HDL-C in plasminogen group mice serum and is given solvent PBS control group, unites Count significant difference (Figure 16).Illustrate that injecting plasminogen can promote the content of serum High Density Lipoprotein Cholesterol to increase, and improve Diabetic dyslipidemia.

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

24-25 week old male db/db mouse 10, experiment are denoted as the 0th day and weigh, divided at random according to weight on the day of starting It is two groups, to plasminogen group and each 5 to solvent PBS control group, and takes 3 db/m as Normal group.It opens within 1st day Begin to plasminogen or PBS, successive administration 31 days.Give plasminogen group tail vein injection people source plasminogen 2mg/0.2ml/ Pcs/day, the PBS of PBS control group tail vein injection same volume, Normal group mouse does not make any processing.Mouse is plucked within 32nd day Eyeball adopts whole blood, and 4 DEG C of 3500r/min are centrifuged 10 minutes, takes supernatant and uses low density lipoprotein cholesterol detection kit Low density lipoprotein cholesterol (LDL-C) concentration in (Bioengineering Research Institute, article No. A113-1 are built up in Nanjing) detection serum.

The results show that diabetic mice continuously injected people source fibrinolysin source after 31 days, plasminogen group mouse blood is given LDL-C contents in clear, which are less than, gives solvent PBS control group, and statistical discrepancy is close to significantly (P=0.1) (Figure 17).Illustrate fibrinolytic Proenzyme can reduce the content of LDL-C in serum.

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

6 week old male ApoE mouse 13 feed high cholesterol diet (Nantong Te Luofei, TP2031) high in fat 16 weeks to lure Lead Atherosclerosis Model^[40,41].Mouse after Cheng Mo continues to feed high cholesterol diet high in fat.Three days every before administration 50 μ l of blood are taken to detect T-CHOL (T-CHO) content, and two groups are randomly divided into according to T-CHO contents, give solvent PBS control group 7, to plasminogen group 6.Start administration to be set to the 1st day, give plasminogen group mouse tail vein injection people source plasminogen It 1mg/0.1ml/ pcs/day, to the PBS of solvent PBS control group tail vein injection same volume, is administered 30 days.Prohibit in the 30th day mouse Food 16 hours extracts eyeball on the 31st day and takes blood, and centrifugation obtains supernatant, and using T-CHOL detection kit, (biological work is built up in Nanjing Journey research institute, article No. A111-1) carry out T-CHOL detection.

Testing result is shown, is significantly lower than to plasminogen group mouse total cholesterol concentration and is given solvent PBS control groups, and Statistical discrepancy is significantly (P=0.014) (Figure 18).Illustrate that plasminogen can reduce ApoE Atherosclerosis Model mice serums The content of middle T-CHOL improves the blood fat disorder of atherosclerosis.

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

6 week old male ApoE mouse 13 feed high cholesterol diet (Nantong Te Luofei, TP2031) high in fat 16 weeks to lure Lead Atherosclerosis Model^[40,41].Mouse after Cheng Mo continues to feed high cholesterol diet high in fat.Three days every before administration 50 μ l of blood are taken to detect T-CHOL (T-CHO) content, and two groups are randomly divided into according to T-CHO contents, give solvent PBS control group 7, to plasminogen group 6.Start administration to be denoted as the 1st day, give plasminogen group mouse tail vein injection people source plasminogen It 1mg/0.1ml/ pcs/day, to the PBS of solvent PBS control group tail vein injection same volume, is administered 30 days.Prohibit in the 30th day mouse Food 16 hours plucks eyeball on the 31st day and takes blood, and centrifugation obtains supernatant, and using triglycerides detection kit, (bioengineering is built up in Nanjing Research institute, article No. A110-1) carry out triglycerides detection.

Testing result is shown, is significantly lower than to plasminogen group mouse triglyceride concentration and is given solvent PBS control groups, and Statistical discrepancy is significantly (P=0.013) (Figure 19).Illustrate that plasminogen can reduce ApoE Atherosclerosis Model mice serums The content of middle triglycerides improves atherosclerosis blood fat disorder.

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

6 week old male ApoE mouse 13 feed high cholesterol diet (Nantong Te Luofei, TP2031) high in fat 16 weeks to lure Lead Atherosclerosis Model^[40,41].Mouse after Cheng Mo continues to feed high cholesterol diet high in fat.Three days every before administration 50 μ l of blood are taken to detect T-CHOL (T-CHO) content, and two groups are randomly divided into according to T-CHO contents, give solvent PBS control group 7, to plasminogen group 6.Start administration to be denoted as the 1st day, give plasminogen group mouse tail vein injection people source plasminogen It 1mg/0.1ml/ pcs/day, to the PBS of solvent PBS control group tail vein injection same volume, is administered 30 days.Mouse fasting in 30th day It 16 hours, plucks within the 31st day eyeball and takes blood, centrifugation obtains supernatant and uses low density lipoprotein cholesterol (LDL-C) detection kit (Bioengineering Research Institute, article No. A113-1 are built up in Nanjing) carries out LDL-C detections.

Solvent PBS control group, and statistical discrepancy are given the results show that being significantly lower than to plasminogen group mouse LDL-C concentration Significantly (P=0.017) (Figure 20).Illustrate that plasminogen can reduce low density lipoprotein in ApoE Atherosclerosis Model mice serums It is disorderly to improve Atherosclerosis Model lipid of mice for protein cholesterol content.

21 plasminogen of embodiment improves ApoE atherosclerosis mouse liver lipidosis

6 week old male ApoE mouse 13 feed high cholesterol diet (Nantong Te Luofei, TP2031) high in fat 16 weeks to lure Lead Atherosclerosis Model^[40,41].Mouse after Cheng Mo continues to feed high cholesterol diet high in fat.Three days every before administration 50 μ l of blood are taken to detect T-CHOL (T-CHO) content, and two groups are randomly divided into according to T-CHO contents, give solvent PBS control group 7, to plasminogen group 6.Start administration to be denoted as the 1st day, give plasminogen group mouse tail vein injection people source plasminogen It 1mg/0.1ml/ pcs/day, to the PBS of solvent PBS control group tail vein injection same volume, is administered 30 days.It was put to death in the 31st day small Mouse, materials liver organization are fixed 24-48 hours in 4% paraformaldehyde, are sunk to the bottom overnight respectively at 4 DEG C in 15%, 30% sucrose, OCT is embedded, 8 μm of frozen section thickness, oil red O stain 15min, and 75% alcohol breaks up 5 seconds, bush uniformly dyeing core 30s, glycerin gelatine Mounting.Slice is in 400 times of optical microphotograph Microscopic observations.

Coloration result is shown, is considerably less than to plasminogen group (Figure 21 B) mouse liver fat deposition and is given solvent PBS control Group (Figure 21 A), and quantitative analysis statistical discrepancy is significantly (P=0.02) (Figure 21 C).Illustrate that plasminogen can reduce fat dynamic Deposition in pulse atherosclerosis model mice liver.

22 plasminogen of embodiment improves deposition of the lipid in ApoE atherosclerosis mouse aorta sinus

6 week old male ApoE mouse 13 feed high cholesterol diet (Nantong Te Luofei, TP2031) high in fat 16 weeks to lure Lead Atherosclerosis Model^[40,41].Mouse after Cheng Mo continues to feed high cholesterol diet high in fat.Three days every before administration 50 μ l of blood are taken to detect T-CHOL (T-CHO) content, and two groups are randomly divided into according to T-CHO contents, give solvent PBS control group 7, to plasminogen group 6.Start administration to be denoted as the 1st day, give plasminogen group mouse tail vein injection people source plasminogen It 1mg/0.1ml/ pcs/day, to the PBS of solvent PBS control group tail vein injection same volume, is administered 30 days.It was put to death in the 31st day small Mouse, dirty 4% paraformaldehyde that is organized in of coring are fixed 24-48 hours, are sunk to the bottom overnight respectively at 4 DEG C in 15%, 30% sucrose, OCT Embedding, 8 μm of frozen section thickness, oil red O stain 15min, 75% alcohol break up 5 seconds, bush uniformly dyeing core 30s, glycerin gelatine envelope Piece.Slice is in 40 times of optical microphotograph Microscopic observations.

Coloration result is shown, is considerably less than to plasminogen group (Figure 22 B) mouse aorta sinus fat deposition and is given solvent PBS Control group (Figure 22 A).Illustrate that plasminogen can reduce deposition of the lipid in Atherosclerosis Model mouse aorta sinus.

23 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, is cored dirty in 4% paraformaldehyde fixation 24-48 hours.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 (23A, 23B), 200 times (23C, 23D) optical microphotograph Microscopic observation.

The results show that give plasminogen group (Figure 23 B, 23D) aortic sinus blood vessel collagen deposition (arrow mark Know) area be significantly less than and give solvent PBS control group (Figure 23 A, 23C), illustrate that plasminogen can cut down hyperlipemia model Mouse aorta sinus Fibrosis levels.

24 plasminogen of embodiment improves ApoE atherosclerosis mouse heart compensatory hypertrophies

6 week old male ApoE mouse 13 feed high cholesterol diet (Nantong Te Luofei, TP2031) high in fat 16 weeks to lure Lead Atherosclerosis Model^[40,41].Mouse after Cheng Mo before administration three days every 50 μ l of blood is taken to detect T-CHOL (T- CHO) content, and two groups are randomly divided into according to T-CHO contents, to solvent PBS control group 7, to plasminogen group 6.Start Administration is set to the 1st day, to 1mg/0.1mL/ pcs/day of plasminogen group mouse tail vein injection people source plasminogen, to solvent The PBS of PBS control group tail vein injection same volume.Administration 30 days, continues to feed high cholesterol diet high in fat during administration.In to Mouse is put to death in the 31st day of medicine after weighing, dirty weigh, and calculate heart coefficient of coring.Heart coefficient (%)=cardiac weight/body Weight × 100.

The results show that it is significantly lower than to plasminogen group mouse heart coefficient to solvent PBS control group (Figure 24).Explanation It is loose that plasminogen can mitigate Cardiac compensation of the ApoE Atherosclerosis Model mouse caused by heart injury.

25 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.It gives 30 days medicine periods put to death mouse in the 31st day, kidney are taken to fix 24-48 hours in 4% paraformaldehyde.Tissue after fixation is through wine Paraffin embedding is carried out after smart serial dehydration and dimethylbenzene are transparent.Renal tissue slice thickness is 3 μm, is washed after slice dewaxing rehydration 1 time, 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 results show that it is considerably less than to plasminogen group (Figure 25 C) renal collagen proteinosis (arrow logo) to solvent PBS control group (Figure 25 B), and statistical discrepancy is significantly (Figure 25 D)；Normal level is substantially returned to plasminogen group fibrosis (Figure 25 A).Illustrate that plasminogen can effectively reduce by 3% cholesterol hyperlipemia model mouse kidney fibrosis.

26 plasminogen of embodiment reduces by 3% cholesterol hyperlipemia model mouse kidney fat deposition

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, is given Medicine 30 days.It puts to death mouse within 31st day, kidney is taken to fix 24-48 hours in 4% paraformaldehyde, respectively at 4 in 15%, 30% sucrose It DEG C sinks to the bottom overnight, OCT embeddings, 8 μm of frozen section thickness, oil red O stain 15min, 75% alcohol breaks up 5 seconds, bush uniformly dyeing core 30 seconds, glycerin gelatine mounting.Slice is in 400 times of optical microphotograph Microscopic observations.

The results show that it is considerably less than to plasminogen group (Figure 26 C) mouse kidney fat deposition (arrow logo) to solvent PBS control group (Figure 26 B), and quantitative analysis statistical discrepancy is significantly (Figure 26 D)；In addition, give plasminogen group lipidosis level It is similar to blank control group mouse (Figure 26 A).Illustrate that plasminogen can cut down fat in hyperlipemia model mouse kidney Deposition, so as to the dirty damage of kidney caused by reducing fat deposition.

Bibliography

[1] Zhao Kejian edits modern pharmacy noun handbook Beijing：China Medical Science Press .2004. pages 533

[2]Shafrir E.,Raz.I.Diabetes:Mellitus or Lipidus[J].Diabetologia, 2003,46:433-440.

[3]Mooradian AD.Cardiovascular disease in type 2diabetes mellitus: Current management guidelines[J].Arch Intern Med.2003,163:33-40.

[4]Appel G.Lipid adnormalities in renal disease[J].Kidney Int,1991, 39:169.

[5]J Zah,Kov,H Varerkov,et al.,Dislipoproteinemia and Chronic Kidney Failure[J].Vnitr Lek.2000,46(9):539-46.

[6]Effat Razeghi,Mohammadreza Shafipour et al.Lipid Disturbances Before and After Renal Transplant.Effat Razeghi et al/Experimental and Clinical Transplantation(2011)4:230-235

[7]Grone HJ.Glomerular lipids in non-hereditary forms of glomerulopathy/glomerulo nephritis[J].Nephrol.Dial Transplant.1999,14:1595- 1598.

[8]Larking RC.Dunlop ME,The link between hyperglycaemia and diabetic nephropathy[J] Athrosclerosis,2001,156(2):25-33.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[33]Dimitrios Davalos,Katerina Akassoglou.Fibrinogen as a key regulator of inflammation in disease.Seminars in Immunopathology,2012.34(1): 43-62.

[34]Valvi D,Mannino DM,Mullerova H,et al.Fibrinogen,chronic obstructive pulmonary disease(COPD)and outcomes in two United States cohorts.Int J Chron Obstruct Pulmon Dis 2012；7:173–82.

[35]Zhang M,Takahashi K,Alicot EM,Vorup-Jensen T,Kessler B,et al. (2006)Activation of the lectin pathway by natural IgM in a model of ischemia reperfusion injury.J Immunol 177: 4727–4734.

[36]Kim SJ,Gershov D,Ma X,Brot N,Elkon KB(2002)I-PLA2 Activation during Apoptosis Promotes the Exposure of Membrane Lysophosphatidylcholine Leading to Binding by Natural Immunoglobulin M Antibodies and Complement Activation.The Journal of Experimental Medicine 196:655–665.

[37]R.Langhorn and J.L.Willesen.Cardiac Troponins in Dogs and Cats.J Vet Intern Med 2016；30:36–50.

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

[39]Ametov AS,Kulidzhanian NK.Diabetes mellitus is an independent risk factor for cardiovascular disease.Ter Arkh.2012；84(8):91-4.

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

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

Sequence table

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

<120>A kind of method for preventing and treating hyperlipidemia

<130> PDK03572

<160> 14

<170> PatentIn version 3.5

<210> 1

<211> 2376

<212> DNA

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

<400> 1

gagcctctgg atgactatgt gaatacccag ggggcttcac tgttcagtgt cactaagaag 60

cagctgggag caggaagtat agaagaatgt gcagcaaaat gtgaggagga cgaagaattc 120

acctgcaggg cattccaata tcacagtaaa gagcaacaat gtgtgataat ggctgaaaac 180

aggaagtcct ccataatcat taggatgaga gatgtagttt tatttgaaaa gaaagtgtat 240

ctctcagagt gcaagactgg gaatggaaag aactacagag ggacgatgtc caaaacaaaa 300

aatggcatca cctgtcaaaa atggagttcc acttctcccc acagacctag attctcacct 360

gctacacacc cctcagaggg actggaggag aactactgca ggaatccaga caacgatccg 420

caggggccct ggtgctatac tactgatcca gaaaagagat atgactactg cgacattctt 480

gagtgtgaag aggaatgtat gcattgcagt ggagaaaact atgacggcaa aatttccaag 540

accatgtctg gactggaatg ccaggcctgg gactctcaga gcccacacgc tcatggatac 600

attccttcca aatttccaaa caagaacctg aagaagaatt actgtcgtaa ccccgatagg 660

gagctgcggc cttggtgttt caccaccgac cccaacaagc gctgggaact ttgtgacatc 720

ccccgctgca caacacctcc accatcttct ggtcccacct accagtgtct gaagggaaca 780

ggtgaaaact atcgcgggaa tgtggctgtt accgtgtccg ggcacacctg tcagcactgg 840

agtgcacaga cccctcacac acataacagg acaccagaaa acttcccctg caaaaatttg 900

gatgaaaact actgccgcaa tcctgacgga aaaagggccc catggtgcca tacaaccaac 960

agccaagtgc ggtgggagta ctgtaagata ccgtcctgtg actcctcccc agtatccacg 1020

gaacaattgg ctcccacagc accacctgag ctaacccctg tggtccagga ctgctaccat 1080

ggtgatggac agagctaccg aggcacatcc tccaccacca ccacaggaaa gaagtgtcag 1140

tcttggtcat ctatgacacc acaccggcac cagaagaccc cagaaaacta cccaaatgct 1200

ggcctgacaa tgaactactg caggaatcca gatgccgata aaggcccctg gtgttttacc 1260

acagacccca gcgtcaggtg ggagtactgc aacctgaaaa aatgctcagg aacagaagcg 1320

agtgttgtag cacctccgcc tgttgtcctg cttccagatg tagagactcc ttccgaagaa 1380

gactgtatgt ttgggaatgg gaaaggatac cgaggcaaga gggcgaccac tgttactggg 1440

acgccatgcc aggactgggc tgcccaggag ccccatagac acagcatttt cactccagag 1500

acaaatccac gggcgggtct ggaaaaaaat tactgccgta accctgatgg tgatgtaggt 1560

ggtccctggt gctacacgac aaatccaaga aaactttacg actactgtga tgtccctcag 1620

tgtgcggccc cttcatttga ttgtgggaag cctcaagtgg agccgaagaa atgtcctgga 1680

agggttgtag gggggtgtgt ggcccaccca cattcctggc cctggcaagt cagtcttaga 1740

acaaggtttg gaatgcactt ctgtggaggc accttgatat ccccagagtg ggtgttgact 1800

gctgcccact gcttggagaa gtccccaagg ccttcatcct acaaggtcat cctgggtgca 1860

caccaagaag tgaatctcga accgcatgtt caggaaatag aagtgtctag gctgttcttg 1920

gagcccacac gaaaagatat tgccttgcta aagctaagca gtcctgccgt catcactgac 1980

aaagtaatcc cagcttgtct gccatcccca aattatgtgg tcgctgaccg gaccgaatgt 2040

ttcatcactg gctggggaga aacccaaggt acttttggag ctggccttct caaggaagcc 2100

cagctccctg tgattgagaa taaagtgtgc aatcgctatg agtttctgaa tggaagagtc 2160

caatccaccg aactctgtgc tgggcatttg gccggaggca ctgacagttg ccagggtgac 2220

agtggaggtc ctctggtttg cttcgagaag gacaaataca ttttacaagg agtcacttct 2280

tggggtcttg gctgtgcacg ccccaataag cctggtgtct atgttcgtgt ttcaaggttt 2340

gttacttgga ttgagggagt gatgagaaat aattaa 2376

<210> 2

<211> 791

<212> PRT

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

<400> 2

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

Glu Gly Val Met Arg Asn Asn

785 790

<210> 3

<211> 2433

<212> DNA

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

<400> 3

atggaacata aggaagtggt tcttctactt cttttatttc tgaaatcagg tcaaggagag 60

cctctggatg actatgtgaa tacccagggg gcttcactgt tcagtgtcac taagaagcag 120

ctgggagcag gaagtataga agaatgtgca gcaaaatgtg aggaggacga agaattcacc 180

tgcagggcat tccaatatca cagtaaagag caacaatgtg tgataatggc tgaaaacagg 240

aagtcctcca taatcattag gatgagagat gtagttttat ttgaaaagaa agtgtatctc 300

tcagagtgca agactgggaa tggaaagaac tacagaggga cgatgtccaa aacaaaaaat 360

ggcatcacct gtcaaaaatg gagttccact tctccccaca gacctagatt ctcacctgct 420

acacacccct cagagggact ggaggagaac tactgcagga atccagacaa cgatccgcag 480

gggccctggt gctatactac tgatccagaa aagagatatg actactgcga cattcttgag 540

tgtgaagagg aatgtatgca ttgcagtgga gaaaactatg acggcaaaat ttccaagacc 600

atgtctggac tggaatgcca ggcctgggac tctcagagcc cacacgctca tggatacatt 660

ccttccaaat ttccaaacaa gaacctgaag aagaattact gtcgtaaccc cgatagggag 720

ctgcggcctt ggtgtttcac caccgacccc aacaagcgct gggaactttg tgacatcccc 780

cgctgcacaa cacctccacc atcttctggt cccacctacc agtgtctgaa gggaacaggt 840

gaaaactatc gcgggaatgt ggctgttacc gtgtccgggc acacctgtca gcactggagt 900

gcacagaccc ctcacacaca taacaggaca ccagaaaact tcccctgcaa aaatttggat 960

gaaaactact gccgcaatcc tgacggaaaa agggccccat ggtgccatac aaccaacagc 1020

caagtgcggt gggagtactg taagataccg tcctgtgact cctccccagt atccacggaa 1080

caattggctc ccacagcacc acctgagcta acccctgtgg tccaggactg ctaccatggt 1140

gatggacaga gctaccgagg cacatcctcc accaccacca caggaaagaa gtgtcagtct 1200

tggtcatcta tgacaccaca ccggcaccag aagaccccag aaaactaccc aaatgctggc 1260

ctgacaatga actactgcag gaatccagat gccgataaag gcccctggtg ttttaccaca 1320

gaccccagcg tcaggtggga gtactgcaac ctgaaaaaat gctcaggaac agaagcgagt 1380

gttgtagcac ctccgcctgt tgtcctgctt ccagatgtag agactccttc cgaagaagac 1440

tgtatgtttg ggaatgggaa aggataccga ggcaagaggg cgaccactgt tactgggacg 1500

ccatgccagg actgggctgc ccaggagccc catagacaca gcattttcac tccagagaca 1560

aatccacggg cgggtctgga aaaaaattac tgccgtaacc ctgatggtga tgtaggtggt 1620

ccctggtgct acacgacaaa tccaagaaaa ctttacgact actgtgatgt ccctcagtgt 1680

gcggcccctt catttgattg tgggaagcct caagtggagc cgaagaaatg tcctggaagg 1740

gttgtagggg ggtgtgtggc ccacccacat tcctggccct ggcaagtcag tcttagaaca 1800

aggtttggaa tgcacttctg tggaggcacc ttgatatccc cagagtgggt gttgactgct 1860

gcccactgct tggagaagtc cccaaggcct tcatcctaca aggtcatcct gggtgcacac 1920

caagaagtga atctcgaacc gcatgttcag gaaatagaag tgtctaggct gttcttggag 1980

cccacacgaa aagatattgc cttgctaaag ctaagcagtc ctgccgtcat cactgacaaa 2040

gtaatcccag cttgtctgcc atccccaaat tatgtggtcg ctgaccggac cgaatgtttc 2100

atcactggct ggggagaaac ccaaggtact tttggagctg gccttctcaa ggaagcccag 2160

ctccctgtga ttgagaataa agtgtgcaat cgctatgagt ttctgaatgg aagagtccaa 2220

tccaccgaac tctgtgctgg gcatttggcc ggaggcactg acagttgcca gggtgacagt 2280

ggaggtcctc tggtttgctt cgagaaggac aaatacattt tacaaggagt cacttcttgg 2340

ggtcttggct gtgcacgccc caataagcct ggtgtctatg ttcgtgtttc aaggtttgtt 2400

acttggattg agggagtgat gagaaataat taa 2433

<210> 4

<211> 810

<212> PRT

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

<400> 4

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

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

785 790 795 800

Thr Trp Ile Glu Gly Val Met Arg Asn Asn

805 810

<210> 5

<211> 2145

<212> DNA

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

<400> 5

aaagtgtatc tctcagagtg caagactggg aatggaaaga actacagagg gacgatgtcc 60

aaaacaaaaa atggcatcac ctgtcaaaaa tggagttcca cttctcccca cagacctaga 120

ttctcacctg ctacacaccc ctcagaggga ctggaggaga actactgcag gaatccagac 180

aacgatccgc aggggccctg gtgctatact actgatccag aaaagagata tgactactgc 240

gacattcttg agtgtgaaga ggaatgtatg cattgcagtg gagaaaacta tgacggcaaa 300

atttccaaga ccatgtctgg actggaatgc caggcctggg actctcagag cccacacgct 360

catggataca ttccttccaa atttccaaac aagaacctga agaagaatta ctgtcgtaac 420

cccgataggg agctgcggcc ttggtgtttc accaccgacc ccaacaagcg ctgggaactt 480

tgtgacatcc cccgctgcac aacacctcca ccatcttctg gtcccaccta ccagtgtctg 540

aagggaacag gtgaaaacta tcgcgggaat gtggctgtta ccgtgtccgg gcacacctgt 600

cagcactgga gtgcacagac ccctcacaca cataacagga caccagaaaa cttcccctgc 660

aaaaatttgg atgaaaacta ctgccgcaat cctgacggaa aaagggcccc atggtgccat 720

acaaccaaca gccaagtgcg gtgggagtac tgtaagatac cgtcctgtga ctcctcccca 780

gtatccacgg aacaattggc tcccacagca ccacctgagc taacccctgt ggtccaggac 840

tgctaccatg gtgatggaca gagctaccga ggcacatcct ccaccaccac cacaggaaag 900

aagtgtcagt cttggtcatc tatgacacca caccggcacc agaagacccc agaaaactac 960

ccaaatgctg gcctgacaat gaactactgc aggaatccag atgccgataa aggcccctgg 1020

tgttttacca cagaccccag cgtcaggtgg gagtactgca acctgaaaaa atgctcagga 1080

acagaagcga gtgttgtagc acctccgcct gttgtcctgc ttccagatgt agagactcct 1140

tccgaagaag actgtatgtt tgggaatggg aaaggatacc gaggcaagag ggcgaccact 1200

gttactggga cgccatgcca ggactgggct gcccaggagc cccatagaca cagcattttc 1260

actccagaga caaatccacg ggcgggtctg gaaaaaaatt actgccgtaa ccctgatggt 1320

gatgtaggtg gtccctggtg ctacacgaca aatccaagaa aactttacga ctactgtgat 1380

gtccctcagt gtgcggcccc ttcatttgat tgtgggaagc ctcaagtgga gccgaagaaa 1440

tgtcctggaa gggttgtagg ggggtgtgtg gcccacccac attcctggcc ctggcaagtc 1500

agtcttagaa caaggtttgg aatgcacttc tgtggaggca ccttgatatc cccagagtgg 1560

gtgttgactg ctgcccactg cttggagaag tccccaaggc cttcatccta caaggtcatc 1620

ctgggtgcac accaagaagt gaatctcgaa ccgcatgttc aggaaataga agtgtctagg 1680

ctgttcttgg agcccacacg aaaagatatt gccttgctaa agctaagcag tcctgccgtc 1740

atcactgaca aagtaatccc agcttgtctg ccatccccaa attatgtggt cgctgaccgg 1800

accgaatgtt tcatcactgg ctggggagaa acccaaggta cttttggagc tggccttctc 1860

aaggaagccc agctccctgt gattgagaat aaagtgtgca atcgctatga gtttctgaat 1920

ggaagagtcc aatccaccga actctgtgct gggcatttgg ccggaggcac tgacagttgc 1980

cagggtgaca gtggaggtcc tctggtttgc ttcgagaagg acaaatacat tttacaagga 2040

gtcacttctt ggggtcttgg ctgtgcacgc cccaataagc ctggtgtcta tgttcgtgtt 2100

tcaaggtttg ttacttggat tgagggagtg atgagaaata attaa 2145

<210> 6

<211> 714

<212> PRT

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

<400> 6

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

Thr Trp Ile Glu Gly Val Met Arg Asn Asn

705 710

<210> 7

<211> 1245

<212> DNA

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

<400> 7

gagcctctgg atgactatgt gaatacccag ggggcttcac tgttcagtgt cactaagaag 60

cagctgggag caggaagtat agaagaatgt gcagcaaaat gtgaggagga cgaagaattc 120

acctgcaggg cattccaata tcacagtaaa gagcaacaat gtgtgataat ggctgaaaac 180

aggaagtcct ccataatcat taggatgaga gatgtagttt tatttgaaaa gaaagtgtat 240

ctctcagagt gcaagactgg gaatggaaag aactacagag ggacgatgtc caaaacaaaa 300

aatggcatca cctgtcaaaa atggagttcc acttctcccc acagacctag attctcacct 360

gctacacacc cctcagaggg actggaggag aactactgca ggaatccaga caacgatccg 420

caggggccct ggtgctatac tactgatcca gaaaagagat atgactactg cgacattctt 480

gagtgtgaag aggcggcccc ttcatttgat tgtgggaagc ctcaagtgga gccgaagaaa 540

tgtcctggaa gggttgtagg ggggtgtgtg gcccacccac attcctggcc ctggcaagtc 600

agtcttagaa caaggtttgg aatgcacttc tgtggaggca ccttgatatc cccagagtgg 660

gtgttgactg ctgcccactg cttggagaag tccccaaggc cttcatccta caaggtcatc 720

ctgggtgcac accaagaagt gaatctcgaa ccgcatgttc aggaaataga agtgtctagg 780

ctgttcttgg agcccacacg aaaagatatt gccttgctaa agctaagcag tcctgccgtc 840

atcactgaca aagtaatccc agcttgtctg ccatccccaa attatgtggt cgctgaccgg 900

accgaatgtt tcatcactgg ctggggagaa acccaaggta cttttggagc tggccttctc 960

aaggaagccc agctccctgt gattgagaat aaagtgtgca atcgctatga gtttctgaat 1020

ggaagagtcc aatccaccga actctgtgct gggcatttgg ccggaggcac tgacagttgc 1080

cagggtgaca gtggaggtcc tctggtttgc ttcgagaagg acaaatacat tttacaagga 1140

gtcacttctt ggggtcttgg ctgtgcacgc cccaataagc ctggtgtcta tgttcgtgtt 1200

tcaaggtttg ttacttggat tgagggagtg atgagaaata attaa 1245

<210> 8

<211> 414

<212> PRT

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

<400> 8

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410

<210> 9

<211> 1104

<212> DNA

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

<400> 9

gtcaggtggg agtactgcaa cctgaaaaaa tgctcaggaa cagaagcgag tgttgtagca 60

cctccgcctg ttgtcctgct tccagatgta gagactcctt ccgaagaaga ctgtatgttt 120

gggaatggga aaggataccg aggcaagagg gcgaccactg ttactgggac gccatgccag 180

gactgggctg cccaggagcc ccatagacac agcattttca ctccagagac aaatccacgg 240

gcgggtctgg aaaaaaatta ctgccgtaac cctgatggtg atgtaggtgg tccctggtgc 300

tacacgacaa atccaagaaa actttacgac tactgtgatg tccctcagtg tgcggcccct 360

tcatttgatt gtgggaagcc tcaagtggag ccgaagaaat gtcctggaag ggttgtaggg 420

gggtgtgtgg cccacccaca ttcctggccc tggcaagtca gtcttagaac aaggtttgga 480

atgcacttct gtggaggcac cttgatatcc ccagagtggg tgttgactgc tgcccactgc 540

ttggagaagt ccccaaggcc ttcatcctac aaggtcatcc tgggtgcaca ccaagaagtg 600

aatctcgaac cgcatgttca ggaaatagaa gtgtctaggc tgttcttgga gcccacacga 660

aaagatattg ccttgctaaa gctaagcagt cctgccgtca tcactgacaa agtaatccca 720

gcttgtctgc catccccaaa ttatgtggtc gctgaccgga ccgaatgttt catcactggc 780

tggggagaaa cccaaggtac ttttggagct ggccttctca aggaagccca gctccctgtg 840

attgagaata aagtgtgcaa tcgctatgag tttctgaatg gaagagtcca atccaccgaa 900

ctctgtgctg ggcatttggc cggaggcact gacagttgcc agggtgacag tggaggtcct 960

ctggtttgct tcgagaagga caaatacatt ttacaaggag tcacttcttg gggtcttggc 1020

tgtgcacgcc ccaataagcc tggtgtctat gttcgtgttt caaggtttgt tacttggatt 1080

gagggagtga tgagaaataa ttaa 1104

<210> 10

<211> 367

<212> PRT

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

<400> 10

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

<210> 11

<211> 750

<212> DNA

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

<400> 11

gccccttcat ttgattgtgg gaagcctcaa gtggagccga agaaatgtcc tggaagggtt 60

gtaggggggt gtgtggccca cccacattcc tggccctggc aagtcagtct tagaacaagg 120

tttggaatgc acttctgtgg aggcaccttg atatccccag agtgggtgtt gactgctgcc 180

cactgcttgg agaagtcccc aaggccttca tcctacaagg tcatcctggg tgcacaccaa 240

gaagtgaatc tcgaaccgca tgttcaggaa atagaagtgt ctaggctgtt cttggagccc 300

acacgaaaag atattgcctt gctaaagcta agcagtcctg ccgtcatcac tgacaaagta 360

atcccagctt gtctgccatc cccaaattat gtggtcgctg accggaccga atgtttcatc 420

actggctggg gagaaaccca aggtactttt ggagctggcc ttctcaagga agcccagctc 480

cctgtgattg agaataaagt gtgcaatcgc tatgagtttc tgaatggaag agtccaatcc 540

accgaactct gtgctgggca tttggccgga ggcactgaca gttgccaggg tgacagtgga 600

ggtcctctgg tttgcttcga gaaggacaaa tacattttac aaggagtcac ttcttggggt 660

cttggctgtg cacgccccaa taagcctggt gtctatgttc gtgtttcaag gtttgttact 720

tggattgagg gagtgatgag aaataattaa 750

<210> 12

<211> 249

<212> PRT

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

<400> 12

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Trp Ile Glu Gly Val Met Arg Asn Asn

245

<210> 13

<211> 684

<212> DNA

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

<400> 13

gttgtagggg ggtgtgtggc ccacccacat tcctggccct ggcaagtcag tcttagaaca 60

aggtttggaa tgcacttctg tggaggcacc ttgatatccc cagagtgggt gttgactgct 120

gcccactgct tggagaagtc cccaaggcct tcatcctaca aggtcatcct gggtgcacac 180

caagaagtga atctcgaacc gcatgttcag gaaatagaag tgtctaggct gttcttggag 240

cccacacgaa aagatattgc cttgctaaag ctaagcagtc ctgccgtcat cactgacaaa 300

gtaatcccag cttgtctgcc atccccaaat tatgtggtcg ctgaccggac cgaatgtttc 360

atcactggct ggggagaaac ccaaggtact tttggagctg gccttctcaa ggaagcccag 420

ctccctgtga ttgagaataa agtgtgcaat cgctatgagt ttctgaatgg aagagtccaa 480

tccaccgaac tctgtgctgg gcatttggcc ggaggcactg acagttgcca gggtgacagt 540

ggaggtcctc tggtttgctt cgagaaggac aaatacattt tacaaggagt cacttcttgg 600

ggtcttggct gtgcacgccc caataagcct ggtgtctatg ttcgtgtttc aaggtttgtt 660

acttggattg agggagtgat gaga 684

<210> 14

<211> 228

<212> PRT

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

<400> 14

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Gly Val Met Arg

225

Claims (10)

1. a kind of method for preventing and/or treating subject's hyperlipidemia and its associated disease is susceptible to suffer from high in fat including administration Mass formed by blood stasis, with hyperlipidemia or suffer from Other diseases and with hyperlipidemia subject prevention and/or therapeutically effective amount fibre Lyase is former.

2. the method for claim 1 wherein the hyperlipidemia is endocrine disturbance disease, glycometabolism disease, liver diseases, kidney Dirty disease, angiocardiopathy, intestines problem, thyroid disease, gall-bladder or disease of biliary tract obesity, are drunk, drug therapy initiation It is or adjoint.

3. the method for claim 2, wherein the hyperlipidemia is hypertension, diabetes, chronic hepatitis, hepatic sclerosis, injury of kidney, Chronic glomerulonephritis, chronic pyelonephritis, nephrotic syndrome, renal insufficiency, kidney transplant, uremia, thyroid function are low Under, obstructive cholecystitis, obstructive cholangitis, estrin treatment cause or it is adjoint.

4. the method for any one of claim 1-3, wherein the hyperlipidemia is I types, IIa types, IIb types, type III or IV types are high Pionemia.

5. the method for any one of claim 1-4, wherein the hyperlipidemia is chosen from the followings one or more：Serum glycerine Three esters (TG) raising, serum low-density fat egg (LDL) raising, very low density lipoprotein (VLDL) raising.

6. a kind of method for being used to prevent subject artery's atherosis by improving hyperlipidemia, including being applied to subject A effective amount of plasminogen.

7. a kind of be used for by improving hyperlipidemia prevention and/or cutting down what subject's fat was deposited in bodily tissue organs abnormality Method, including applying a effective amount of plasminogen to subject.

8. one kind is used to lead in bodily tissue organs abnormality deposition by improving hyperlipidemia prevention and/or treatment subject's fat The method of the disease of cause, including applying a effective amount of plasminogen to subject.

9. the method for claim 7 or 8, wherein the fat refers to fat at subcutaneous group in bodily tissue organs abnormality deposition Knit, around vascular wall, internal organs, organ, the abnormal deposition of mesenterium, peritonaeum.

10. the method for claim 9, wherein fat disease caused by bodily tissue organs abnormality deposition includes obesity Disease, fatty liver, the lipid mechanism harm of heart, the lipid mechanism harm of kidney, atherosclerosis.