CN109925507A - A kind of method and drug prevented or treat osteoarthritis - Google Patents

Abstract

The present invention relates to the methods and drug of preventing or treat osteoarthritis, specifically disclose a kind of by using the method for a effective amount of plasminogen treatment osteoarthritis and for the drug comprising plasminogen of this method.The invention further relates to plasminogens in preparation treatment joint injury, Regeneration of Articular Cartilage, prevention joint injury is promoted to develop the purposes in the drug for being osteoarthritis.

Description

A kind of method and drug prevented or treat osteoarthritis

Technical field

The present invention relates to the methods and drug of preventing or treat osteoarthritis by using plasminogen.

Background technique

Osteoarthritis, also known as Osteoarthritis, degenerative arthritis, degenerative joint disease, osteoarthropathy are most common Arthritis form, influence about 2.37 hundred million (3.3%) population and even disabled one of the leading reason of arthralgia^[1], In 60 years old or more people, there are about the women of 10% male and 18% to be affected^[2].Osteoarthritis, which is that one kind is multifactor, draws Rise, the different substantiality disease characterized by articular cartilage progressive decomposes, these factors include wound, abnormal mechanical load, Nutrition supply deficiency and hereditary predisposition etc., there are also metabolic factors and subpatellar fat pad.The abnormal Biological Strength of past research concern It learns on subchondral bone, articular cartilage integrality and the physiopathologic influence of cartilage cell；Recent evidence show that osteoarthritis Clinical symptoms not only influence articular cartilage, but also influence the integrality of a variety of joint tissues, including synovial membrane, bone, ligament, support Muscle and meniscus etc.^[3]。

The basic reason of osteoarthritis is not that lesion has occurred in bone, modern medical research has found that, the basic original of illness Because being forfeiture of the cartilage etc. " joint protection system " to joint protective capability.It is in particular in: the hair of all kinds of bone and joint diseases Life often starts from the lesions of synovium contributed, cartilage is damaged or denaturation；Take cartilage damage caused by certain anti-inflammatory classes, hormone medicine It is also one of the main reason of many bone and joint diseases.Due to the missing of synovium of joint, the damage of cartilage and knuckle synovia, lead Joint bone is caused to lack necessary protection, so that the bone of joint is due to a lack of necessary " cartilage protection " when physical activity Violent rigid friction directly occurs, and causes a variety of symptoms such as patient articular's pain, swelling, deformation, spur hyperplasia^[4].Therefore, Damaged articular cartilage, synovial membrane and the power of regeneration for promoting cartilage, synovial membrane are repaired, knuckle synovia is expedited the emergence of, to restore articular organs " cartilage protection layer " be treat bone and joint diseases key point.This research proves that plasminogen can improve the knot of Bones and joints Structure integrality enhances local joint osteoblast activity and/or reduces the activity of osteoclast, promotes under regenerating bone or cartilage and cartilage Bone bone remoulding and pain etc. is effectively relieved, is that the treatment of the joint injury disease including osteoarthritis opens new way Diameter.

Summary of the invention

Summary of the invention

The present invention relates to the methods and drug of preventing or treat osteoarthropathy by using plasminogen.Present invention demonstrates that fine Lyase original can improve the structural intergrity of Bones and joints, enhance local joint osteoblast activity and/or reduce osteoclast Activity promotes regenerating bone or cartilage and subchondral bone bone remoulding, and the symptoms such as pain can be effectively relieved, and is the treatment of bone and joint diseases Open new approach.

In particular it relates to:

1. a kind of method for treating osteoarthritis, including administration a effective amount of plasminogen of subject.

2. 1 method, the plasminogen increases the amount of articular cartilage and/or promotes articular cartilage damage reparation.

3. 1 or 2 method, the plasminogen improves joint synovitis disease situation.

4. the method for any one of 1-3, the plasminogen promotes the subchondral bone bone remoulding in joint.

5. the method for any one of 1-4, the plasminogen improves inflammatory conditions, pain and/or the improvement joint in joint Function.

6. the method for any one of 1-5, wherein the plasminogen mitigates arthroncus and pain.

7. a kind of method for promoting osteoarthritis subject Regeneration of Articular Cartilage, including administration a effective amount of fibrinolytic of subject Proenzyme.

8. a kind of method for promoting the reparation of subject's joint injury, including administration a effective amount of plasminogen of subject.

9. 8 method, the plasminogen promotes Regeneration of Articular Cartilage and/or subchondral bone bone remoulding.

10. 8 or 9 method, wherein the subject is osteoarthritis subject.

11. the method for any one of 8-10, wherein the plasminogen improves inflammatory conditions and/or the mitigation of joint tissue Arthralgia.

12. the method for any one of 1-11, wherein the plasminogen and sequence 2,6,8,10 or 12 have at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity, and still have plasminogen living Property.

13. the method for any one of 1-11, the plasminogen is that have comprising activities of endothelial tissue plasminogen segment and still The protein of activities of endothelial tissue plasminogen.

14. the method for any one of 1-11, the plasminogen is selected from Glu- plasminogen, Lys- plasminogen, small fibre Lyase original, Microplasminogen, delta- plasminogen or their variant for retaining activities of endothelial tissue plasminogen.

15. the method for any one of 1-11, the plasminogen is natural or synthetic human plasminogen or it is still protected Stay the variant or segment of activities of endothelial tissue plasminogen.

16. the method for any one of 1-11, the plasminogen is from the people of primate or rodent fibre Lyase original is directly to homologue or its variant or segment for still retaining activities of endothelial tissue plasminogen.

17. the method for any one of 1-11, the amino acid of the plasminogen is as shown in sequence 2,6,8,10 or 12.

18. the method for any one of 1-11, wherein the plasminogen is naive plasminogen.

19. the method for any one of 1-18, wherein the subject is people.

20. the method for any one of 1-19, wherein the subject lacks or missing plasminogen.

21. the method for any one of 1-20, wherein the plasminogen and one or more joint injury therapeutic agents or Method or one or more osteoarthritis treatment drugs or method use in conjunction.

22. a kind of plasminogen of the method for any one of item 1-21.

23. a kind of pharmaceutical composition, it includes pharmaceutically acceptable carrier and for side described in any one of item 1-21 The plasminogen of method.

24. a kind of preventative or therapeutic agent box, it includes: the fibre of (i) for any one of item 1-21 the method Lyase original and (ii) are used to deliver the component (means) of the plasminogen to the subject.

25. according to kit described in item 24, wherein the component is syringe or bottle.

26. 24 or 25 kit also includes label or operation instructions, the label or operation instructions instruction will The plasminogen administers the subject with any one of practical matter 1-21 the method.

27. a kind of product, it includes:

Container containing label；With

For the plasminogen of any one of item 1-21 the method or include the pharmaceutical composition of plasminogen comprising (i) Object, wherein the plasminogen or composition are administered the subject with any one of practical matter 1-21 institute by label instruction State method.

28. the kit of any one of 24-26 or the product of item 27, also comprising other one or more components or appearance Device contains one or more other joint injury therapeutic agents or articles or one or more other in the component or container Osteoarthritis treatment drug or articles.

29. the kit of any one of 24-26 or the product of item 27, further include the Other diseases concurrent with osteoarthritis Drug.

30. the medicament for being used to treat osteoarthritis comprising plasminogen.

31. the medicament for being used to treat joint injury comprising plasminogen.

32. the pharmaceutical composition for being used to treat osteoarthritis comprising plasminogen, kit, product.

33. the pharmaceutical composition for being used to treat joint injury comprising plasminogen, kit, product.

34. purposes of the plasminogen in the drug, product or kit of preparation treatment osteoarthritis.

35. 34 purposes, the plasminogen increases the amount of articular cartilage and/or promotes articular cartilage damage reparation.

36. 34 or 35 purposes, the plasminogen improves joint synovitis disease situation.

37. the purposes of any one of 34-36, the plasminogen promotes the subchondral bone bone remoulding in joint.

38. the purposes of any one of 34-37, inflammatory conditions, pain and/or the improvement that the plasminogen improves joint are closed Save function.

39. the purposes of any one of 34-38, wherein the plasminogen mitigates arthroncus and pain.

40. purposes of the plasminogen in the drug that preparation promotes osteoarthritis subject Regeneration of Articular Cartilage.

41. purposes of the plasminogen in the drug that preparation promotes the reparation of subject's joint injury.

42. 41 purposes, the plasminogen promotes Regeneration of Articular Cartilage and/or subchondral bone bone remoulding.

43. 41 or 42 purposes, wherein the subject is osteoarthritis subject.

44. the purposes of any one of 41-43, wherein the plasminogen improves the inflammatory conditions of joint tissue and/or subtracts Light arthralgia.

45. the method for any one of 34-44, wherein the plasminogen and sequence 2,6,8,10 or 12 have at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity, and still there is fibrinolysin Former activity.

46. the purposes of any one of 34-44, the plasminogen is that have comprising activities of endothelial tissue plasminogen segment and still There is the protein of activities of endothelial tissue plasminogen.

47. the purposes of any one of 34-44, the plasminogen is selected from Glu- plasminogen, Lys- plasminogen, small fibre Lyase original, Microplasminogen, delta- plasminogen or their variant for retaining activities of endothelial tissue plasminogen.

48. the purposes of any one of 34-44, the plasminogen be natural or synthetic human plasminogen or its still Retain the variant or segment of activities of endothelial tissue plasminogen.

49. the purposes of any one of 34-44, the plasminogen is from the people of primate or rodent fibre Lyase original is directly to homologue or its variant or segment for still retaining activities of endothelial tissue plasminogen.

50. the purposes of any one of 34-44, the amino acid of the plasminogen is as shown in sequence 2,6,8,10 or 12.

51. the purposes of any one of 34-44, wherein the plasminogen is naive plasminogen.

52. the purposes of any one of 34-51, wherein the subject is people.

53. the purposes of any one of 34-52, wherein the plasminogen and one or more joint injury therapeutic agents or Purposes or one or more osteoarthritis treatment drugs or method use in conjunction.

Detailed description of the invention

The definition of term and the description of technical solution

The connection tissue of " joint " between bone has lacuna in the connection tissue, can do different degrees of activity.Joint master The structure wanted have joint capsule, articular cavity, articular cartilage, synovia and ligament, the muscle and tendon of periarticular, plica synovialis, Synovial bursa, meniscus, subchondral bone etc..Joint capsule is the connective tissue membrane vesicle for wrapping two opposite epiphysis of joint.In it is divided into Outer two layers: outer layer is fibrous layer, is made of dense connective tissue, thick and tough and tensile, mainly plays the fastness for maintaining joint and steady Qualitative effect；Internal layer is stratum synoviale, thin and pliable, is made of the loose connective tissue of rich blood vessel, and the interior of fibrous layer is covered on Face forms the capsule around synovial membrane chamber.Articular cavity is the tissue lacuna surrounded by joint capsule and articular surface.Under normal circumstances, articular cavity A little sticky liquid (i.e. synovia) is inside had, has the function of lubrication and nutrition joint.Articular cartilage, which refers to, is covered on joint The very thin cartilage of that layer on surface.It is covered on articular surface, has elasticity well, to be reduced friction, buffering concussion With the effect of impact force.Synovia, that is, joint fluid, as secreted by synovial membrane.Normal synovia is limpid, sticky.It is sliding when there is inflammation in joint Liquid measure increased significantly, and joint cavity pressure increases, thus generate partial gall.

Some embodiments of the invention are related to the method for plasminogen treatment joint injury and promote joint injury tested The method of person's joint injury reparation, including administration a effective amount of plasminogen of subject.In some embodiments, the pass Section damage includes joint injury caused by inflammation, retrogression pathological changes, dysbolism or wound.In some embodiments, described Plasminogen treats joint injury by promotion Regeneration of Articular Cartilage and/or subchondral bone bone remoulding, or promotes joint injury It repairs.In some embodiments, plasminogen improves the inflammation of the joint tissue of joint injury subject and/or mitigates joint Pain.In some embodiments, the inflammation of the joint tissue includes the inflammation of synovium of joint.In some embodiments, The plasminogen mitigates joint injury subject arthroncus and pain.

In some embodiments, the present invention relates to the method for promoting Regeneration of Articular Cartilage, including administration subject are effective The plasminogen of amount.In some embodiments, the present invention relates to plasminogens by promoting regenerating bone or cartilage to promote joint injury Reparation and treatment joint injury disease.

In above-mentioned plasminogen treatment joint injury, some embodiments for promoting joint injury reparation, the joint Damage is joint injury caused by osteoarthritis.

In above-mentioned plasminogen treatment joint injury, some embodiments for promoting joint injury reparation, the fibrinolytic Proenzyme enables joint injury to repair, is developed by joint injury by promoting Regeneration of Articular Cartilage and/or subchondral bone bone remoulding The arthritic process of skeletonization is truncated.Therefore, the present invention also relates to the methods that plasminogen is used to prevent osteoarthritis, including A effective amount of plasminogen is applied to the subject of osteoarticular injury.

In above-mentioned all plasminogen treatment joint injuries, promote joint injury reparation, promotion regenerating bone or cartilage, prevention joint Lesion development is in the method for osteoarthritis, and the plasminogen can be administered alone, can also be with one or more other medicines Object or method are administered in combination.It is described combined administration be included in one or more other medicines or method application before, while or it After apply plasminogen.

" osteoarthritis (osteoarthritis, OA) " is a kind of retrogression pathological changes, is due to increasing age, obesity, strain, wound The damage of articular cartilage degeneration caused by the factors such as wound, joint birth defect, joint deformity, joint margins and subchondral bone Reactive hyperplasia, also known as osteoarthropathy, degenerative arthritis, senescent arthritis etc..Clinical manifestation is the joint slowly developed Pain, tenderness, stiff, arthroncus, limitation of activity and joint deformity etc..

Osteoarthritis is the retrogression pathological changes of Bones and joints, weight-bearing joints and the more joint of activity is apt to occur in, for example, neck Vertebra, lumbar vertebrae, knee joint, hip joint etc..It excessively bears a heavy burden or uses these joints, can promote the generation of degeneration variation.It is clinical Show as the arthralgia slowly developed, tenderness, stiff, arthroncus, limitation of activity and joint deformity etc..Imageology packet The abnormal change of articular cartilage and subchondral bone matter is included, joint space is narrow (it is thinning that this shows that articular cartilage has started), sclerotin Calcification, joint margins come to a point, and spur is formed and/or subchondral bone blister cavities is formed." osteoarthritis " of the present invention covers The osteoarthritis that parts of body occurs, osteoarthritis or related regression including cervical vertebra, lumbar vertebrae, knee joint, hip region Venereal disease becomes.

Some embodiments of the invention be related to plasminogen treatment osteoarthritis method, including administration subject it is effective The plasminogen of amount.In some embodiments, the plasminogen promotes Regeneration of Articular Cartilage and/or subchondral bone bone weight It builds.In some embodiments, plasminogen improves the inflammatory conditions of joint tissue and/or mitigates arthroncus and/or pain. In some embodiments, plasminogen improves the inflammation of synovium of joint and/or mitigates arthroncus and/or pain.Some In embodiment, the osteoarthritis is cervical vertebra, lumbar vertebrae, knee joint, the osteoarthritis of hip region or associated degenerative disease Become.

Above-mentioned plasminogen treatment osteoarthritis embodiment in, " treatment " include mitigate, alleviate, improvement or Eliminate following one or more symptom or sign: arthralgia, tenderness, stiff, arthroncus, joint function disturbance or by Limit, articular cartilage or subchondral bone matter abnormal change, joint space are narrow, and sclerotin calcification, joint margins come to a point, spur, cartilage Sending down the fishbone blister cavities.In the embodiment of the plasminogen treatment osteoarthritis, the osteoarthritis includes cervical vertebra, waist Vertebra, knee joint, the osteoarthritis of hip joint and its associated degenerative lesion.

" bone remoulding " is also referred to as " bone remodeling ", refer to mature bone tissue removed from bone (the referred to as process of bone resorption) and Form new bone tissue (the referred to as process of ossification or new bone formation).These processes also control fracture equivalent damage after bone again at Shape or displacement, and the micro-damage occurred during normal activity.Remold the functional requirement also responsive to mechanical load.The knot of bone Structure and the sufficient of calcium are supplied in requisition for two kinds of cell types of osteoblast (secreting new bone) and osteoclast (osteoclastic) and bone remoulding The close fit of other cell masses (such as immunocyte etc.) existing for position, and dependent on complicated signal transduction path and control Making mechanism realizes growth appropriate and rate of differentiation.

In some embodiments, the present invention relates to plasminogens to promote Regeneration of Articular Cartilage and/or subchondral bone bone weight It builds.In some embodiments, the present invention relates to plasminogens to promote (enhancing) osteoarthritis subject's osteoarthritis illness portion The activity of position osteoblast and/or the activity for reducing osteoclast.

In some embodiments, the present invention relates to the methods that plasminogen promotes the reparation of subject's joint injury, including A effective amount of plasminogen of subject is administered.In some embodiments, the joint injury includes inflammation, degenerative disease Become, joint injury caused by dysbolism or wound.In some embodiments, the plasminogen is by promoting articular cartilage Regeneration and/or subchondral bone bone remoulding treat joint injury, or promote the reparation of joint injury.In some embodiments, institute Plasminogen is stated to promote the activity of (enhancing) joint injury subject's joint injury position osteoblast and/or reduce osteoclast Activity.In some embodiments, the joint injury is joint injury caused by osteoarthritis.In some embodiments In, the joint injury is joint injury caused by inflammation, dysbolism or wound.

" improvement " joint synovitis disease (situation), the inflammation (situation) in " improvement " joint, the inflammation of " improvement " joint tissue The inflammation of (situation) or " improvement " joint tissue, which refers to that inflammation or inflammatory conditions are treated by administration plasminogen, to be improved, scorching Disease reaction is final to be alleviated, subsides.

In the embodiment of above-mentioned all plasminogen treatment osteoarthritis, the plasminogen can be administered alone, It can also be administered in combination with one or more other medicines or method.The combined administration is included in one or more other medicines Or method application before, simultaneously or after apply plasminogen.

Fibrinolysin is the key component of plasminogen activating system system (PA system).It is a kind of protease of wide spectrum, can Several components of hydrolyzed cellular epimatrix (ECM), including fibrin, gelatin, fibronectin, laminin and albumen are poly- Sugar^[5].In addition, fibrinolysin, which can activate some metalloprotein enzyme precursors (pro-MMPs), forms active metalloproteinases (MMPs).Therefore fibrinolysin is considered as an important upstream regulation object of extracellular proteolysis effect^[6,7].Fibrinolysin be by Plasminogen passes through the PAs: tissue-type plasminogen activator (tPA) or urokinase type plasminogen activator of two kinds of physiologicals (uPA) proteolysis is formed.Since relative level is higher in blood plasma and other body fluid for plasminogen, conventionally PA system 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, there is also the specific physiological inhibitors of fibrinolysin and PAs.The main suppression of fibrinolysin Preparation is α 2- antiplasmin (α 2-antiplasmin).The activity of PAs is inhibited by the plasminogen activator of uPA and tPA simultaneously Agent -1 (PAI-1) inhibits and the main plasminogen activator agent inhibitor -2 (PAI-2) for inhibiting uPA is adjusted.Certain cell surfaces With the active uPA specific cell surface receptor (uPAR) of direct hydrolysis^[8,9]。

Plasminogen is a single chain glycoprotein, is made of 791 amino acid, molecular weight is about 92kDa^[10,11].Fibrinolytic Proenzyme is mainly synthesized in liver, is largely present in extracellular fluid.Content of plasminogen is about 2 μM in blood plasma.Therefore plasminogen It is a huge potential source of proteolytic activity in tissue and body fluid^[12,13].There are two kinds of molecule shapes for plasminogen Formula: glutamic acid-plasminogen (Glu-plasminogen) and lysine-plasminogen (Lys-plasminogen).Natural point Secreting has amino terminal (end a N-) glutamic acid, therefore referred to as glutamic acid-fibrinolytic with the plasminogen of uncracked form Proenzyme.However, glutamic acid-plasminogen hydrolyzes at Lys76-Lys77 becomes lysine-fibrinolysin in the presence of fibrinolysin It is former.Compared with glutamic acid-plasminogen, lysine-plasminogen and fibrin have higher affinity, and can be higher Rate activated by PAs.The Arg560-Val561 peptide bond of the plasminogen of both forms can be cut by uPA or tPA, be caused The formation of the dichain proteins enzyme fibrinolysin of disulfide bond connection^[14].The amino terminus portion of plasminogen includes five homologous tricyclics, I.e. so-called kringles, carboxy-terminal sections include protease domain.Some kringles contain mediate 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 dissolution is the pass preventing pathologic thrombus and being formed Key^[15].Fibrinolysin also has the substrate specificity to the several components of ECM, including laminin, fibronectin, proteoglycans And gelatin, show fibrinolysin ECM reconstruction in also play an important role [^11,16,17].Indirectly, fibrinolysin can also pass through conversion Certain protease precursors are active protease come the other components for the ECM that degrades, including MMP-1, MMP-2, MMP-3 and MMP-9.Cause This, it is thus proposed that, fibrinolysin may be an important upstream regulator of extracellular proteolysis^[18].In addition, fibrinolysin has There is the ability for the growth factor for activating certain potential forms^[19-21].In vitro, fibrinolysin can also hydrolyze the component of complement system simultaneously Discharge chemotactic complement fragment.

" fibrinolysin " is to be present in the very important enzyme of one of 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 source of people plasminogen amino acid sequence (sequence 4) calculating is made of 810 amino acid, and molecular weight is about 90kD, mainly in liver Dirty middle synthesis and the glycoprotein that can be recycled in blood, encode the cDNA sequence of the amino acid sequence as shown in sequence 3.Overall length Plasminogen include seven structural domains: positioned at the serine protease domain of C-terminal, the Pan Apple (PAp) of N-terminal Structural domain and 5 Kringle structural domains (Kringle1-5).Referring 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 Residue Glu184-Cys262 is included, Kringle3 includes residue Cys275-Cys352, and Kringle4 includes residue Cys377- Cys454, Kringle5 include residue Cys481-Cys560.According to NCBI data, serine protease domain includes residue Val581-Arg804。

Glu- plasminogen is the plasminogen of Native full-length, be made of 791 amino acid (without containing 19 amino acid Signal peptide), the cDNA sequence of the sequence is encoded as shown in sequence 1, and amino acid sequence is as shown in sequence 2.In vivo, there is also One is the Lys- plasminogens hydrolyzed from the 76-77 amino acids of Glu- plasminogen to be formed, such as 6 institute of sequence Show, encodes the cDNA sequence of the amino acid sequence as shown in sequence 5.Delta- plasminogen (δ-plasminogen) is overall length Plasminogen has lacked the segment of Kringle2-Kringle5 structure, only contains Kringle1 and serine protease Domain^[22,23], there is the amino acid sequence (sequence 8) of document report delta- plasminogen^[23], encode the amino acid sequence CDNA sequence such as sequence 7.Miniplasminogen (Mini-plasminogen) is made of Kringle5 and serine protease domain, is had Document report it include that residue Val443-Asn791 (is with the Glu residue of Glu- plasminogen sequence without containing signal peptide Beginning amino acid)^[24], amino acid sequence encodes the cDNA sequence of the amino acid sequence as shown in sequence 9 as shown in sequence 10. And Microplasminogen (Micro-plasminogen) only contains serine protease domain, there is its amino acid sequence of document report Column include that residue A la543-Asn791 (is starting amino with the Glu residue of the Glu- plasminogen sequence without containing signal peptide Acid)^[25], also there is patent document CN102154253A to report that its sequence includes residue Lys531-Asn791 (without containing signal peptide Glu- plasminogen sequence Glu residue 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 " of the invention, " fibrinoclase " are used interchangeably, and meaning is identical； " plasminogen " is used interchangeably with " plasminogen ", " plasminogen ", and meaning is identical.

In this application, the meaning of the plasminogen " shortage " or activity are the content ratio of plasminogen in subject's body Normal person is low, down to being enough to influence the normal physiological function of the subject；The meaning or activity of the plasminogen " missing " Be substantially less than normal person for the content of plasminogen in subject's body, in addition activity or expression it is atomic, external source offer is only provided 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 closed nonactive conformation, 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 domain of plasminogen includes the weight for maintaining plasminogen to be in nonactive closing conformation Want determinant, and KR structural domain then can be present in conjunction with the lysine residue on receptor and substrate.It is known a variety of to make For the enzyme of plasminogen activator, comprising: 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, in conjunction with the target sequence in substrate and can play The active fragment of proteolysis function.The present invention relates to the technical solutions of plasminogen to cover 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%, the protein of the amino acid sequence of 95%, 96%, 97%, 98%, 99% homology.Therefore, fibrinolytic of the present invention Proenzyme includes containing the activities of endothelial tissue plasminogen segment and still maintaining the albumen of the activities of endothelial tissue plasminogen.

Currently, including: to tissue plasminogen activator's activity for plasminogen in blood and its activity determination method 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 it is living Detection, the detection of Plasma Tissue-Type Plasminogen Activitor mortifier antigen, plasma fibrin lyase-antifibrinolysin of property Multienzyme complex detects (PAP).The detection method of most common of them be Chromogenic assay: in Xiang Shoujian blood plasma plus streptokinase (SK) and Chromophoric substrate is transformed into PLM by the PLG in inspection blood plasma under the action of SK, and the latter acts on chromophoric substrate, then with light splitting Photometric determination, absorbance increase directly proportional to activities of endothelial tissue plasminogen.Furthermore immuno-chemical method can also be used, gel electrophoresis, exempt from Epidemic disease turbidimetry, 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, has both included that albumen is same 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.Plasminogen of the invention includes the natural plasminogen of people, further includes from not Infraspecific plasminogen ortholog thing with activities of endothelial tissue plasminogen or lineal homologue.

" conservative substitution variant " refers to that one of them given amino acid residue changes but do not change the whole of protein or enzyme Body conformation and function, this include but is 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 replacement.Therefore, two albumen of identity function or the similitude of amino acid sequence may not Together.For example, 70% to 99% similarity (identity) based on MEGALIGN algorithm." conservative substitution variant " further includes passing through BLAST or fasta algorithm determine the polypeptide or enzyme of the amino acid identities with 60% or more, if can be more preferable up to 75% or more, Preferably up to 85% or more, or even up to 90% the above are best, and have compared with natural or parent protein or enzyme identical Or substantially similar property or function." separation " plasminogen refers to the fibrinolysin for separating and/or recycling from its natural surroundings Former albumen.In some embodiments, the plasminogen can purify (1) to more than 90%, greater than 95% or greater than 98% Purity (by weight), as by determined by Lowry method, such as more than 99% (by weight), (2) to be enough by using Rotating cup sequenator obtains the degree of at least 15 residues of N-terminal or internal amino acid sequence, or (3) are somebody's turn to do to homogeney Homogeney is the sodium dodecyl sulfate-polypropylene by using Coomassie blue or silver staining under reproducibility or non-reducing conditions What acrylamide gel electrophoresis (SDS-PAGE) determined.Isolated plasminogen also includes by biotechnology from recombinant cell system Plasminogen that is standby, and being separated by least one purification step.

Term " polypeptide ", " peptide " and " protein " is used interchangeably herein, and refers to the polymerization of the amino acid of any length Form, may include genetic coding and non-genetic coding amino acid, chemistry or biochemical modification or derivatization Amino acid, and the polypeptide with modified peptide backbone.The term includes fusion protein, including but not limited to has heterologous amino The fusion protein of acid sequence, with 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 (%) " referring to polypeptide sequence It is candidate after realizing maximum percentage sequence identity, and when any conservative substitution not being considered as a part of sequence identity The percentage of amino acid residue identical with the amino acid residue in reference polypeptide sequence in sequence.To measure percent amino acid The comparison of sequence identity purpose can realize with the various ways within the scope of art technology, such as using publicly available 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, any algorithm including realizing maximum contrast needs to institute's full length of comparison sequence are used for.However, For the purposes of the present invention, amino acid sequence identity percent value is to compare computer program ALIGN-2 generation using sequence 's.

In the case where using ALIGN-2 come comparing amino acid sequence, amino acid sequence A is given relative to given amino acid Sequence B % amino acid sequence identity (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 identity of B) it calculates as follows:

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 unequal situation of length of base acid sequence B, A can be not equal to B relative to A relative to the % amino acid sequence identity of B % amino acid sequence identity.Unless expressly stated otherwise, all % amino acid sequence identity values used herein are all It is to be obtained according to described in the preceding paragraph using ALIGN-2 computer program.

As used in this article, term " treatment " and " processing ", which refer to, obtains desired pharmacology and/or physiologic effect.The effect Fruit can be prevention disease or its symptom completely or partially, and/or partially or completely cure disease and/or its symptom, and wrap It includes: (a) preventing disease and occur in subject's body, 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 to mammal or other subjects application 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 separated 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 to be suitble to the chemically synthesized method of plasminogen.Various forms of SPPS, such as Fmoc and Boc can be used for synthesizing fibrinolysin It is former.Technical description for synthesis in solid state is in Barany and Solid-Phase Peptide Synthesis；The 3-284 pages in Volume 2: Special Methods in Peptide of The Peptides:Analysis, Synthesis, Biology. Synthesis, Part A., Merrifield wait J.Am.Chem.Soc., 85:2149-2156 (1963)；Stewart etc., Solid Phase Peptide Synthesis,2nd ed.Pierce Chem.Co.,Rockford,Ill.(1984)；With The Ganesan A.2006Mini 2005Protein such as Rev.Med Chem.6:3-10 and Camarero JA Pept In Lett.12:723-8.In short, handling small insoluble porous bead with the functional element for being built with peptide chain thereon.In idol After connection/de-protected repetitive cycling, the free N-terminal amine of the solid phase of attachment is coupled with the Amino Acid Unit individually protected by N.So Afterwards, this element is deprotected, expose can and other amino acid attachment new N-terminal amine.Peptide remains fixed in solid phase, it It is cut away afterwards.

Standard recombinant methods can be used to produce plasminogen of the invention.For example, by the nucleic acid of encoding plasminogen It is inserted into expression vector, is operatively connected it with the regulating and controlling sequence in expression vector.Expression regulation sequence includes but is not limited to Promoter (such as naturally associated or heterologous promoter), signal sequence, enhancer element and transcription terminator.Expression Regulation 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 expression vector is usually in host organisms as episome or as the integration portion of host chromosome DNA Divide duplication.In general, expression vector contain selection marker (such as amicillin resistance, hygromycin resistance, tetracyclin resistance, Kalamycin resistance or neomycin resistance) it is detected with helping those of to convert cell with desired DNA sequence dna to external source.

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, also it can be generated 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 promoter system System, or the promoter systems from phageλ.Promoter would generally control expression, optionally in the case where operator sequence, And there is 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 benefit The promoter of enzyme.

Outside microorganism, mammalian cell (such as the mammalian cell cultivated in cell culture in vitro) can also For expressing and generating anti-Tau antibody of the invention (such as polynucleotides of the anti-Tau antibody of encoding schemes).Referring to Winnacker,From Genes to Clones,VCH Publishers,N.Y.,N.Y.(1987).Suitable mammal Host cell includes CHO cell line, various Cos cell lines, HeLa cell, myeloma cell line and transformed B cell or miscellaneous Hand over tumor.Expression vector for these cells may include expression control sequence, such as replication orgin, promoter and enhancer (Queen etc., Immunol.Rev.89:49 (1986)) and required machining information site, such as ribosome bind site, RNA splice site, 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 according to the standard schedule of this field, including ammonium sulfate precipitation Column, column chromatography, high performance liquid chroma- tography (HPLC), gel electrophoresis etc. purify plasminogen of the present invention.The plasminogen Be 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 preparation 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- amylalcohol；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；At salt counter ion such as sodium；Metal Compound (such as zinc-albumen composition)；And/or nonionic surfactant, such as TWEENTM, PLURONICSTM or poly- second Glycol (PEG).It is preferred that the anti-VEGF antibodies preparaton being lyophilized describes in WO 97/04801, it includes herein as ginseng It examines.

Preparaton of the invention contains needed for the specific illness that need to treat more than one reactive compound, preferably It complementary activities and those of is free from side effects between each other.For example, antihypertensive drug, antiarrhythmic drug, are controlled Treat the drug etc. of diabetes.

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

Plasminogen of the invention can prepare sustained release preparation.The appropriate example of sustained release preparation includes with certain shapes and containing There are the penetrating matrix of solid hydrophobic polymers half of glycoprotein, such as film or microcapsules.Sustained-release matrix example includes polyester, hydrogel (such as poly- (2- hydroxyethyl-methacrylate) (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 22:547 (1983)) of Pidolidone and γ ethyl-L-glutamate, 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 acid.Polymer such as ethylene-second Vinyl acetate and lactic-co-glycolic acid energy sustained release molecule 100 days or more, and the time of some hydrogels release proteins compared with It is short.Can be designed according to Related Mechanism makes protein stabilized rational strategy.For example, if it find that the mechanism of cohesion is to pass through sulphur Intermolecular S -- S is formed for Disulfide interchange, then can by modification sulfhydryl residue, from acid solution be lyophilized, control humidity, Stabilization is realized using suitable additive and the specific polymer matrix composition of exploitation.

Administration and dosage

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

Prepared product for parenteral administration includes sterile aqueous or non-aqueous solution, suspension and emulsion.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 Include water, alcohol/aqueous solution, emulsion or suspension, including salt water and buffer medium.Parenteral medium includes that sodium chloride is molten 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, figure, body surface area, age including patient, the particular compound to be applied, gender, application Number and path, general health and the other medicines being administered simultaneously.The present invention includes the dosage of the pharmaceutical composition of plasminogen Range can be, for example, such as daily about 0.0001 to 2000mg/kg, or about 0.001 to 500mg/kg (such as 0.02mg/kg, 0.25mg/kg, 0.5mg/kg, 0.75mg/kg, 10mg/kg, 50mg/kg etc.) subject's weight.For example, dosage can be 1mg/kg weight or 50mg/kg weight or the range in 1-50mg/kg, or at least 1mg/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 medicament administration of the 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 this hairs that can be used for treating osteoarthritis Bright plasminogen or fibrinolysin.The product preferably includes a container, label or package insert.Container appropriate has bottle, Bottle, syringe etc..Container can be made of a variety of materials such as glass or plastics.The container contains composition, the composition Disease or illness of the invention can effectively be treated and there is sterile access port (such as the container can be intravenous solution packet or small Bottle, containing can be by the plug that hypodermic needle penetrates).At least one of composition activating agent is plasminogen/fibre Lyase.On the container or appended label illustrates the composition for treating osteoarthritis of the present invention.The product It can further include the second container containing pharmaceutically acceptable buffer, the salt water of such as phosphate-buffered, Ringer's solution and Portugal Grape sugar juice.It can further include required other materials from the point of view of business and user's angle, including other buffers, dilute Release agent, filtrate, needle and syringe.In addition, the product includes the package insert with operation instruction, including for example indicate institute The user of composition is stated by the other medicines administered patient of the adjoint disease of plasminogen composition and treatment.

Detailed description of the invention

Figure 1A-D shows 0.5 μ g/kg vitamin D exhausted mining areas knee joint Safranin O coloration result.A is blank Control group, B are to give solvent PBS control group, and C is to give plasminogen group, and D is quantitative analysis results.The results show that giving plasminogen Group knee cartilage (arrow logo), which is significantly more than, gives solvent PBS control group, and statistical discrepancy is significant (* indicates P < 0.05), and with It is compared to solvent PBS control group and is more nearly blank control mouse to plasminogen group knee cartilage amount.Illustrate plasminogen Vitamin D exhausted mining areas knee cartilage loss can be substantially reduced.

Fig. 2A-E gives the osteoarthritis mouse knee joint Safranin of plasminogen Type Ⅱ collagen enzyme induction in 14 days O dyes appraisal result.A, C is to give solvent PBS control group, and B, D are to give plasminogen group, and E is quantitative analysis results.As a result it shows Show, be significantly lower than to plasminogen group knee joint pathological score and give solvent PBS control group, and extremely significant (the * * table of statistical discrepancy Show P < 0.01).Illustrate that plasminogen can reduce the kneed damage of osteoarthritis mouse of Type Ⅱ collagen enzyme induction.

Fig. 3 A-D gives the osteoarthritis Plg of plasminogen Type Ⅱ collagen enzyme induction in 14 days^-/-Mouse knee joint Safranin O dyes representative picture.A, C is to give solvent PBS control group, and B, D are to give plasminogen group.The results show that molten Matchmaker's PBS control group cartilaginous tissue (thin arrow logo) structural arrangement disorder, cell number significantly reduce, and Safranin O coloring is bright Aobvious to reduce, bone trabecula (block arrow mark) attenuates, is broken；To plasminogen group relative to solvent PBS control group, cartilaginous tissue Structure is relatively neat, and cell number is relatively more at cartilage, Safranin O color range it is relatively wide.Illustrate plasminogen energy Enough reduce the osteoarthritis Plg of Type Ⅱ collagen enzyme induction^-/-The kneed damage of mouse.

Fig. 4 A-B gives the osteoarthritis mouse knee joint Safranin O of plasminogen ligament cutting induction in 14 days Dye representative picture.A is to give solvent PBS control group, and B is to give plasminogen group.The results show that soft to solvent PBS control group Bone (triangle mark) is lost seriously, and bone trabecula (arrow logo) attenuates, and the bone-free girder marrow of larger area occurs in fracture Chamber；Plasminogen group is given, compared to PBS control group, bone trabecula continuity is preferable, not more serious fracture, without larger face Long-pending bone-free trabecular region, cartilaginous tissue are relatively more.Illustrate that plasminogen can improve the osteoarthritis of ligament cutting induction Model mice knee joint institutional framework situation.

Fig. 5 A-E gives the osteoarthritis mouse knee joint alkaline phosphatase of plasminogen ligament cutting induction in 14 days Dye fruit.A, C is to give solvent PBS control group, and B, D are to give plasminogen group, and E is quantitative analysis results.The results show that giving fibrinolytic Proenzyme group mouse knee cartilage surface (thin arrow logo) and the coloring of growth plate (block arrow mark) alkaline phosphatase are more than Solvent PBS control group is given, and statistical discrepancy is significant (* indicates P < 0.05).Illustrate that plasminogen can remarkably promote ligament cutting The increase of the osteoarthritis mouse knee joint alkaline phosphatase activities of induction, i.e. plasminogen promote knee joint osteoblast Activity obviously increases.

Fig. 6 A-D gives plasminogen 28 days MIA osteoarthritis mouse knee joint II Collagen Type VI immunohistochemical staining knots Fruit.A is sham-operation group, and B is to vehicle control group, and C is to give plasminogen group, and D is quantitative analysis results.The results show that artificial hand Art group mouse knee joint has a certain amount of II Collagen Type VI (arrow logo), to vehicle control group knee joint II Collagen Type VI amount and artificial hand Art group is significantly more than to plasminogen group knee joint II Collagen Type VI amount to vehicle control group and sham-operation group without significant difference, And average optical density quantitative analysis results statistical discrepancy is significant (* indicates P < 0.05).The result shows that plasminogen can promote bone The regeneration of arthritis model mouse knee cartilage.

Fig. 7 A-D gives plasminogen 28 days MIA osteoarthritis mouse knee joint Safranin O dyeing representative diagrams Piece.A is sham-operation group, and B is to vehicle control group, and C is to give plasminogen group, and D is pathological score result.The results show that artificial hand Art group mouse knee joint is with the presence of a certain amount of cartilage (arrow logo)；It is significantly reduced simultaneously to vehicle control group knee cartilage amount And histological scores obviously increase, and illustrate that MIA success inducing osteoarthritis is scorching；It is significantly more than to plasminogen group knee cartilage amount To vehicle control group, histological scores are also significantly lower than to vehicle control group, and statistical difference is significant (* indicates P < 0.05). It should be the result shows that plasminogen can promote regenerating bone or cartilage, improvement osteoarthritic lesions.

Fig. 8 A-D gives knee joint femoral surface Safranin O dye on the left of 28 days osteoarthritis mouse of plasminogen Color result.A is sham-operation group, and B is to vehicle control group, and C is to give plasminogen group, and D is average optical density quantitative analysis results. The results show that femoral surface has a small amount of cartilage (arrow logo) at sham-operation group mouse knee joint, vehicle control group femur is given Surface cartilage amount and sham-operation group are significantly more than to plasminogen group femoral surface cartilage amount to Vehicle controls without significant difference Group and sham-operation group, and average optical density quantitative analysis results statistical discrepancy is significant (* indicates P < 0.05).The result shows that fibrinolysin Proper energy enough promotes osteoarthritis mouse femur surface cartilage to regenerate.

Fig. 9 gives plasminogen 7 days MIA osteoarthritis rat pain testing results.The results show that sham-operation group is big Mouse pain threshold with higher；It is substantially reduced to vehicle control group rat or so pain threshold that has a pain in the leg, hence it is evident that be lower than sham-operation Group；Having a pain in the leg to plasminogen group rat or so, pain threshold is significantly raised, and compared with to vehicle control group, left leg statistical discrepancy is close Significantly (P=0.08), right leg statistical discrepancy are significant (* indicates P < 0.05).The result shows that plasminogen being capable of substantially reduced bone Arthritis ache.

Figure 10 gives plasminogen 28 days osteoarthritis mice pain testing results.The results show that sham-operation group is small Mouse pain threshold is relatively high；It is substantially reduced to vehicle control group mice pain threshold value, hence it is evident that be lower than sham-operation group mouse；To fibre Lyase original group mice pain threshold value is significantly raised, hence it is evident that is higher than and gives vehicle control group mouse, and the close significant (P=of statistical discrepancy 0.09).It should be the result shows that plasminogen can reduce osteo-arthritic pain.

Figure 11 A-F gives knee joint Safranin O coloration result on the left of 28 days osteoarthritis mouse of plasminogen. The results show that there are a certain amount of articular cartilage (arrow logos) for knee joint femoral and femoral surface on the left of sham-operation group, to molten Matchmaker's control group shin bone and femoral surface cartilage amount and sham-operation group difference are unobvious, are significantly more than and give to plasminogen group cartilage amount Vehicle control group and sham-operation group.Illustrate that plasminogen can promote osteoarthritis mouse Regeneration of Articular Cartilage.

Figure 12 A-D shows that plasminogen inhibits osteoarthritis mouse bone resorption.The results show that sham-operation group (figure 12A) there are a certain amount of acid phosphatase (arrow logos) for knee joint；Give vehicle control group (Figure 12 B) knee joint acid phosphatase Enzyme increases, hence it is evident that is more than sham-operation group；It is considerably less than to plasminogen group (Figure 12 C) knee joint acid phosphatase to Vehicle controls Group, and statistical discrepancy is significant (* indicates P < 0.05) (Figure 12 D).It should be the result shows that plasminogen can reduce osteoarthritic knee joint Acid phosphatase reduces osteoclast activity, inhibits bone resorption.

Figure 13 A-F shows that plasminogen promotes 9 Positive Stem Cells quantity of MIA osteoarthritic knee joint ligament tibia end Sox to increase Add.The results show that sham-operation group (Figure 13 A, D) knee joint tibial is there are a certain amount of 9 Positive Stem Cells of Sox (arrow logo), It is significantly reduced to 9 Positive Stem Cells quantity of vehicle control group (Figure 13 B, E) knee joint tibial Sox, gives plasminogen group (figure 13C, F) knee joint tibial Sox 9 Positive Stem Cells quantity be significantly more than to vehicle control group.It should be the result shows that plasminogen can Promote 9 Positive Stem Cells quantity of osteoarthritic knee joint ligament tibia end Sox to increase, repairs osteoarthritis to kneed damage.

Figure 14 A-C shows that plasminogen improves MIA osteoarthritis mouse gonarthromeningitis disease.The results show that false Operation group (Figure 14 A) knee joint synovial membrane has no apparent inflammatory cell infiltration, can to vehicle control group (Figure 14 B) knee joint synovial membrane See apparent inflammatory cell infiltration (arrow logo), it is obvious to plasminogen group (Figure 14 C) gonarthromeningitis disease cellular infiltration Less than to vehicle control group.It should be the result shows that plasminogen can improve osteoarthritic knee joint synovial membrane inflammation.

Embodiment

The osteoporosis mouse knee cartilage that 1 plasminogen of embodiment reduces vitamin D induction is lost

Male C57 mouse 15 of 5-6 week old are taken, 3 groups are randomly divided into after weighing, blank control group gives plasminogen group With give solvent PBS control group, every group each 5.50 μ l corn oils are injected intraperitoneally in blank control group mouse daily；Give plasminogen group It is injected intraperitoneally vitamin D (Sigma Aldrich), induces daily according to 0.5 μ g/kg weight with to solvent PBS control group mouse Osteoporosis model^[26,27].Mouse starts to be administered at the same time, gives plasminogen group mouse tail vein injection source of people plasminogen 1mg/0.1mL/ pcs/day, to the PBS of solvent PBS control group tail vein injection same volume, blank control group mouse is not done at administration Reason, continuous modeling are administered 28 days.All mouse feed low calcium feed during administration.Start modeling administration to be set to the 1st day, the 29th day Materials knee joint fixes 24 hours in 4% paraformaldehyde after execution, then decalcification three weeks in 10%EDTA, and graded sucrose solutions are washed It washs, the above operation need to carry out under the conditions of 4 DEG C.Then paraffin embedding, 8 μm of slice row Safranin O dyeing.Slice is at 100 times Optical microphotograph is under the microscope.

It is red that cartilage dyeing liquor (safranin O method) dyeing theory is that the cartilage of basophilla is presented in conjunction with basic dye safranin O Color, safranin O are a kind of polyanionic dyes of positive ion of combination, display cartilage be based on yin in the dye of positive ion and polysaccharide from Subbase group (chondroitin sulfate or keratan sulfate) combines.Safranin O coloring is approximate with the concentration of anion proportional, indirectly Reflect the content and distribution of proteoglycan in matrix.

Safranin O dyeing is sarranine dyeing, the main acidic protein chitosan component shown in cartilaginous tissue, Neng Gouxian Show Subchondral drilling^[28]。

Solvent PBS control is given the results show that being significantly more than to plasminogen group (Fig. 1 C) knee cartilage (arrow logo) Group (Figure 1B), statistical discrepancy is significant (* indicates P < 0.05) (Fig. 1 D), and gives plasminogen group compared with to solvent PBS control group Knee cartilage is more nearly blank control mouse (Figure 1A).Illustrate that plasminogen can substantially reduce vitamin D osteoporosis Model mice knee cartilage is lost.

The osteoarthritis mouse injury of knee joint of 2 plasminogen of embodiment reduction Type Ⅱ collagen enzyme induction

The C57 male mice of 10 week old 12, according to 50mg/kg weight be injected intraperitoneally yellow Jackets, anesthetized mice, so Intramuscular injection Tolfedine (0.1ml/kg) afterwards.Open notch at right side of mice knee joint, 90 degree of knee joint bending, mouse According to 5 μ g/6 μ l/ intraarticular injection Type Ⅱ collagen enzymes (C6885, sigma)；Left side knee joint and right side knee joint operate It is identical, but only inject the physiological saline of same volume^[29,30].Mouse is randomly divided into two groups according to weight respectively after injection 7 days, gives Solvent PBS control group and plasminogen group is given, every group each 6, and start to be administered.To plasminogen group according to 1mg/0.1ml/ Pcs/day tail vein injection gives human plasminogen, to the PBS of solvent PBS control group tail vein injection same volume, successive administration 14 days.Starting administration and is set to the 1st day, materials knee joint fixes 24 hours in 4% paraformaldehyde after putting to death within the 15th day, and then 10% Decalcification three weeks in EDTA, graded sucrose solutions washing, the above operation need to carry out under the conditions of 4 DEG C.Then paraffin embedding, 8 μm are cut Piece row Safranin O dyeing.Slice presses table 1 to knee joint in 40 (A, B) and 100 (C, D) times of optical microphotograph microscopic observation Carry out pathological score^[31]。

Table 1

Solvent PBS control group (figure is given the results show that being significantly lower than to plasminogen group knee joint (Fig. 2 B, D) pathological score 2A, C), and statistical discrepancy is extremely significant (* * indicates P < 0.01) (Fig. 2 E).Illustrate that plasminogen can significantly mitigate Type Ⅱ collagen The osteoarthritis of enzyme induction reduces the loss of articular cartilage to kneed damage.

The osteoarthritis Plg of 3 plasminogen of embodiment improvement Type Ⅱ collagen enzyme induction^-/-Mouse knee joint knot of tissue Structure state

The Plg of 10 week old^-/-Male mice 10, according to 50mg/kg weight be injected intraperitoneally yellow Jackets, anesthetized mice, Then intramuscular injection Tolfedine (0.1ml/kg).A notch is opened at right side of mice knee joint, it is 90 degree of knee joint bending, small Mouse is according to 5 μ g/6 μ l/ intraarticular injection Type Ⅱ collagen enzymes (C6885, sigma)；Left side knee joint and right side knee actuator Make physiological saline that is identical, but only injecting same volume^[29,30].Mouse is randomly divided into two groups according to weight respectively after injection 7 days, To solvent PBS control group and plasminogen group is given, every group each 5, and start to be administered.To plasminogen group according to 1mg/0.1ml/ Pcs/day tail vein injection gives human plasminogen, to the PBS of solvent PBS control group tail vein injection same volume, successive administration 14 days.Starting administration and is set to the 1st day, materials knee joint fixes 24 hours in 4% paraformaldehyde after putting to death within the 15th day, and then 10% Decalcification three weeks in EDTA, graded sucrose solutions washing, the above operation need to carry out under the conditions of 4 DEG C.Then paraffin embedding, 4 μm are cut Piece row Safranin O dyeing.Slice is in 40 (A, B) and 100 (C, D) times of optical microphotograph microscopic observation.

The results show that giving solvent PBS control group (Fig. 3 A, C) cartilaginous tissue (thin arrow logo) structural arrangement disorder, cell Number significantly reduces, and Safranin O coloring significantly reduces, and bone trabecula (block arrow mark) attenuates, is broken；Give plasminogen group (Fig. 3 B, D) relative to solvent PBS control group is given, Cartilage tissue constructs are relatively neat, and cell number is relatively more at cartilage, Safranin O color range it is relatively wide.Illustrate that plasminogen can reduce the osteoarthritis Plg of Type Ⅱ collagen enzyme induction^-/- The kneed damage of mouse.

4 plasminogen of embodiment improves the osteoarthritis mouse knee joint institutional framework state of ligament cutting induction

The C57 male mice of 9-10 week old 10.Yellow Jackets, anesthetized mice is injected intraperitoneally according to 50mg/kg weight. Knee joint peripheral hair is sloughed, mouse muscle injects Tolfedine (0.1ml/kg) analgestic before perioperatively.Mouse is placed on Under disecting microscope, knee cap distal end and tibial plateau opening on right side separate patellar ligament, exposure articular cavity；Blunt separation stock The intercondylar fat pad of bone enables preceding right-angled intersection ligament to be observed；With micro- blade break before right-angled intersection ligament and Medial collateral ligament sutures joint capsule and skin, establishes osteoarthritis^[32,33].Left side knee joint only expose articular cavity, not into Row rupturing operation.It is postoperative to be not fixed limbs it should be noted that avoiding damage to articular cartilage in art, it is move freely in cage.First after operation It, every mouse muscle injection Tolfedine (0.1mg/kg) in 12 hours and benzylpenicillin potassium (40,000 units/kg).Mouse after two weeks Two groups are randomly divided into according to weight, to solvent PBS control group and gives plasminogen group, every group each 5, and start to be administered.To fibre Lyase original group gives human plasminogen according to 1mg/0.1ml/ pcs/day of tail vein injection, gives solvent PBS control group tail vein injection The PBS of same volume, successive administration 14 days.Start administration to be set to the 1st day, materials knee joint is in 4% poly after putting to death within the 15th day Formaldehyde fixes 24 hours, then decalcification three weeks in 10%EDTA, and graded sucrose solutions washing, the above operation need to be under the conditions of 4 DEG C It carries out.Then paraffin embedding, 4 μm of slice row Safranin O dyeing.Slice is in 40 times of optical microphotographs microscopic observations.

The results show that losing serious, bone trabecula (arrow mark to solvent PBS control group (Fig. 4 A) cartilage (triangle mark) Know) attenuate, it is broken, the bone-free girder ossis of larger area occurs；Plasminogen group (Fig. 4 B) is given, compared to PBS control group, Bone trabecula continuity is preferable, not more serious fracture, not the bone-free trabecular region of larger area, and cartilaginous tissue is relatively It is more.Illustrate that plasminogen can improve the osteoarthritis mouse knee joint institutional framework situation of ligament cutting induction.

5 plasminogen of embodiment increases the osteoarthritis mouse knee joint alkaline phosphatase activities of ligament cutting induction

The C57 male mice of 9-10 week old 10.Yellow Jackets, anesthetized mice is injected intraperitoneally according to 50mg/kg weight. Knee joint peripheral hair is sloughed, mouse muscle injects Tolfedine (0.1ml/kg) analgestic before perioperatively.Mouse is placed on Under disecting microscope, knee cap distal end and tibial plateau opening on right side separate patellar ligament, exposure articular cavity；Blunt separation stock The intercondylar fat pad of bone enables preceding right-angled intersection ligament to be observed；Right-angled intersection ligament before being broken with micro- blade, seam Joint capsule and skin are closed, osteoarthritis is established^[32,33].Left side knee joint only exposes articular cavity, without rupturing operation.Art In it should be noted that avoid damage to articular cartilage, it is postoperative to be not fixed limbs, move freely in cage.Hand first post-operative day, every 12 hours Mouse muscle injects Tolfedine (0.1mg/kg) and benzylpenicillin potassium (40,000 units/kg).Mouse is random according to weight after two weeks It is divided into two groups, to solvent PBS control group and gives plasminogen group, every group each 5, and start to be administered.To plasminogen group according to 1mg/0.1ml/ pcs/day of tail vein injection gives human plasminogen, to solvent PBS control group tail vein injection same volume PBS, successive administration 14 days.Start administration to be set to the 1st day, materials knee joint is fixed in fixer after putting to death within the 15th day.It is fixed Formula of liquid: 2% paraformaldehyde, 0.075mol/L lysine, 0.01mol/L sodium periodate.4 DEG C of PBS washing lotion gradients are washed after fixation It washs each 12 hours, is subsequently placed in 4 DEG C of decalcifying Fluids decalcification 2 weeks, changes a decalcifying Fluid within every 5 days.4 DEG C of PBS washing lotions after the completion of decalcification Gradient wash 12 hours, knee joint carried out paraffin embedding after alcohol serial dehydration and dimethylbenzene are transparent.It is sliced 5um, dewaxing is multiple Water, 4 DEG C of magnesium chloride buffer overnight incubations.Alkaline phosphatase substrate solution is incubated at room temperature 1 hour, and haematoxylin is redyed 2 minutes.Stream Water rinses 5 minutes, and 60 DEG C 30 minutes roasting, neutral gum mounting, and slice is in 200 times of optical microphotographs microscopic observations.

Alkaline phosphatase (alkaline phosphatase, ALP) is the mark of osteoblast early differentiation^[32]。

The results show that being coloured to the plasminogen group mouse knee cartilage surface (Fig. 5 B, D) and growth plate alkaline phosphatase (arrow logo), which is more than, to be given solvent PBS control group (Fig. 5 A, C), and statistical discrepancy is significant (* indicates P < 0.05) (Fig. 5 E).It says Bright plasminogen can remarkably promote the increasing of the osteoarthritis mouse knee joint alkaline phosphatase activities of ligament cutting induction Add, i.e., plasminogen promotes the osteoblast activity of knee joint endoprosthesis cartilage to obviously increase.

6 plasminogen of embodiment promotes the regeneration of MIA osteoarthritis mouse knee cartilage

Take 8-10 weeks C57 male mice 25, weighing is randomly divided into two groups by weight, sham-operation group 5, model group 20 Only.The anesthesia of 3% yellow Jackets is injected intraperitoneally according to 50mg/kg weight in all mouse, and model group mouse sloughs left side after anesthesia Knee hair, 70% alcohol and iodine tincture disinfection, 90 degree of left side knee joint bending, along knee level operation syringe Needle (in order to avoid piercing through skin), the gap below discovery kneecap.With the slight pressure mark region, then vertically lift needle Head and syringe, insert the needle into marked region, across the kneecap tendon perpendicular to shin bone, intraarticular injection MIA (iodoacetic acid, Monoiodoacetic acid, MIA) normal saline solution 0.1mg/10 μ l；10 μ l physiology of joint cavity injection on the left of sham-operation group Salt water, after injection, massaging knee, to ensure to be uniformly distributed^[34].Right side knee joint is not handled.After MIA is injected 3 days in articular cavity, Model group mouse carries out pain test, is randomly divided into two groups according to test result mouse, to vehicle control group and gives plasminogen Group, every group each 10, and start to be administered, it is denoted as administration the 1st day, to plasminogen group mouse according to 1mg/0.1ml/ pcs/day of tail It is injected intravenously human plasminogen, gives the daily tail vein injection same volume solvent PBS buffer solution of vehicle control group mouse, it is continuous to inject 28 days.Sham-operation group mouse is not cooked drug treatment.MIA solution is prepared: MIA powder (Sigma, 57858-5G) is dissolved in physiology salt In water, concentration 10mg/ml, then 0.22 μm of membrane filtration, matching while using.29th day execution mouse materials left side knee joint in Fixed in PLP fixer, then decalcification three weeks in 10%EDTA, graded sucrose solutions wash, then paraffin embedding.Histotomy With a thickness of 5 μm, washed 1 time after slice dewaxing rehydration.PAP are irised out tissue, with 3% dioxygen water incubation 15 minutes, 0.01M PBS It washes 2 times, every time 5 minutes.5% normal sheep serum liquid (Vector laboratories, Inc., USA) is closed 30 minutes；When Between arrive after, reject sheep blood serum liquid, be added dropwise rabbit source it is anti-4 DEG C of II Collagen Type VI antibody (Abcam, ab34712) overnight incubation, 0.01M PBS 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 0.01M PBS is washed 2 times, every time 5 minutes.It develops the color by DAB kit (Vector laboratories, Inc., USA), haematoxylin is multiple after washing 3 times Dye 30 seconds, flowing water rinse 5 minutes.Gradient alcohol dehydration, dimethylbenzene is transparent and neutral gum mounting, slice are aobvious in 200 times of optics Micro- microscopic observation is taken pictures, and is handled with Image-Pro software and collected data.

Iodoacetic acid (monoiodoacetic acid, MIA) plays the role of destroying articular cartilage and surrounding synovial membrane ligament, will It is injected into articular cavity, can change the original stable state of articular cavity, can also cause intra-articular inflammatory reaction, thus change cartilage and The metabolism of subchondral bone destroys the stabilization of intra-articular environment, makes articular chondrocyte apoptosis, cartilage wear.Studies have shown that iodine second Acid is able to suppress articular chondrocyte metabolism, it is dead to cause cartilage cell, and then cartilage matrix is caused to be degraded, induces arthritis Cartilage changes and osteoarthritis.II Collagen Type VI is one of main component of cartilage matrix, in the maintenance of cartilage mechanical property It plays a significant role^[35]。

The results show that sham-operation group (Fig. 6 A) mouse knee joint has a certain amount of II Collagen Type VI (arrow logo), solvent is given Control group (Fig. 6 B) knee joint II Collagen Type VI amount and sham-operation group give plasminogen group (Fig. 6 C) knee joint II without significant difference Collagen Type VI amount is significantly more than to vehicle control group and sham-operation group, and the significant (* of average optical density quantitative analysis results statistical discrepancy Indicate P < 0.05) (Fig. 6 D).The result shows that plasminogen can promote osteoarthritis mouse knee cartilage to regenerate.

The osteoarthritis improvement result that 7 plasminogen of embodiment induces MIA

Take 8-10 weeks C57 male mice 25, weighing is randomly divided into two groups by weight, sham-operation group 5, model group 20 Only.The anesthesia of 3% yellow Jackets is injected intraperitoneally according to 50mg/kg weight in all mouse, and model group mouse sloughs left side after anesthesia Knee hair, 70% alcohol and iodine tincture disinfection, 90 degree of left side knee joint bending, along knee level operation syringe Needle (in order to avoid piercing through skin), the gap below discovery kneecap.With the slight pressure mark region, then vertically lift needle Head and syringe, insert the needle into marked region, across the kneecap tendon perpendicular to shin bone, intraarticular injection MIA physiology salt is water-soluble Liquid 0.1mg/10 μ l；10 μ l physiological saline of joint cavity injection on the left of sham-operation group, after injection, massaging knee, to ensure uniformly to divide Cloth^[34].Right side knee joint is not handled.After MIA is injected 3 days in articular cavity, model group mouse carries out pain test, is tied according to test Fruit mouse is randomly divided into two groups, to vehicle control group and gives plasminogen group, and every group each 10, and start to be administered, it is denoted as administration It is 1st day, small to vehicle control group to plasminogen group mouse according to 1mg/0.1ml/ pcs/day of tail vein injection human plasminogen The daily tail vein injection same volume solvent PBS buffer solution of mouse, continuous injection 28 days.Sham-operation group mouse is not cooked drug treatment.MIA Solution is prepared: MIA powder (Sigma, 57858-5G) is dissolved in physiological saline, concentration 10mg/ml, then 0.22 μm of filter membrane Filtering, matching while using.Execution mouse materials left side knee joint is fixed in PLP fixer within 29th day, then takes off in 10%EDTA Calcium three weeks, graded sucrose solutions washed, then paraffin embedding.5 μm of slice row Safranin O dyeing.Slice is aobvious in 40 times of optics Micro- microscopic observation is taken pictures, and carries out histological scores according to table 1.

The results show that sham-operation group (Fig. 7 A) mouse knee joint is with the presence of a certain amount of cartilage (arrow logo)；To solvent Control group (Fig. 7 B) knee cartilage amount significantly reduces and histological scores obviously increase, and illustrates MIA success inducing osteoarthritis It is scorching；To plasminogen group (Fig. 7 C) knee cartilage amount be significantly more than to vehicle control group, histological scores be also significantly lower than to Vehicle control group, and statistical difference is significant (* indicates P < 0.05) (Fig. 7 D).It should be the result shows that plasminogen can promote cartilage Damage of the osteoarthritis to joint is repaired in regeneration.

8 plasminogen of embodiment promotes the regeneration of osteoarthritis mouse knee cartilage

Taking 11-15 week old db/db female mice 18, weighing is randomly divided into two groups by weight, and sham-operation group 3, model Group 15.Model group mouse according to 50mg/kg weight be injected intraperitoneally 3% yellow Jackets anesthesia, slough back two sides hair with 70% alcohol and iodine tincture disinfection is cut skin, muscle of back and peritonaeum, is gently drawn white shiny cellulite with pincet Out outside notch, fractionation of fatty group can see ovary.Ovary lower end fallopian tubal is ligatured with silk thread first, then extracts ovary.Artificial hand Skin, muscle of back and peritonaeum are only cut after art group mouse anesthesia, do not extract ovary.Every after all operation mouse suture disinfections Intramuscular injection antibiotic (5000U/ is only), is subcutaneously injected anodyne (2mg/kg), continuous injection 3 days.After ovary is cut off 65 days, institute There is mouse peritoneal injection yellow Jackets anesthesia, sloughs right side knee hair, 70% alcohol and iodine tincture disinfection, right side knee 90 degree of arthrogryposis, accurate injection site is found, the needle (in order to avoid piercing through skin) of syringe is run along knee level, until It was found that the gap below kneecap.With the slight pressure mark region, then vertically lifts syringe needle and syringe, insert the needle into Marked region, across the kneecap tendon perpendicular to shin bone, it should imperceptible any resistance, model group right side of mice intraarticular injection 5 μ g/6 μ l II Collagenase Type normal saline solutions, 6 μ l physiological saline of sham-operation group right side of mice intraarticular injection^[36,37]。 After injection, massaging knee, to ensure to be uniformly distributed.Left side knee joint does not do injection treatment.After Collagenase injection 7 days, Suo You little Mouse weighing, model group mouse are randomly divided into two groups according to weight, to vehicle control group 7 and to plasminogen group 8, and start Administration is denoted as administration the 1st day, to plasminogen group mouse according to 1mg/0.1ml/ pcs/day of tail vein injection human plasminogen, gives The daily tail vein injection same volume solvent PBS buffer solution of vehicle control group mouse, continuous injection 28 days.Sham-operation group mouse is not cooked Drug treatment.Execution mouse materials left side knee joint is fixed in PLP fixer within 29th day, then decalcification three in 10%EDTA Week, graded sucrose solutions washing.Then paraffin embedding, 5 μm of slice row Safranin O dyeing.Slice is in 100 times of optical microphotographs It takes pictures under the microscope, and is handled with Image-Pro software and collect data.

The results show that femoral surface has a small amount of cartilage (arrow logo) at sham-operation group (Fig. 8 A) mouse knee joint, give Vehicle control group (Fig. 8 B) femoral surface cartilage amount and sham-operation group give plasminogen group (Fig. 8 C) femur table without significant difference Thin-skinned bone amount is significantly more than to vehicle control group and sham-operation group, and the significant (* of average optical density quantitative analysis results statistical discrepancy Indicate P < 0.05) (Fig. 8 D).The result shows that plasminogen can promote osteoarthritis mouse femur surface cartilage to regenerate.

9 plasminogen of embodiment mitigates MIA osteoarthritis rat arthritis pain

Taking SD rat 21, weight about 200g~250g, weighing is randomly divided into two groups by weight, and sham-operation group 5, mould Type group 16.All 3% yellow Jackets of rats by intraperitoneal injection (50mg/kg) anesthesia, model group rats slough arranged on left and right sides knee Joint hair, 70% alcohol and iodine tincture disinfection, find accurate injection site, along knee by 90 degree of knee joint bending The needle (in order to avoid piercing through skin) of level run syringe, the gap below discovery kneecap.With the slight pressure mark area Then domain vertically lifts syringe needle and syringe, insert the needle into marked region, across the kneecap tendon perpendicular to shin bone, it should feel Inject MIA normal saline solution 2mg/50 μ l respectively less than any resistance, in bilateral articular cavity；Sham-operation group arranged on left and right sides is closed Save 50 μ l physiological saline of intracavitary administration.After injection, massaging knee, to ensure to be uniformly distributed^[38].MIA solution is prepared: MIA powder (Sigma, 57858-5G) is dissolved in physiological saline, concentration 40mg/ml, 0.22 μm of membrane filtration, matching while using.MIA injection 3 After it, model group rats carry out pain test, are randomly divided into two groups according to test result, to vehicle control group and give plasminogen Group, every group each 8, and start to be administered, it is denoted as the 1st day.Give every rat of vehicle control group daily tail vein injection 0.7ml solvent (10mM sodium citrate buffer solution, 2% R-gene, 3% mannitol, PH7.4), to plasminogen group rat according to 7mg/ 0.7ml/ pcs/day of tail vein injection human plasminogen successive administration 7 days, pays attention to observation animal state during administration.A 8th day left side Right leg carries out pain test^[38]。

The results show that rats in sham-operated group pain threshold is relatively high；It has a pain in the leg pain threshold to vehicle control group rat or so It is substantially reduced, hence it is evident that be lower than sham-operation group；It has a pain in the leg pain threshold apparent increase to plasminogen group rat or so, and to solvent pair It is compared according to group, for left leg statistical discrepancy close to significant (P=0.08), right leg statistical discrepancy is significant (* indicates P < 0.05) (Fig. 9).Knot Fruit shows that plasminogen being capable of substantially reduced relieving pain in osteoarthritis.

10 plasminogen of embodiment mitigates osteoarthritis mouse arthritis pain

7 week old C57 female mices 19 are taken, are weighed, are randomly divided into two groups by weight, sham-operation group 3, model group 16 Only.The anesthesia of 3% yellow Jackets is injected intraperitoneally according to 50mg/kg weight in model group mouse, sloughs back two sides hair with 70% Alcohol and iodine tincture disinfection, cut skin, muscle of back and peritonaeum, gently cut the pull-out of white shiny cellulite with pincet Outside mouthful, fractionation of fatty group can see ovary.Ovary lower end fallopian tubal is ligatured with silk thread first, then extracts ovary.Sham-operation group Skin, muscle of back and peritonaeum are only cut after mouse anesthesia, do not extract ovary.Intramuscular injection after all operation mouse suture disinfections Antibiotic (5000U/ is only), is subcutaneously injected anodyne (2mg/kg), continuous injection 3 days.After ovary is cut off 65 days, all mouse abdomens Chamber injects yellow Jackets anesthesia, sloughs right side knee hair, 70% alcohol and iodine tincture disinfection, right side knee joint bending 90 degree, accurate injection site is found, along the needle (in order to avoid piercing through skin) of knee level operation syringe, until finding kneecap The gap of lower section.With the slight pressure mark region, then vertically lifts syringe needle and syringe, insert the needle into mark zone Domain, across the kneecap tendon perpendicular to shin bone, it should imperceptible any resistance, 5 μ g/6 μ of model group right side of mice intraarticular injection L II Collagenase Type normal saline solution, 6 μ l physiological saline of sham-operation group right side of mice intraarticular injection^[36,37].After injection, Massaging knee, to ensure to be uniformly distributed.Left side knee joint does not do injection treatment.After Collagenase injection 7 days, all mouse weighings, Model group mouse is randomly divided into two groups according to weight, to vehicle control group and gives plasminogen group, every group each 8, and start to Medicine is denoted as administration the 1st day, to plasminogen group mouse according to 1mg/0.1ml/ pcs/day of tail vein injection human plasminogen, to molten The daily tail vein injection same volume solvent PBS buffer solution of matchmaker's control group mice, continuous injection 28 days.Sham-operation group mouse do not do to Medicine processing.Right leg carries out pain test within 29th day^[34]。

The results show that sham-operation group mice pain threshold value is relatively high；It is obviously dropped to vehicle control group mice pain threshold value It is low, hence it is evident that be lower than sham-operation group mouse；It is significantly raised to plasminogen group mice pain threshold value, hence it is evident that be higher than to vehicle control group Mouse, and statistical discrepancy is close to significantly (P=0.09) (Figure 10).It should be the result shows that plasminogen can reduce osteoarthritis pain Bitterly.

11 plasminogen of embodiment promotes the regeneration of osteoarthritis Mouse cartilage

7 week old C57 female mices 19 are taken, are weighed, are randomly divided into two groups by weight, sham-operation group 3, model group 16 Only.The anesthesia of 3% yellow Jackets is injected intraperitoneally according to 50mg/kg weight in model group mouse, sloughs back two sides hair with 70% Alcohol and iodine tincture disinfection, cut skin, muscle of back and peritonaeum, gently cut the pull-out of white shiny cellulite with pincet Outside mouthful, fractionation of fatty group can see ovary.Ovary lower end fallopian tubal is ligatured with silk thread first, then extracts ovary.Sham-operation group Skin, muscle of back and peritonaeum are only cut after mouse anesthesia, do not extract ovary.Intramuscular injection after all operation mouse suture disinfections Antibiotic (5000U/ is only), is subcutaneously injected anodyne (2mg/kg), continuous injection 3 days.After ovary is cut off 65 days, all mouse abdomens Chamber injects yellow Jackets anesthesia, sloughs right side knee hair, 70% alcohol and iodine tincture disinfection, right side knee joint bending 90 degree, accurate injection site is found, along the needle (in order to avoid piercing through skin) of knee level operation syringe, until finding kneecap The gap of lower section.With the slight pressure mark region, then vertically lifts syringe needle and syringe, insert the needle into mark zone Domain, across the kneecap tendon perpendicular to shin bone, it should imperceptible any resistance, 5 μ g/6 μ of model group right side of mice intraarticular injection L II Collagenase Type normal saline solution, 6 μ l physiological saline of sham-operation group right side of mice intraarticular injection^[36,37].After injection, Massaging knee, to ensure to be uniformly distributed.Left side knee joint does not do injection treatment.After Collagenase injection 7 days, all mouse weighings, Model group mouse is randomly divided into two groups according to weight, to vehicle control group and gives plasminogen group, every group each 8, and start to Medicine is denoted as administration the 1st day, to plasminogen group mouse according to 1mg/0.1ml/ pcs/day of tail vein injection human plasminogen, to molten The daily tail vein injection same volume solvent PBS buffer solution of matchmaker's control group mice, continuous injection 28 days.Sham-operation group mouse do not do to Medicine processing.Execution mouse materials two sides knee joint is fixed in PLP fixer within 29th day, then decalcification three weeks in 10%EDTA, Graded sucrose solutions washing.Then paraffin embedding, 5 μm of slice row Safranin O dyeing.Slice is under 100 times of optical microscopies Observation is taken pictures, and is handled with Image-Pro software and collected data.

The results show that there are a certain amount of joint is soft for knee joint femoral and femoral surface on the left of sham-operation group (Figure 11 A, D) Bone (arrow logo), it is unobvious to vehicle control group (Figure 11 B, E) shin bone and femoral surface cartilage amount and sham-operation group difference, it gives Plasminogen group (Figure 11 C, F) cartilage amount is significantly more than to vehicle control group and sham-operation group.Illustrate that plasminogen can promote bone Arthritis model mouse Regeneration of Articular Cartilage.

12 plasminogen of embodiment inhibits osteoarthritis mouse bone resorption

7 week old C57 female mices 19 are taken, are weighed, are randomly divided into two groups by weight, sham-operation group 3, model group 16 Only.The anesthesia of 3% yellow Jackets is injected intraperitoneally according to 50mg/kg weight in model group mouse, sloughs back two sides hair with 70% Alcohol and iodine tincture disinfection, cut skin, muscle of back and peritonaeum, gently cut the pull-out of white shiny cellulite with pincet Outside mouthful, fractionation of fatty group can see ovary.Ovary lower end fallopian tubal is ligatured with silk thread first, then extracts ovary.Sham-operation group Skin, muscle of back and peritonaeum are only cut after mouse anesthesia, do not extract ovary.Intramuscular injection after all operation mouse suture disinfections Antibiotic (5000U/ is only), is subcutaneously injected anodyne (2mg/kg), continuous injection 3 days.After ovary is cut off 65 days, all mouse abdomens Chamber injects yellow Jackets anesthesia, sloughs right side knee hair, 70% alcohol and iodine tincture disinfection, right side knee joint bending 90 degree, accurate injection site is found, along the needle (in order to avoid piercing through skin) of knee level operation syringe, until finding kneecap The gap of lower section.With the slight pressure mark region, then vertically lifts syringe needle and syringe, insert the needle into mark zone Domain, across the kneecap tendon perpendicular to shin bone, it should imperceptible any resistance, 5 μ g/6 μ of model group right side of mice intraarticular injection L II Collagenase Type normal saline solution, 6 μ l physiological saline of sham-operation group right side of mice intraarticular injection^[36,37].After injection, Massaging knee, to ensure to be uniformly distributed.Left side knee joint does not do injection treatment.After Collagenase injection 7 days, all mouse weighings, Model group mouse is randomly divided into two groups according to weight, to vehicle control group and gives plasminogen group, every group each 8, and start to Medicine is denoted as administration the 1st day, to plasminogen group mouse according to 1mg/0.1ml/ pcs/day of tail vein injection human plasminogen, to molten The daily tail vein injection same volume solvent PBS buffer solution of matchmaker's control group mice, continuous injection 28 days.Sham-operation group mouse do not do to Medicine processing.Execution mouse materials left side knee joint is fixed in PLP fixer within 29th day, then decalcification three weeks in 10%EDTA, Graded sucrose solutions washing, is sliced 5 μm, and dewax rehydration, carries out acid phosphatase (TRAP) dyeing.

Acid phosphatase (TRAP) is the specificity marker enzyme of osteoclast, and osteoclast is that the major function of bone resorption is thin Born of the same parents.The results show that there are a certain amount of acid phosphatase (arrow logos) for sham-operation group (Figure 12 A) knee joint；To Vehicle controls Group (Figure 12 B) knee joint acid phosphatase increases, hence it is evident that is more than sham-operation group；Give plasminogen group (Figure 12 C) knee joint acidity Phosphatase is considerably less than to vehicle control group, and statistical discrepancy is significant (* indicates P < 0.05) (Figure 12 D).It should be the result shows that fibrinolytic Proenzyme can reduce osteoarthritic knee joint acid phosphatase, reduce osteoclast activity, inhibit bone resorption.

13 plasminogen of embodiment promotes 9 Positive Stem Cells quantity of MIA osteoarthritic knee joint ligament tibia end Sox to increase

Take 8-10 weeks C57 male mice 25, weighing is randomly divided into two groups by weight, sham-operation group 5, model group 20 Only.The anesthesia of 3% yellow Jackets is injected intraperitoneally according to 50mg/kg weight in all mouse, and model group mouse sloughs left side after anesthesia Knee hair, 70% alcohol and iodine tincture disinfection, 90 degree of left side knee joint bending, along knee level operation syringe Needle (in order to avoid piercing through skin), the gap below discovery kneecap.With the slight pressure mark region, then vertically lift needle Head and syringe, insert the needle into marked region, across the kneecap tendon perpendicular to shin bone, intraarticular injection MIA physiology salt is water-soluble Liquid 0.1mg/10 μ l；10 μ l physiological saline of joint cavity injection on the left of sham-operation group, after injection, massaging knee, to ensure uniformly to divide Cloth^[34].Right side knee joint is not handled.After MIA is injected 3 days in articular cavity, model group mouse carries out pain test, is tied according to test Fruit mouse is randomly divided into two groups, to vehicle control group and gives plasminogen group, and every group each 10, and start to be administered, it is denoted as administration It is 1st day, small to vehicle control group to plasminogen group mouse according to 1mg/0.1ml/ pcs/day of tail vein injection human plasminogen The daily tail vein injection same volume solvent PBS buffer solution of mouse, continuous injection 28 days.Sham-operation group mouse is not cooked drug treatment.MIA Solution is prepared: MIA powder (Sigma, 57858-5G) is dissolved in physiological saline, concentration 10mg/ml, then 0.22 μm of filter membrane Filtering, matching while using.Execution mouse materials left side knee joint is fixed in PLP fixer within 29th day, then takes off in 10%EDTA Calcium three weeks, graded sucrose solutions washed, then paraffin embedding.Histotomy is washed 1 time after slice dewaxing rehydration with a thickness of 5 μm. PAP are irised out tissue, and with 3% dioxygen water incubation 15 minutes, 0.01M PBS was washed 2 times, every time 5 minutes.5% normal sheep serum Liquid (Vector laboratories, Inc., USA) is closed 30 minutes；After time arrives, it is anti-that rabbit source is added dropwise in reject sheep blood serum liquid 4 DEG C of 9 antibody of Sox (Abcam, ab185966) overnight incubations, 0.01M PBS are washed 2 times, every time 5 minutes.Goat anti-rabbit igg (HRP) antibody (Abcam) (secondary antibody) is incubated at room temperature 1 hour, and 0.01M PBS is washed 2 times, every time 5 minutes.By DAB kit (Vector laboratories, Inc., USA) colour developing, haematoxylin is redyed 30 seconds after washing 3 times, and flowing water rinses 5 minutes.Gradient Dehydration of alcohol, dimethylbenzene is transparent and neutral gum mounting, and slice is in 100 (A-C) and 400 (D-F) times of optical microphotograph microscopic observation It takes pictures, and is handled with Image-Pro software and collect data.

Sox 9 is the key transcription factor in cartilage development forming process, determines the aggregation of mescenchymal stem cell and to soft Bone cell differentiation, to cartilage reach maturity and the processes such as injury repair play important adjustment effect^[39]。

The results show that there are a certain amount of 9 Positive Stem Cells (arrows of Sox for sham-operation group (Figure 13 A, D) knee joint tibial Mark), it is significantly reduced to 9 Positive Stem Cells quantity of vehicle control group (Figure 13 B, E) knee joint tibial Sox, gives plasminogen group 9 Positive Stem Cells quantity of (Figure 13 C, F) knee joint tibial Sox is significantly more than to vehicle control group.It should be the result shows that plasminogen It can promote the increase of 9 Positive Stem Cells quantity of osteoarthritic knee joint ligament tibia end Sox, repair osteoarthritis to kneed damage.

14 plasminogen of embodiment improves MIA osteoarthritis mouse gonarthromeningitis disease

Take 8-10 weeks C57 male mice 25, weighing is randomly divided into two groups by weight, sham-operation group 5, model group 20 Only.The anesthesia of 3% yellow Jackets is injected intraperitoneally according to 50mg/kg weight in all mouse, and model group mouse sloughs left side after anesthesia Knee hair, 70% alcohol and iodine tincture disinfection, 90 degree of left side knee joint bending, along knee level operation syringe Needle (in order to avoid piercing through skin), the gap below discovery kneecap.With the slight pressure mark region, then vertically lift needle Head and syringe, insert the needle into marked region, across the kneecap tendon perpendicular to shin bone, intraarticular injection MIA physiology salt is water-soluble Liquid 0.1mg/10 μ l；10 μ l physiological saline of joint cavity injection on the left of sham-operation group, after injection, massaging knee, to ensure uniformly to divide Cloth^[34].Right side knee joint is not handled.After MIA is injected 3 days in articular cavity, model group mouse carries out pain test, is tied according to test Fruit mouse is randomly divided into two groups, to vehicle control group and gives plasminogen group, and every group each 10, and start to be administered, it is denoted as administration It is 1st day, small to vehicle control group to plasminogen group mouse according to 1mg/0.1ml/ pcs/day of tail vein injection human plasminogen The daily tail vein injection same volume solvent PBS buffer solution of mouse, continuous injection 28 days.Sham-operation group mouse is not cooked drug treatment.MIA Solution is prepared: MIA powder (Sigma, 57858-5G) is dissolved in physiological saline, concentration 10mg/ml, then 0.22 μm of filter membrane Filtering, matching while using.Execution mouse materials left side knee joint is fixed in PLP fixer within 29th day, then takes off in 10%EDTA Calcium three weeks, graded sucrose solutions washed, then paraffin embedding (muscle for retaining knee joint periphery).Histotomy with a thickness of 5 μm, Slice dewaxing rehydration simultaneously uses haematoxylin and eosin stains (H&E dyeing), and 1% hydrochloride alcohol breaks up, and ammonium hydroxide returns indigo plant, and alcohol gradient Dehydration, dimethylbenzene is transparent, neutral gum mounting, and slice is in 400 times of optical microphotographs microscopic observations.

The results show that sham-operation group (Figure 14 A) knee joint synovial membrane has no apparent inflammatory cell infiltration, vehicle control group is given The visible apparent inflammatory cell infiltration (arrow logo) of (Figure 14 B) knee joint synovial membrane, it is sliding to plasminogen group (Figure 14 C) knee joint Film inflammation cellular infiltration is considerably less than to vehicle control group.It should be the result shows that plasminogen can improve osteoarthritic knee joint synovial membrane Inflammation.

(people) plasminogen used in all of above embodiment comes from people contributor blood plasma, is based on document^[40-42]It is retouched The method stated simultaneously carries out process optimization, and gained is purified from human plasma.Purity > 95% of plasminogen monomer.

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Sequence table

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

<120>a kind of method and drug of prevention or treatment osteoarthritis

<130> PB00391

<150> 201711350845.3

<151> 2017-12-15

<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

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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>nucleic acid sequence of the natural plasminogen containing signal peptide (deriving from swiss prot)

<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>amino acid sequence of the natural plasminogen containing signal peptide (deriving from swiss prot)

<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- plasminogen) nucleic acid sequence

<400> 5

aaagtgtatc tctcagagtg caagactggg aatggaaaga actacagagg gacgatgtcc 60

aaaacaaaaa atggcatcac ctgtcaaaaa tggagttcca cttctcccca cagacctaga 120

ttctcacctg ctacacaccc ctcagaggga ctggaggaga actactgcag gaatccagac 180

aacgatccgc aggggccctg gtgctatact actgatccag aaaagagata tgactactgc 240

gacattcttg agtgtgaaga ggaatgtatg cattgcagtg gagaaaacta tgacggcaaa 300

atttccaaga ccatgtctgg actggaatgc caggcctggg actctcagag cccacacgct 360

catggataca ttccttccaa atttccaaac aagaacctga agaagaatta ctgtcgtaac 420

cccgataggg agctgcggcc ttggtgtttc accaccgacc ccaacaagcg ctgggaactt 480

tgtgacatcc cccgctgcac aacacctcca ccatcttctg gtcccaccta ccagtgtctg 540

aagggaacag gtgaaaacta tcgcgggaat gtggctgtta ccgtgtccgg gcacacctgt 600

cagcactgga gtgcacagac ccctcacaca cataacagga caccagaaaa cttcccctgc 660

aaaaatttgg atgaaaacta ctgccgcaat cctgacggaa aaagggcccc atggtgccat 720

acaaccaaca gccaagtgcg gtgggagtac tgtaagatac cgtcctgtga ctcctcccca 780

gtatccacgg aacaattggc tcccacagca ccacctgagc taacccctgt ggtccaggac 840

tgctaccatg gtgatggaca gagctaccga ggcacatcct ccaccaccac cacaggaaag 900

aagtgtcagt cttggtcatc tatgacacca caccggcacc agaagacccc agaaaactac 960

ccaaatgctg gcctgacaat gaactactgc aggaatccag atgccgataa aggcccctgg 1020

tgttttacca cagaccccag cgtcaggtgg gagtactgca acctgaaaaa atgctcagga 1080

acagaagcga gtgttgtagc acctccgcct gttgtcctgc ttccagatgt agagactcct 1140

tccgaagaag actgtatgtt tgggaatggg aaaggatacc gaggcaagag ggcgaccact 1200

gttactggga cgccatgcca ggactgggct gcccaggagc cccatagaca cagcattttc 1260

actccagaga caaatccacg ggcgggtctg gaaaaaaatt actgccgtaa ccctgatggt 1320

gatgtaggtg gtccctggtg ctacacgaca aatccaagaa aactttacga ctactgtgat 1380

gtccctcagt gtgcggcccc ttcatttgat tgtgggaagc ctcaagtgga gccgaagaaa 1440

tgtcctggaa gggttgtagg ggggtgtgtg gcccacccac attcctggcc ctggcaagtc 1500

agtcttagaa caaggtttgg aatgcacttc tgtggaggca ccttgatatc cccagagtgg 1560

gtgttgactg ctgcccactg cttggagaag tccccaaggc cttcatccta caaggtcatc 1620

ctgggtgcac accaagaagt gaatctcgaa ccgcatgttc aggaaataga agtgtctagg 1680

ctgttcttgg agcccacacg aaaagatatt gccttgctaa agctaagcag tcctgccgtc 1740

atcactgaca aagtaatccc agcttgtctg ccatccccaa attatgtggt cgctgaccgg 1800

accgaatgtt tcatcactgg ctggggagaa acccaaggta cttttggagc tggccttctc 1860

aaggaagccc agctccctgt gattgagaat aaagtgtgca atcgctatga gtttctgaat 1920

ggaagagtcc aatccaccga actctgtgct gggcatttgg ccggaggcac tgacagttgc 1980

cagggtgaca gtggaggtcc tctggtttgc ttcgagaagg acaaatacat tttacaagga 2040

gtcacttctt ggggtcttgg ctgtgcacgc cccaataagc ctggtgtcta tgttcgtgtt 2100

tcaaggtttg ttacttggat tgagggagtg atgagaaata attaa 2145

<210> 6

<211> 714

<212> PRT

<213>LYS77-PLG(Lys- plasminogen) amino 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- plasminogen) nucleic acid sequence

<400> 7

gagcctctgg atgactatgt gaatacccag ggggcttcac tgttcagtgt cactaagaag 60

cagctgggag caggaagtat agaagaatgt gcagcaaaat gtgaggagga cgaagaattc 120

acctgcaggg cattccaata tcacagtaaa gagcaacaat gtgtgataat ggctgaaaac 180

aggaagtcct ccataatcat taggatgaga gatgtagttt tatttgaaaa gaaagtgtat 240

ctctcagagt gcaagactgg gaatggaaag aactacagag ggacgatgtc caaaacaaaa 300

aatggcatca cctgtcaaaa atggagttcc acttctcccc acagacctag attctcacct 360

gctacacacc cctcagaggg actggaggag aactactgca ggaatccaga caacgatccg 420

caggggccct ggtgctatac tactgatcca gaaaagagat atgactactg cgacattctt 480

gagtgtgaag aggcggcccc ttcatttgat tgtgggaagc ctcaagtgga gccgaagaaa 540

tgtcctggaa gggttgtagg ggggtgtgtg gcccacccac attcctggcc ctggcaagtc 600

agtcttagaa caaggtttgg aatgcacttc tgtggaggca ccttgatatc cccagagtgg 660

gtgttgactg ctgcccactg cttggagaag tccccaaggc cttcatccta caaggtcatc 720

ctgggtgcac accaagaagt gaatctcgaa ccgcatgttc aggaaataga agtgtctagg 780

ctgttcttgg agcccacacg aaaagatatt gccttgctaa agctaagcag tcctgccgtc 840

atcactgaca aagtaatccc agcttgtctg ccatccccaa attatgtggt cgctgaccgg 900

accgaatgtt tcatcactgg ctggggagaa acccaaggta cttttggagc tggccttctc 960

aaggaagccc agctccctgt gattgagaat aaagtgtgca atcgctatga gtttctgaat 1020

ggaagagtcc aatccaccga actctgtgct gggcatttgg ccggaggcac tgacagttgc 1080

cagggtgaca gtggaggtcc tctggtttgc ttcgagaagg acaaatacat tttacaagga 1140

gtcacttctt ggggtcttgg ctgtgcacgc cccaataagc ctggtgtcta tgttcgtgtt 1200

tcaaggtttg ttacttggat tgagggagtg atgagaaata attaa 1245

<210> 8

<211> 414

<212> PRT

<213>delta-plg(delta- plasminogen) amino 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>the small plasminogen of Mini-plg() 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>the small plasminogen of Mini-plg() 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>nucleic acid sequence in serine protease (structure) 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>amino acid sequence in serine protease (structure) 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 treating osteoarthritis, including administration a effective amount of plasminogen of subject.

2. method of claim 1, the plasminogen increases the amount of articular cartilage and/or promotes articular cartilage damage reparation.

3. the method for claims 1 or 2, the plasminogen improves joint synovitis disease situation.

4. the method for any one of claim 1-3, the plasminogen promotes the subchondral bone bone remoulding in joint.

5. the method for any one of claim 1-4, inflammatory conditions, pain and/or the improvement that the plasminogen improves joint are closed Save function.

6. the method for any one of claim 1-5, wherein the plasminogen mitigates arthroncus and pain.

7. a kind of method for promoting osteoarthritis subject Regeneration of Articular Cartilage, including administration a effective amount of fibrinolysin of subject It is former.

8. a kind of method for promoting the reparation of subject's joint injury, including administration a effective amount of plasminogen of subject.

9. method for claim 8, the plasminogen promotes Regeneration of Articular Cartilage and/or subchondral bone bone remoulding.

10. the method for claim 8 or 9, wherein the subject is osteoarthritis subject.