TW202408566A - A method of treating nerve damage and related conditions - Google Patents

Abstract

The present invention relates to a method for treating nerve damage and related diseases, comprising administering a therapeutically effective amount of a fibrinolytic enzyme activation pathway component to a subject. The present invention also relates to a drug, a drug composition, a product, and a reagent kit containing the fibrinolytic enzyme activation pathway component for treating the above-mentioned diseases.

Description

A method of treating nerve damage and related conditions

The present invention relates to a method for treating nerve damage and related diseases, such as paralysis, comprising administering an effective amount of a component of a fibrinolytic enzyme activation pathway or a related compound, such as fibrinolytic enzyme, to a subject to repair damaged nerves and improve clinical symptoms and signs.

The nervous system includes the central nervous system (brain, spinal cord) and the peripheral nervous system (peripheral nervous tissue). Nervous system damage is caused by a variety of factors: (1) physical damage, which directly causes damage to the nervous tissue at the site of injury, such as brain nervous tissue damage or spinal cord damage caused by trauma; (2) temporary or permanent ischemia or hypoxia of part of the nervous system, such as brain nervous tissue damage caused by stroke or cerebral embolism; (3) exposure to neurotoxins, such as chemical agents used to treat cancer; (4) chronic metabolic diseases, such as peripheral nerve damage caused by diabetes or renal dysfunction.

Spinal cord nerve injury is a general term for pathological conditions in which the structure and function of the spinal cord nerves are destroyed or damaged due to trauma or disease. For example, spinal cord trauma, spinal compression fractures, transverse myelitis, spinal cord tumors, vertebral tuberculosis and syringomyelia can cause spinal cord injury and paralysis. Paralysis caused by transverse lesions above the cervical enlargement of the spinal cord is high paralysis, and paralysis caused by spinal cord injury below the third thoracic vertebra is bilateral lower limb paralysis. In the acute stage of spinal cord injury, the bilateral limbs below the injury plane lose sensation, movement, reflexes, etc., and the bladder and anal sphincter function is lost, leading to spinal shock. There is currently no ideal treatment for nerve damage, including spinal cord injury.

The present invention has found that fibrosinogen can significantly promote the repair of nerve damage, promote the remyelination of damaged nerves, and improve related symptoms.

Specifically, the present invention relates to the following:

1. A method of treating nerve damage, comprising administering to a subject a therapeutically effective amount of one or more compounds selected from: components of the plasminogen activation pathway, capable of directly activating plasminogen or by activating plasminogen. Compounds that indirectly activate plasminogen by upstream components of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin, and are capable of upregulating plasminogen or plasminogen Compounds expressed by activators, plasminogen analogs, plasmin analogs, tPA or uPA analogs and antagonists of fibrinolysis inhibitors.

2. The method of claim 1, wherein the components of the plasminogen activation pathway are selected from the group consisting of plasminogen, recombinant human plasmin, Lys-plasminogen, Glu-fiber. Proteinogen, plasmin, plasminogen and plasmin variants and analogs containing one or more kringle domains and protease domains of plasminogen and plasmin, mini-plasminogen, mini-plasmin, micro-plasminogen, micro-plasmin, delta-plasminogen , delta-plasmin (delta-plasmin), plasminogen activator, tPA and uPA.

3. The method of claim 1, wherein the antagonist of the fibrinolytic inhibitor is an inhibitor of PAI-1, leukocyte antigen C1 inhibitor, α2 antifibrinolytic enzyme or α2 macroglobulin, such as an antibody.

4. A method as claimed in any one of claims 1 to 3, wherein the nerve damage comprises spinal cord nerve damage, in particular compressive spinal cord nerve damage.

In some embodiments, the nerve injury is central nerve injury (e.g., spinal nerve injury) or peripheral nerve injury.

5. The method of claim 4, wherein the spinal cord nerve injury includes spinal cord nerve injury caused by spinal compression fracture, transverse myelitis, spinal cord tumor, vertebral tuberculosis or syringomyelia.

6. The method of any of claims 1-5, comprising treating a condition associated with neurological damage.

7. The method of claim 6, wherein the nerve damage-related condition comprises paralysis.

8. The method of claim 7, wherein the paralysis is high paralysis or bilateral lower limb paralysis.

9. The method of any one of claims 1-8, wherein the compound promotes the repair of damaged nerves.

10. The method of any one of claims 1-9, wherein the compound promotes remyelination of nerves.

11. The method of any one of claims 1-10, wherein the compound promotes expression of spinal cord neurofilament proteins.

12. The method of any one of claims 1-11, wherein the compound promotes recovery of sensory nerve function.

13. The method of claim 12, wherein the compound promotes restoration of pain sensation.

14. The method of any one of claims 1-13, wherein the compound is plasminogen.

In some embodiments, the present application relates to one or more activities or functions of plasminogen selected from the group consisting of: promoting the regeneration of nerve myelin sheaths, promoting the expression of spinal neurofilament proteins and nerve fiber regeneration, promoting the recovery of sensory nerve function, or It can restore motor nerve function, promote pain recovery, promote the expression of synaptophysin, and promote the recovery of Nissl body levels in damaged nerve cells.

15. In some embodiments, the plasminogen described above has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to Sequence 2, and Still possess plasminogen activity (e.g., proteolytic activity or lysine-binding activity).

16. In some embodiments, the above-mentioned profibrinolytic enzyme is a protein that contains a profibrinolytic enzyme active fragment and still has profibrinolytic enzyme activity (such as proteolytic activity or lysine binding activity). In some embodiments, the profibrinolytic enzyme active fragment has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the profibrinolytic enzyme active fragment shown in SEQ ID NO: 14, or has 100% amino acid sequence identity with the profibrinolytic enzyme active fragment shown in SEQ ID NO: 14.

17. The method of any one of claims 1-14, wherein the plasminogen is selected from Glu-plasminogen, Lys-plasminogen, small plasminogen, microplasminogen, delta- Plasminogen or their variants that retain plasminogen activity (e.g., proteolytic activity or lysine-binding activity).

18. The method of any one of claims 1 to 14, wherein the fibrinolysinogen is natural or synthetic full-length human fibrinolysinogen, or a variant or fragment thereof that still retains fibrinolysinogen activity (eg, proteolytic activity, or lysine binding activity).

In any of the above embodiments of the invention, the plasminogen may have at least 75%, 80%, 85%, 90%, 95%, 96%, 97 %, 98% or 99% sequence identity and still possess plasminogen activity (e.g., proteolytic activity or lysine-binding activity). In some embodiments, the plasminogen is based on sequence 2, 6, 8, 10 or 12, adding, deleting and/or replacing 1-100, 1-90, 1-80, 1-70 ,1-60,1-50,1-45,1-40,1-35,1-30,1-25,1-20,1-15,1-10,1-5,1-4,1 -3, 1-2, 1 amino acid proteins that still have plasminogen activity, such as proteolytic activity or lysine-binding activity, such as proteolytic activity. In some embodiments, the plasminogen comprises or has the amino acid sequence set forth in Sequence 2, 6, 8, 10, or 12.

In some embodiments, the fibrinolysinogen is a protein comprising a fibrinolysinogen active fragment and still having fibrinolysinogen activity, such as proteolytic activity, or lysine binding activity, or proteolytic activity and lysine binding activity. In some embodiments, the fibrinolysinogen is selected from Glu-fibrinolysinogen, Lys-fibrinolysinogen, minifibrinolysinogen, microfibrinolysinogen, delta-fibrinolysinogen or variants thereof retaining fibrinolysinogen activity (such as proteolytic activity or lysine binding activity). In some embodiments, the fibrinolysinogen is natural or synthetic human fibrinolysinogen, or variants or fragments thereof still retaining fibrinolysinogen activity (such as proteolytic activity or lysine binding activity). In some embodiments, the fibrinolysinogen is a human fibrinolysinogen ortholog from primates or rodents or a variant or fragment thereof that still retains fibrinolysinogen activity. In some embodiments, the amino acids of the fibrinolysinogen are shown in sequence 2, 6, 8, 10 or 12. In some embodiments, the fibrinolysinogen is human native fibrinolysinogen.

In some embodiments, the subject is human. In some embodiments, the subject lacks or lacks plasminogen. In some embodiments, the deficiency or deletion is congenital, secondary, and/or localized.

In some embodiments, the drug composition comprises a pharmaceutically acceptable carrier and a fibronectin for use in the aforementioned method. In some embodiments, the kit may be a preventive or therapeutic kit comprising: (i) fibronectin for use in the aforementioned method and (ii) means for delivering the fibronectin to the subject. In some embodiments, the means is a syringe or a vial. In some embodiments, the kit further comprises a label or instructions for use, which indicates that the fibronectin is administered to the subject to implement any of the aforementioned methods.

In some embodiments, the article of manufacture comprises: a container containing a label; and (i) plasminogen for use in the foregoing method or a pharmaceutical composition comprising plasminogen, wherein the label indicates that the plasminogen is to be administered The lysinogen or composition is administered to the subject to perform any of the foregoing methods.

In some embodiments, the kit or product further comprises one or more additional components or containers containing other drugs.

In some embodiments of the foregoing methods, the plasminogen is administered systemically or locally, preferably by intravenous, intramuscular, subcutaneous or intrathecal administration of plasminogen for treatment. In some embodiments of the foregoing methods, the plasminogen is administered in combination with an appropriate polypeptide carrier or stabilizer. In some embodiments of the aforementioned methods, the plasminogen is administered at 0.0001-2000 mg/kg, 0.001-800 mg/kg, 0.01-600 mg/kg, 0.1-400 mg/kg, 1-200 mg/kg, 1-100mg/kg, 10-100mg/kg (calculated per kilogram of body weight) or 0.0001-2000mg/cm2, 0.001-800 mg/cm2, 0.01-600 mg/cm2, 0.1-400 mg/cm2, 1-200 mg /cm2, 1-100 mg/cm2, 10-100 mg/cm2 (calculated per square centimeter of body surface area), preferably repeated at least once, preferably at least daily.

The present invention explicitly covers all combinations of technical features belonging to the embodiments of the present invention, and the technical solutions after these combinations have been explicitly disclosed in this application, just as the above-mentioned technical solutions have been individually and explicitly disclosed. In addition, the present invention also explicitly covers the combination between various embodiments and their elements, and the technical solutions after these combinations are explicitly disclosed in this article.

Fibrinolytic system, also known as fibrinolytic system, is a system composed of a series of chemical substances involved in the process of fibrinolysis (fibrinolysis), mainly including fibrinolytic enzyme (plasminogen), plasmin , plasminogen activator, fibrinolysis inhibitor. Plasminogen activators include tissue plasminogen activator (t-PA) and urokinase plasminogen activator (u-PA). t-PA is a serine protease synthesized by vascular endothelial cells. t-PA activates plasminogen, and this process is mainly performed on fibrin; urokinase-type plasminogen activator (u-PA) is produced by renal tubular epithelial cells and vascular endothelial cells and can directly activate plasminogen. and does not require fibrin as a cofactor. Plasminogen (PLG) is synthesized by the liver. When blood coagulates, a large amount of PLG is adsorbed on the fibrin mesh. Under the action of t-PA or u-PA, it is activated into plasmin, which promotes fibrinolysis. Plasmin (PL) is a serine protease with the following functions: degrades fibrin and fibrinogen; hydrolyzes various coagulation factors V, VIII, X, VII, XI, II, etc.; converts plasminogen into plasmin; Hydrolyzed complement, etc. Fibrinolysis inhibitors: including plasminogen activator inhibitors (PAI) and α2 antiplasmin (α2-AP). PAI mainly has two forms, PAI-1 and PAI-2, which can specifically bind to t-PA in a 1:1 ratio, thereby inactivating it and activating PLG at the same time. α2-AP is synthesized by the liver and combines with PL in a 1:1 ratio to form a complex, inhibiting PL activity; FXXIII binds α2-AP to fibrin with a covalent bond, weakening the sensitivity of fibrin to the action of PL. Substances that inhibit the activity of the fibrinolytic system in the body: PAI-1, complement C1 inhibitor; α2 antiplasmin; α2 macroglobulin.

The term "components of the plasminogen activation pathway" of the present invention encompasses:

1. Plasminogen, Lys-plasminogen, Glu-plasminogen, micro-plasminogen, delta-plasminogen; their variants or analogs;

2. Plasmin and its variants or analogs; and

3. Plasminogen activators, such as tPA and uPA and tPA or uPA variants and analogs containing one or more domains of tPA or uPA (such as one or more kringle domains and proteolytic domains) .

"Variants" of the above-mentioned fibrinolysinogen, fibrinolysin, tPA and uPA include all naturally occurring human genetic variants and other mammalian forms of these proteins, as well as proteins that still have fibrinolysinogen, fibrinolysin, tPA or uPA activity by adding, deleting and/or substituting, for example, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2, 1 amino acids. For example, "variants" of fibrinolysin, fibrinolysin, tPA, and uPA include mutant variants of these proteins obtained by, e.g., 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2, 1 conservative amino acid substitutions.

"Plasminogen variants" of the invention encompass at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99 of sequence 2, 6, 8, 10 or 12. % sequence identity and still possess plasminogen activity (such as proteolytic activity, or lysine-binding activity, or proteolytic activity and lysine-binding activity). For example, the "plasminogen variant" of the present invention can be based on sequence 2, 6, 8, 10 or 12, with 1-100, 1-90, 1-80, 1- added, deleted and/or substituted. 70, 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, Proteins that contain 1-3, 1-2, or 1 amino acid and still have plasminogen activity (such as proteolytic activity, or lysine-binding activity, or proteolytic activity and lysine-binding activity). In particular, plasminogen variants of the present invention include all naturally occurring human genetic variants as well as other mammalian forms of these proteins, as well as those modified by conservative amino acid substitutions such as 1-100, 1-90, 1-80, 1- 70, 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, Mutated variants of these proteins obtained from 1-3, 1-2, and 1 amino acid.

The plasminogen of the invention may be an ortholog of human plasminogen from primates or rodents or it may still retain plasminogen activity (e.g., proteolytic activity, or lysine-binding activity, or proteolytic activity and lysine-binding activity), such as the plasminogen shown in sequence 2, 6, 8, 10 or 12, such as the human native plasminogen shown in sequence 2.

The "analogs" of the above-mentioned fibrinolysinogen, fibrinolysin, tPA and uPA include compounds that provide substantially similar effects to fibrinolysinogen, fibrinolysin, tPA or uPA, respectively.

"Variants" and "analogs" of plasminogen, plasmin, tPA and uPA as described above encompass fibers containing one or more domains (eg, one or more kringle domains and proteolytic domains) "Variants" and "analogues" of proteases, plasmin, tPA and uPA. For example, "variants" and "analogs" of plasminogen encompass plasminogens that include one or more plasminogen domains (eg, one or more kringle domains and proteolytic domains) Variants and analogs, such as mini-plasminogen. "Variants" and "analogs" of plasmin encompass plasmin "variants" that include one or more plasmin domains (eg, one or more kringle domains and proteolytic domains) and "analogs" such as mini-plasmin and delta-plasmin.

Whether the above-mentioned "variants" or "analogs" of fibrinolysinogen, fibrinolysin, tPA or uPA have the activity of fibrinolysinogen, fibrinolysin, tPA or uPA, respectively, or whether they provide substantially similar effects to fibrinolysinogen, fibrinolysin, tPA or uPA, respectively, can be detected by methods known in the art, for example, by measuring the level of activated fibrinolysin activity based on enzymography, ELISA (enzyme-linked immunosorbent assay) and FACS (fluorescence-activated cell sorting method), for example, it can be measured by referring to the method selected from the following literature: Ny, A., Leonardsson, G., Hagglund, A.C, Hagglof, P., Ploplis, V.A., Carmeliet, P. and Ny, T. (1999). Ovulation inplasminogen-deficient mice. Endocrinology 140，5030-5035；Silverstein RL, Leung LL, Harpel PC, Nachman RL (November 1984). "Complex formation of platelet thrombospondin with plasminogen. Modulation of activation by tissue activator". J. Clin. Invest. 74 (5): 1625–33；Gravanis I, Tsirka SE (February 2008). "Tissue-type plasminogen activator as a therapeutic target in stroke". Expert Opinion on Therapeutic Targets. 12 (2): 159–70；Geiger M, Huber K, Wojta J, Stingl L, Espana F, Griffin JH, Binder BR (Aug 1989). "Complex formation between urokinase and plasma protein C inhibitor in vitro and in vivo". Blood. 74 (2): 722–8.

In some embodiments of the invention, the "component of the plasminogen activation pathway" of the invention is plasminogen, selected from Glu-plasminogen, Lys-plasminogen, small plasmin Pro, microplasminogen, delta-plasminogen, or variants thereof that retain plasminogen activity (e.g., proteolytic activity, or lysine-binding activity, or proteolytic activity and lysine-binding activity) . In some embodiments, the plasminogen is natural or synthetic human plasminogen, or conservative mutant variants thereof that still retain plasminogen activity, or fragments thereof. In some embodiments, the plasminogen is a human plasminogen ortholog from a primate or rodent, or a conservative mutant variant thereof that still retains plasminogen activity, or a fragment thereof. In some embodiments, the plasminogen amino acid is represented by sequence 2, 6, 8, 10, or 12. In some embodiments, the plasminogen is human native plasminogen. In some embodiments, the plasminogen is human native plasminogen as set forth in Sequence 2.

"Compounds capable of activating plasminogen directly or indirectly by activating upstream components of the plasminogen activation pathway" means compounds capable of activating plasminogen directly or by activating plasminogen Any compound that indirectly activates plasminogen by activating components upstream of the pathway, such as tPA, uPA, streptokinase, salupulase, alteplase, reteplase, tenecteplase, anisteplase, Monteplase, lanoteplase, pamiplase, staphylokinase.

The "antagonist of fibrinolysis inhibitor" of the present invention is a compound that antagonizes, weakens, blocks or prevents the action of fibrinolysis inhibitor, such as PAI-1, leuprorelin C1 inhibitor, α2 antifibrinolytic enzyme and α2 macroglobulin. The antagonists are, for example, antibodies to PAI-1, complement C1 inhibitor, α2 antifibrinolytic enzyme or α2 macroglobulin, or antisense RNA or small RNA that blocks or downregulates the expression of, for example, PAI-1, complement C1 inhibitor, α2 antifibrinolytic enzyme or α2 macroglobulin, or compounds that occupy the binding site of PAI-1, complement C1 inhibitor, α2 antifibrinolytic enzyme or α2 macroglobulin but have no function of PAI-1, complement C1 inhibitor, α2 antifibrinolytic enzyme or α2 macroglobulin, or compounds that block the binding domain and/or active domain of PAI-1, complement C1 inhibitor, α2 antifibrinolytic enzyme or α2 macroglobulin.

Fibrinolytic enzymes are key components of the fibrinolytic enzyme activation system (PA system). They are broad-spectrum proteases that can hydrolyze several components of the extracellular matrix (ECM), including fibroin, gelatin, fibronectin, laminin, and proteoglycans. In addition, fibrinolytic enzymes can activate some metalloproteinase precursors (pro-MMPs) to form active metalloproteinases (MMPs). Therefore, fibrinolytic enzymes are considered to be an important upstream regulator of extracellular proteolysis. Fibrinolytic enzymes are formed by proteolysis of fibrinolytic enzymes by two physiological PAs: tissue-type fibrinolytic enzyme activator (tPA) or urokinase-type fibrinolytic enzyme activator (uPA). Due to the relatively high levels of fibrinolysins in plasma and other body fluids, it has traditionally been believed that the PA system is regulated primarily through the synthesis and activity levels of PAs. The synthesis of the PA system components is tightly regulated by different factors, such as hormones, growth factors, and cytokines. In addition, there are specific physiological inhibitors of fibrinolysins and PAs. The major inhibitor of fibrinolysins is α2-antiplasmin. The activity of PAs is also regulated by the fibrinolysinogen activator inhibitor-1 (PAI-1) for both uPA and tPA, and the lysozyme activator inhibitor-2 (PAI-2) which primarily inhibits uPA. Certain cells have a specific cell surface receptor for uPA (uPAR) that has direct hydrolytic activity.

Plasminogen is a single-chain glycoprotein consisting of 791 amino acids with a molecular weight of approximately 92 kDa. Plasminogen is mainly synthesized in the liver and exists in large amounts in extracellular fluid. Plasminogen content in plasma is approximately 2 μM. Plasminogen is therefore a huge potential source of proteolytic activity in tissues and body fluids. Plasminogen exists in two molecular forms: glutamate-plasminogen (Glu-plasminogen) and lysine-plasminogen (Lys-plasminogen). The naturally secreted and uncleaved form of plasminogen has an amino-terminal (N-terminal) glutamate and is therefore called glutamate-plasminogen. However, in the presence of plasmin, glutamate-plasminogen is hydrolyzed to lysine-plasminogen at Lys76-Lys77. Compared with glutamate-plasminogen, lysine-plasminogen has a higher affinity for fibrin and can be activated by PAs at a higher rate. The Arg560-Val561 peptide bond of both forms of plasminogen can be cleaved by uPA or tPA, resulting in the formation of the disulfide-linked double-chain protease plasmin. The amino-terminal part of plasminogen contains five homologous tricycles, the so-called kringles, and the carboxyl-terminal part contains the protease domain. Some kringles contain lysine-binding sites that mediate specific interactions of plasminogen with fibrin and its inhibitor α2-AP. A recently discovered 38 kDa fragment of plasminogen, which includes kringles1-4, is a potent inhibitor of angiogenesis. This fragment, named angiostatin, is produced by hydrolysis of plasminogen by several proteases.

The main substrate of fibrinolytic enzymes is fibrin, and the dissolution of fibrin is the key to preventing pathological thrombosis. Fibrinolytic enzymes also have substrate specificity for several components of the ECM, including laminin, fibronectin, proteoglycans, and gelatin, indicating that fibrinolytic enzymes also play an important role in ECM reconstruction. Indirectly, fibrinolytic enzymes can also degrade other components of the ECM, including MMP-1, MMP-2, MMP-3, and MMP-9, by converting certain protease precursors into active proteases. Therefore, it has been suggested that fibrinolytic enzymes may be an important upstream regulator of extracellular proteolysis. In addition, fibrinolytic enzymes have the ability to activate certain latent forms of growth factors. In vitro, fibrinolytic enzymes can also hydrolyze components of the complement system and release proteolytic fragments.

"Plasmin" is a very important enzyme present in the blood that can hydrolyze fibrin clots into fibrin degradation products and D-dimer.

"Plasminogen" is the zymogen form of plasmin. According to the sequence in swiss prot, it is composed of 810 amino acids based on the amino acid sequence of natural human plasminogen containing the signal peptide (sequence 4). The molecular weight is about 90kD, a glycoprotein mainly synthesized in the liver and able to circulate in the blood. The cDNA sequence encoding this amino acid sequence is shown in Sequence 3. Full-length plasminogen contains seven domains: a serine protease domain at the C-terminus, a Pan Apple (PAp) domain at the N-terminus, and five Kringle domains (Kringle1-5). Referring to the sequence in swiss prot, its signal peptide includes residues Met1-Gly19, PAp includes residues Glu20-Val98, Kringle1 includes residues Cys103-Cys181, Kringle2 includes residues Glu184-Cys262, Kringle3 includes residues Cys275-Cys352, and Kringle4 Includes residues Cys377-Cys454, Kringle5 includes residues Cys481-Cys560. According to NCBI data, the serine protease domain includes residues Val581-Arg804.

Glu-plasminogen is the natural full-length plasminogen of human beings, consisting of 791 amino acids (excluding the 19-amino acid signal peptide). The cDNA sequence encoding the sequence is shown in SEQ ID NO: 1, and its amino acid sequence is shown in SEQ ID NO: 2. In vivo, there is also a Lys-plasminogen formed by hydrolysis of the 76th-77th amino acids of Glu-plasminogen, as shown in SEQ ID NO: 6, and the cDNA sequence encoding the amino acid sequence is shown in SEQ ID NO: 5. Delta-plasminogen (δ-plasminogen) is a fragment of the full-length plasminogen that lacks the Kringle2-Kringle5 structure and contains only Kringle1 and the serine protease domain (also known as the protease domain (PD)). The amino acid sequence of delta-plasminogen has been reported in the literature (SEQ ID NO: 8), and the cDNA sequence encoding the amino acid sequence is shown in SEQ ID NO: 7. Mini-plasminogen is composed of Kringle5 and serine protease domains. Literature reports that it includes residues Val443-Asn791 (with the Glu residue of the Glu-plasminogen sequence without a signal peptide as the starting amino acid). Its amino acid sequence is shown in SEQ ID NO: 10, and the cDNA sequence encoding the amino acid sequence is shown in SEQ ID NO: 9. Micro-plasminogen contains only a serine protease domain. Some literature reports that its amino acid sequence includes residues Ala543-Asn791 (with the Glu residue of the Glu-plasminogen sequence without a signal peptide as the starting amino acid), and some patent document CN102154253A reports that its sequence includes residues Lys531-Asn791 (with the Glu residue of the Glu-plasminogen sequence without a signal peptide as the starting amino acid). In this patent application, the sequence of micro-plasminogen refers to patent document CN102154253A, and its amino acid sequence is shown in SEQ ID NO: 12. The cDNA sequence encoding the amino acid sequence is shown in SEQ ID NO: 11.

The structure of full-length plasminogen is also described in the article of Aisina et al. (Aisina RB, Mukhametova L I. Structure and function of plasminogen/plasmin system[J]. Russian Journal of Bioorganic Chemistry, 2014, 40(6):590-605) middle. In this article, Aisina et al. described that plasminogen includes Kringle1, 2, 3, 4, and 5 domains and a serine protease domain (also known as protease domain (PD)), among which Kringles is responsible for plasmin Protogen binds to low and high molecular weight ligands, causing plasminogen to convert into a more open configuration and thus easier to be activated. The protease domain (PD) is residues Val562-Asn791, specific for tPA and UPA. Cleaves the Arg561-Val562 activation bond of plasminogen, thereby forming plasmin from plasminogen.

In the present invention, "fibrinolytic enzyme" and "fibrinolytic enzyme" can be used interchangeably and have the same meaning; "fibrinolytic enzyme" and "fibrinolytic enzyme progen" can be used interchangeably and have the same meaning.

In the present application, the meaning or activity of the "lack" of fibrinolysin is that the content of fibrinolysin in the subject's body is lower than that of a normal person, low enough to affect the normal physiological function of the subject; the meaning or activity of the "absence" of fibrinolysin is that the content of fibrinolysin in the subject's body is significantly lower than that of a normal person, or even the activity or expression is extremely low, and normal physiological function can only be maintained through exogenous supply.

Those with ordinary knowledge in the art can understand that all technical solutions for plasminogen of the present invention are applicable to plasmin, and therefore, the technical solutions described in the present invention cover both plasminogen and plasmin. During circulation, plasminogen adopts a closed, inactive conformation, but when bound to thrombus or cell surface, it changes to an open conformation mediated by plasminogen activator (PA) Conformation of active plasmin. Active plasmin can further hydrolyze the fibrin clot into fibrin degradation products and D-dimer, thereby dissolving the thrombus. The PAp domain of plasminogen contains important determinants that maintain plasminogen in an inactive closed conformation, while the KR domain can bind to lysine residues present on the receptor and substrate. Several enzymes are known to act as plasminogen activators, including: tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), kallikrein, and coagulation factor XII (Hage Mann factor) etc.

"Fibrinolytic enzyme active fragment" refers to a fragment having the activity of binding to lysine in a substrate target sequence (lysine binding activity), or the activity of exerting a proteolytic function (proteolytic activity), or a fragment having both proteolytic activity and lysine binding activity. The technical scheme of the present invention involving fibrinolytic enzyme covers the technical scheme of replacing fibrinolytic enzyme with a fibrinolytic enzyme active fragment. In some embodiments, the profibrinolytic enzyme active fragment of the present invention comprises the serine protease domain of profibrinolytic enzyme or consists of the serine protease domain of profibrinolytic enzyme. Preferably, the profibrinolytic enzyme active fragment of the present invention comprises sequence 14, or an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with sequence 14, or consists of sequence 14, or an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with sequence 14. In some embodiments, the fibrinolytic enzyme active fragment of the present invention comprises a region selected from one or more of Kringle 1, Kringle 2, Kringle 3, Kringle 4, and Kringle 5, or consists of a region selected from one or more of Kringle 1, Kringle 2, Kringle 3, Kringle 4, and Kringle 5. In some embodiments, the fibrinolytic enzyme of the present invention includes a protein containing the above-mentioned fibrinolytic enzyme active fragment.

At present, the methods for measuring fibrinolysinogen and its activity in the blood include: detection of tissue fibrinolysinogen activator activity (t-PAA), detection of plasma tissue fibrinolysinogen activator antigen (t-PAAg), detection of plasma tissue fibrinolysinogen activity (plgA), detection of plasma tissue fibrinolysinogen antigen (plgAg), detection of plasma tissue fibrinolysinogen activator inhibitor activity, detection of plasma tissue fibrinolysinogen activator inhibitor antigen, and detection of plasma fibrinolysin-antifibrinolysin complex (PAP). The most commonly used detection method is the chromogenic substrate method: chain kinase (SK) and chromogenic substrate are added to the test plasma. Under the action of SK, PLG in the test plasma is converted into PLM, which acts on the chromogenic substrate and is then measured by a spectrophotometer. The increase in absorbance is proportional to the activity of fibrinolytic enzyme. In addition, immunochemical methods, gel electrophoresis, immunoturbidimetry, radioimmunoassay, etc. can also be used to measure the activity of fibrinolytic enzyme in the blood.

"Orthologs" refer to homologs between different species, including both protein homologs and DNA homologs, also known as orthologs and vertical homologs. Specifically, they refer to proteins or genes evolved from the same ancestral gene in different species. The fibrinolysinogen of the present invention includes human natural fibrinolysinogen, and also includes fibrinolysinogen orthologs or orthologs derived from different species and having fibrinolysinogen activity.

"Conservative substitution variants" refer to those in which a given amino acid residue is changed but does not change the overall conformation and function of the protein or enzyme. This includes but is not limited to those with similar properties (such as acidic, basic, hydrophobic, etc.) Amino acids replace amino acids in the amino acid sequence of the parent protein. Amino acids with similar properties are well known. For example, arginine, histidine, and lysine are hydrophilic basic amino acids and are interchangeable. Likewise, isoleucine is a hydrophobic amino acid and can be replaced by leucine, methionine or valine. Therefore, the similarity of two proteins or amino acid sequences with similar functions may differ. For example, 70% to 99% similarity (identity) based on the MEGALIGN algorithm. "Conservative substitution variants" also include polypeptides or enzymes that have more than 60% amino acid identity determined by BLAST or FASTA algorithms. It is better if it can reach more than 75%, preferably more than 85%, or even more than 90%. Optimal and having the same or substantially similar properties or functions as the native or parent protein or enzyme.

"Isolated" plasminogen refers to plasminogen protein separated and/or recovered from its natural environment. In some embodiments, the plasminogen is purified (1) to a purity (by weight) of greater than 90%, greater than 95%, or greater than 98%, as determined by the Lowry method, for example, greater than 99% (by weight), (2) to a sufficient extent to obtain at least 15 residues of the N-terminal or internal amino acid sequence by using a spin cup sequence analyzer, or (3) to homogeneity, which was determined by using Cooma Determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) stained with blue or silver under reducing or non-reducing conditions. Isolated plasminogen also includes plasminogen prepared from recombinant cells by bioengineering techniques and separated by at least one purification step.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymeric form of amino acids of any length, which may include genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones. The term includes fusion proteins, including but not limited to fusion proteins with heterologous amino acid sequences, fusions with heterologous and homologous leader sequences (with or without an N-terminal methionine residue); and the like.

"Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after introducing gaps, if necessary, to achieve maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. One of ordinary skill in the art can determine appropriate parameters for aligning sequences, including any algorithm needed to achieve maximum alignment over the full length of the compared sequences. However, for the purposes of the present invention, percentage amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2.

In the case of using ALIGN-2 to compare amino acid sequences, the % amino acid sequence identity of a given amino acid sequence A relative to a given amino acid sequence B (or may be expressed as a given amino acid sequence A having or comprising a certain % amino acid sequence identity relative to, with, or to a given amino acid sequence B) is calculated as follows:

Multiply the fraction X/Y by 100

where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A. Unless expressly stated otherwise, all % amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

As used herein, the terms "treatment" and "treatment" refer to obtaining a desired pharmacological and/or physiological effect. The effect may be a complete or partial prevention of the disease or its symptoms, and/or a partial or complete cure of the disease and/or its symptoms, and include: (a) preventing the disease from occurring in a subject, who may have A predisposing factor for a disease, but not yet diagnosed as having a disease; (b) inhibiting the disease, i.e. arresting its development; and (c) alleviating the disease and/or its symptoms, i.e. causing the regression of the disease and/or its symptoms.

The terms "individual", "subject" and "patient" are used interchangeably herein to refer to mammals, including but not limited to murine (rat, mouse), non-human primates, humans, dogs, cats, Hoofed animals (such as horses, cattle, sheep, pigs, goats), etc.

A "therapeutically effective amount" or "effective amount" refers to an amount of plasminogen sufficient to effect the prevention and/or treatment of a disease when administered to a mammal or other subject to treat a disease. The "therapeutically effective amount" will vary depending on the plasminogen used, the severity of the disease and/or symptoms thereof, and age, weight, etc. of the subject to be treated.

Preparation of plasminogen of the present invention

Fibrinolysinogen can be isolated from nature and purified for further therapeutic use, or it can be synthesized by standard chemical peptide synthesis techniques. When the peptide is synthesized chemically, the synthesis can be carried out via liquid phase or solid phase. Solid phase peptide synthesis (SPPS), in which the C-terminal amino acid of the sequence is attached to an insoluble support, followed by the sequential addition of the remaining amino acids in the sequence, is a suitable method for the chemical synthesis of fibrolysinogen. Various forms of SPPS, such as Fmoc and Boc, can be used to synthesize fibrolysinogen. Techniques for solid phase synthesis are described in Barany and Solid-Phase Peptide Synthesis; pages 3-284 in The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A., Merrifield, et al. J. Am. Chem. Soc., 85: 2149-2156 (1963); Stewart et al., Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co., Rockford, Ill. (1984); and Ganesan A. 2006 Mini Rev. Med Chem. 6:3-10 and Camarero JA et al. 2005 Protein Pept Lett. 12:723-8. Briefly, small insoluble porous beads are treated with functional units on which peptide chains are constructed. After repeated cycles of coupling/deprotection, the attached solid phase free N-terminal amine is coupled to a single N-protected amino acid unit. This unit is then deprotected, exposing a new N-terminal amine that can be attached to another amino acid. The peptide remains fixed to the solid phase and is later cleaved off.

Standard recombinant methods can be used to produce plasminogen of the invention. For example, a nucleic acid encoding plasminogen is inserted into an expression vector so that it is operably linked to regulatory sequences in the expression vector. Expression control sequences include, but are not limited to, promoters (eg, naturally associated or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences. Expression control can be a eukaryotic promoter system in a vector capable of transforming or transfecting eukaryotic host cells (eg COS or CHO cells). Once the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high-level expression of the nucleotide sequence and collection and purification of plasminogen.

Suitable expression vectors typically replicate in the host organism as episomes or as integrated parts of the host chromosomal DNA. Typically, the expression vector contains a selectable marker (eg, ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance, or neomycin resistance) to facilitate transformation of the exogenous source with the desired DNA sequence of those cells are tested.

Escherichia coli is an example of a prokaryotic host cell that can be used to replicate the subject antibody encoding polynucleotide. Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts, expression vectors can also be generated, which usually contain expression control sequences (e.g., replication origins) that are compatible with the host cells. In addition, there are many well-known promoters, such as the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter system, or the promoter system from bacteriophage λ. The promoter generally controls expression, optionally in the case of an operator gene sequence, and has a ribosome binding site sequence, etc., to initiate and complete transcription and translation.

Other microorganisms, such as yeast, can also be used for expression. Yeast (e.g., S. cerevisiae) and Pichia are examples of suitable yeast host cells, with suitable vectors having expression control sequences (e.g., promoters), replication origins, termination sequences, etc. as required. Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, in particular, promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for maltose and galactose utilization.

In addition to microorganisms, mammalian cells (eg, mammalian cells cultured in in vitro cell culture) can also be used to express and produce the anti-Tau antibodies of the invention (eg, polynucleotides encoding the subject anti-Tau antibodies). See Winnacker, From Genes to Clones, VCH Publishers, N.Y., N.Y. (1987). Suitable mammalian host cells include CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, and transformed B cells or hybridomas. Expression vectors for use in these cells may contain expression control sequences, such as origins of replication, promoters, and enhancers (Queen et al., Immunol. Rev. 89:49 (1986)), as well as necessary processing information sites, such as ribosome binding sites, RNA splicing sites, polyadenylation sites, and transcription terminator sequences. Examples of suitable expression control sequences are promoters derived from the white immunoglobulin gene, SV40, adenovirus, bovine papillomavirus, cytomegalovirus, and the like. See Co et al., J. Immunol. 148:1149 (1992).

Once synthesized (chemically or recombinantly), the fibers of the present invention can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, high performance liquid chromatography (HPLC), gel electrophoresis, etc. Lysogen. The plasminogen is substantially pure, such as at least about 80% to 85% pure, at least about 85% to 90% pure, at least about 90% to 95% pure, or 98% to 99% pure or purer, e.g., free of contaminants such as cellular debris, macromolecules other than the subject antibodies, and the like.

pharmaceutical preparations

Therapeutic formulations can be prepared by mixing lyophilized preparations or aqueous solutions of fibrolytic enzymes of the desired purity with optional pharmaceutical carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)). Acceptable carriers, excipients, and stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexadecylamine chloride; benzalkonium chloride; chloride), benzathine chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens such as methyl or propyl parabens; o-catechol; resorcinol; cyclohexanol; 3-pentanol; m-cresol); low molecular weight polypeptides (less than about 10 residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, fucose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., zinc-protein complexes); and/or nonionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). Preferred lyophilized anti-VEGF antibody formulations are described in WO 97/04801, which is incorporated herein by reference.

The formulations according to the invention may also contain more than one active compound required for the specific condition to be treated, preferably those whose activities are complementary and which do not have adverse effects on each other. For example, antihypertensive drugs, antiarrhythmic drugs, drugs to treat diabetes, etc.

The lysinogen of the present invention can be encapsulated in microcapsules prepared by, for example, coacervation techniques or interfacial polymerization, for example, hydroxymethylcellulose or gel-microcapsules and poly-(methyl methacrylate) microcapsules placed in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. These techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Plasminogens of the present invention for in vivo administration must be sterile. This can be easily accomplished by filtration through a sterile membrane before or after freeze drying and reconstitution.

The plasminogen of the present invention can be prepared into sustained-release preparations. Suitable examples of sustained release formulations include shaped solid hydrophobic polymeric semipermeable matrices containing glycoproteins, such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels such as poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res., 15: 167-277 (1981); Langer, Chem Tech., 12:98-105 (1982)) or poly(vinyl alcohol), polylactide (U.S. Patent 3,773,919, EP 58,481), copolymer of L-glutamic acid and γ-ethyl-L-glutamic acid (Sidman, et al., Biopolymers 22:547 (1983)), non-degradable ethylene-vinyl acetate (Langer, et al., supra), or degradable lactic acid-glycolic acid copolymers such as Lupron DepotTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and polyD-(-)-3-hydroxybutyric acid. Polymers such as ethylene- Vinyl acetate and lactic-glycolic acid can sustain the release of molecules for more than 100 days, while some hydrogels release proteins for a shorter time. Rational strategies to stabilize proteins can be designed based on the relevant mechanisms. For example, if the mechanism of aggregation is found to be The formation of intermolecular S-S bonds through thiodisulfide exchange can be stabilized by modifying sulfhydryl residues, freeze-drying from acidic solutions, controlling humidity, using appropriate additives, and developing specific polymer matrix compositions.

Administration and dosage

Administration of the pharmaceutical composition of the present invention can be achieved by different ways, for example, intravenously, intraperitoneally, subcutaneously, intracranially, intrathecally, intraarterially (eg, via the carotid artery), intramuscularly.

Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, or fixed oils. Intravenous vehicles include fluid and nutritional supplements, electrolyte supplements, and more. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases, among others.

Your healthcare provider will determine the dosage regimen based on a variety of clinical factors. As is well known in the medical arts, the dosage for any given patient depends on a variety of factors, including the patient's size, body surface area, age, the specific compound being administered, gender, the number and route of administration, general health, and other drugs being administered concurrently. The dosage range of the pharmaceutical composition containing plasminogen of the present invention can be, for example, about 0.0001 to 2000 mg/kg, or about 0.001 to 500 mg/kg (for example, 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg per day). kg, 0.75 mg/kg, 10 mg/kg, 50 mg/kg, etc.) Subject weight. For example, the dose may be 1 mg/kg body weight or 50 mg/kg body weight or in the range of 1-50 mg/kg, or at least 1 mg/kg. Doses above or below this illustrative range are also contemplated, particularly in view of the factors noted above. Doses intermediate within the above ranges are also included within the scope of the invention. Subjects may administer such doses daily, every other day, weekly, or according to any other schedule determined by empirical analysis. An exemplary dosing schedule includes 1-10 mg/kg on consecutive days. It is necessary to evaluate the therapeutic effect and safety in real time during the administration of the drug of the present invention.

products or kits

One embodiment of the invention relates to a preparation or kit comprising plasminogen or plasmin of the invention useful in the treatment of cardiovascular diseases caused by diabetes and related disorders. The article preferably includes a container, label or packaging insert. Suitable containers are bottles, vials, syringes, etc. Containers can be made from various materials such as glass or plastic. The container contains a composition effective to treat a disease or condition of the invention and has a sterile access port (for example, the container may be an intravenous solution packet or vial containing a stopper penetrable by a hypodermic needle) ). At least one active agent in the composition is plasminogen/plasmin. The label on or attached to the container states that the composition is used to treat cardiovascular disease caused by diabetes and its related conditions according to the present invention. The article of manufacture may further comprise a second container containing a pharmaceutically acceptable buffer, such as phosphate buffered saline, Ringer's solution, and dextrose solution. It may further contain other materials required from a commercial and user perspective, including other buffers, diluents, filters, needles and syringes. Additionally, the article of manufacture includes a package insert with instructions for use, including, for example, instructions to a user of the composition to administer the plasminogen composition to the patient as well as other medications for the treatment of concomitant diseases.

Example

The human plasminogen used in the following examples was obtained from human donor plasma based on the methods described in the following references: Kenneth C Robbins, Louis Summaria, David Elwyn et al. Further Studies on the Purification and Characterization of Human Plasminogen and Plasmin. Journal of Biological Chemistry, 1965, 240 (1): 541-550; Summaria L, Spitz F, Arzadon L et al. Isolation and characterization of the affinity chromatography forms of human Glu- and Lys-plasminogens and plasmins. J Biol Chem. 1976 Jun 25; 251 (12): 3693-9; HAGAN JJ, ABLONDI FB, DE RENZO EC. Purification and biochemical properties of human plasminogen. J Biol Chem. 1960 Apr;235:1005-10, and the process was optimized and purified from human donor plasma, in which human fibronectin monomer was >98%.

Example 1 Fibrolysinogen promotes the repair of nerve damage in mice with spinal cord compression injury model

Fifteen 8-week-old C57 female mice were weighed and randomly divided into three groups: sham operation group, vehicle PBS control group, and fibrinolysin group, with 5 mice in each group. All mice were anesthetized with intraperitoneal injection of sodium pentobarbital solution (50 mg/kg), and the dorsal midline skin was incised, the dorsal T7-T9 muscles were separated, and the T7-T9 vertebral lamina were removed. Microvascular clamps (3.5×1.0 mm, Reward) were clamped at the T8 spinal cord for 1 minute in the vehicle PBS control group and the fibrinolysin group; only the vertebral lamina was removed in the sham operation group to avoid dura mater damage. All mice were sutured immediately after surgery, and Tolfedine (0.1 mg/kg) and penicillin potassium (40,000 units/kg) were injected intramuscularly and placed on a heating pad until they woke up. To prevent infection during the operation, mice were manually assisted to urinate after the operation until they could urinate autonomously ^[1] . Medication was started 30 minutes after the operation. Mice in the fibroblast group were injected with fibroblast at a dose of 1 mg/0.1 ml/mouse/day via the caudal vein. The vehicle PBS control group was given the same volume of PBS in the same manner. Mice in the sham operation group were not given medication. Medication began on the first day, and the medication cycle was 7 days. On the eighth day of medication, the mice were dissected and the spinal cord T7-T9 segments were fixed in 4% paraformaldehyde. The fixed tissue samples were dehydrated with alcohol gradient and transparentized with xylene before being embedded in paraffin. The thickness of the spinal cord tissue cross-section was 4 μm. The sections were dewaxed and rehydrated and stained with hematoxylin and eosin (HE staining). After differentiation with 1% hydrochloric acid alcohol, the sections were blued with ammonia water and dehydrated with alcohol gradient and sealed. The sections were observed under a 200x optical microscope.

The results showed that the neurons and nerve fibers in the sham surgery group (Figure 1A) were normal in appearance, with red cytoplasm staining. The normal nerves and the junction of injured nerves were collected from the vehicle PBS control group (Figure 1B) and the fibronectin group. In the vehicle PBS control group, a larger injury cavity (marked by a triangle) was observed in the junction area, while the nerve repair degree of the injured part in the fibronectin group (Figure 1C) was significantly better than that in the vehicle PBS control group, and no obvious cavity was observed. This indicates that fibronectin can significantly promote the repair of injured nerves in the spinal cord compression injury model mice.

Example 2 Plasminogen promotes remyelination of nerves

Fifteen 8-week-old C57 female mice were weighed and randomly divided into three groups: a sham operation group, a PBS-administered vehicle control group, and a plasminogen-administered group, with 5 mice in each group. All mice were anesthetized by intraperitoneal injection of pentobarbital sodium solution (50 mg/kg), the midline skin of the back was incised, the back T7-T9 muscles were separated, and the T7-T9 laminae were removed. In the vehicle PBS control group and the plasminogen administration group, a microvascular clip (3.5×1.0mm, Reward) was clamped on the T8 spinal cord for 1 minute; in the sham operation group, only the lamina was removed to avoid dural damage. . All mice were sutured immediately after surgery, intramuscularly injected with Tolfedine (0.1 mg/kg) and penicillin potassium (40,000 units/kg), and then placed on a heating pad until they woke up. Infection was prevented during the operation, and the mice were manually assisted to urinate until they urinate spontaneously after surgery ^[1] . Administration was started 30 minutes after the operation. The mice in the plasminogen group were given plasminogen by tail vein injection at 1mg/0.1ml/mouse/day. The mice in the vehicle PBS control group were given the same volume of PBS in the same way. Sham surgery The mice in the group were not treated with medication. On the first day of administration, the administration cycle is 7 days. On the 8th day after administration, the mice were dissected and the spinal cord T7-T9 segments were fixed in 4% paraformaldehyde. Fixed tissue samples were dehydrated through alcohol gradients and cleared in xylene before being embedded in paraffin. The thickness of the cross-sectioned spinal cord tissue was 4 μm. After deparaffinization to water, LFB staining was performed with myelin staining solution. Dehydrate with graded alcohol, clear with xylene, and mount with neutral gum. Observe and take pictures under an optical microscope.

LFB (luxol fast blue) staining uses fast blue staining to stain myelin. The degree of blue represents the amount of myelin. LFB staining is an effective method for studying the positioning of corticospinal tracts, myelin lesions, injury and morphological observation of regeneration and repair ^[2,3] .

The results showed that in the sham operation group (Figure 2A), the nerve myelin sheath structure was intact and continuous; in the control group given vehicle PBS (Figure 2B), the nerve myelin sheath showed obvious disintegration in a larger area (marked by arrows), with lighter blue and myelin sheath. The sheath was broken; compared with the vehicle PBS control group, the myelin structure of the plasminogen-administered group (Figure 2C) was obviously more complete and the blue color was darker. This shows that plasminogen can promote myelin regeneration in spinal cord compression model mice.

Embodiment 3 Plasminogen promotes spinal neurofilament protein expression

Fifteen 8-week-old C57 female mice were weighed and randomly divided into three groups: a sham operation group, a PBS-administered vehicle control group, and a plasminogen-administered group, with 5 mice in each group. All mice were anesthetized by intraperitoneal injection of pentobarbital sodium solution (50 mg/kg), the midline skin of the back was incised, the back T7-T9 muscles were separated, and the T7-T9 laminae were removed. In the vehicle PBS control group and the plasminogen administration group, a microvascular clip (3.5×1.0mm, Reward) was clamped on the T8 spinal cord for 1 minute; in the sham operation group, only the lamina was removed to avoid dural damage. . All mice were sutured immediately after surgery, intramuscularly injected with Tolfedine (0.1 mg/kg) and penicillin potassium (40,000 units/kg), and then placed on a heating pad until they woke up. Infection was prevented during the operation, and the mice were manually assisted to urinate until they urinate spontaneously after the operation. Administration was started 30 minutes after the operation. The mice in the plasminogen group were given plasminogen by tail vein injection at 1mg/0.1ml/mouse/day. The mice in the vehicle PBS control group were given the same volume of PBS in the same way. Sham surgery The mice in the group were not treated with medication. On the first day of administration, the administration cycle is 7 days. On the 8th day after administration, the mice were dissected and spinal cord T7-T9 were fixed in 4% paraformaldehyde. Fixed tissue samples were dehydrated through alcohol gradients and cleared in xylene before being embedded in paraffin. The thickness of tissue sections was 3 μm, and the sections were dewaxed, rehydrated and washed once. Repair with citric acid for 30 minutes, cool at room temperature for 10 minutes and then rinse gently with water. Incubate with 3% hydrogen peroxide for 15 minutes and circle the tissue with a PAP pen. 10% sheep serum (Vector laboratories, Inc., USA) was used to block for 1 hour; after the time was up, the sheep serum liquid was discarded. Rabbit-derived anti-neurofilament protein (NFP) antibody (Abcam, ab207176) was incubated overnight at 4°C, and washed twice with PBS for 5 minutes each time. Goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody was incubated at room temperature for 1 hour, and washed twice with PBS for 5 minutes each time. The color was developed according to the DAB kit (Vector laboratories, Inc., USA), washed three times with water, counterstained with hematoxylin for 30 seconds, returned to blue with running water for 5 minutes, and then washed once with PBS. Gradient dehydration was used to clear and seal the sections, and the sections were observed under a 200x optical microscope.

Neurofilament protein (NFP) is a protein that constitutes the intermediate filaments of nerve cell axons. Its function is to provide elasticity to make nerve fibers easy to stretch and prevent breakage. It is of great significance in maintaining the cytoskeleton, stabilizing cell morphology and axonal transport ^[4] .

The results showed that the expression of NFP (indicated by arrows) in the fibrinolytic group (Figure 3C) was significantly higher than that in the PBS control group (Figure 3B), and the expression of NFP was closer to that in the sham surgery group (Figure 3A). This indicates that fibrinolytic can promote the expression of NFP and promote the regeneration of spinal cord nerve fibers in mice with spinal cord compression injury model.

Embodiment 4 Plasminogen promotes repair of nerve damage

Ten 8-week-old Plg-/- female mice were weighed and randomly divided into two groups: a control group given vehicle PBS and a group given plasminogen, with 5 mice in each group. All mice were anesthetized by intraperitoneal injection of pentobarbital sodium solution (50 mg/kg), the midline skin of the back was incised, the back T7-T9 muscles were separated, and the T7-T9 laminae were removed. A microvascular clip (3.5 × 1.0 mm, Reward) was clamped on the T8 spinal cord for 1 minute in the vehicle PBS control group and the plasminogen group. All mice were sutured immediately after surgery, intramuscularly injected with Tolfedine (0.1 mg/kg) and penicillin potassium (40,000 units/kg), and then placed on a heating pad until they woke up. Infection was prevented during the operation, and the mice were manually assisted to urinate until they urinate spontaneously after surgery ^[1] . Administration was started 30 minutes after surgery. The mice in the plasminogen group were given plasminogen by tail vein injection at 1mg/0.1ml/mouse/day, and the mice in the vehicle PBS control group were given the same volume of PBS in the same way. On the first day of administration, administration was continued for 7 days. On the 8th day after administration, the mice were dissected and the spinal cord T7-T9 segments were fixed in 4% paraformaldehyde. Fixed tissue samples were dehydrated through alcohol gradients and cleared in xylene before being embedded in paraffin. The thickness of the transverse sections of the spinal cord tissue was 4 μm. The sections were dewaxed, rehydrated, and stained with hematoxylin and eosin (H&E staining). They were differentiated with 1% hydrochloric acid alcohol, returned to blue with ammonia water, and dehydrated in gradient alcohol and sealed. The sections were examined under a 200x optical microscope. Observe below.

The results showed that in the sham surgery group (Figure 4A), neurons and neurofibrils were normal in appearance, with red cytoplasm staining. In the vehicle PBS control group (Figure 4B) and the fibronectin group (Figure 4C), normal nerves and the junction of injured nerves were collected. It was found that the normal neuron and neurofibril structures disappeared at the injured site in both groups, and were replaced by repaired fibers, lightly stained cytoplasm, and inflammatory cell infiltration. However, in the vehicle PBS control group, a larger area of injured cavities (marked by triangles) was seen at the junction area, while the degree of repair of the injured site in the fibronectin group was significantly better than that in the vehicle PBS control group, with no obvious cavities and significantly reduced inflammatory cell infiltration. This indicates that fibrolytic enzyme can significantly promote the repair of spinal cord injury in the spinal cord compression nerve injury model Plg-/- mice.

Example 5 Fibrolysinogen promotion Plg-/- Regeneration of myelin sheath in mice with spinal cord compression injury

Ten 8-week-old Plg-/- female mice were weighed and randomly divided into two groups: a control group given vehicle PBS and a group given plasminogen, with 5 mice in each group. All mice were anesthetized by intraperitoneal injection of pentobarbital sodium solution (50 mg/kg), the midline skin of the back was incised, the back T7-T9 muscles were separated, and the T7-T9 laminae were removed. A microvascular clip (3.5 × 1.0 mm, Reward) was clamped on the T8 spinal cord for 1 minute in the vehicle PBS control group and the plasminogen group. All mice were sutured immediately after surgery, intramuscularly injected with Tolfedine (0.1 mg/kg) and penicillin potassium (40,000 units/kg), and then placed on a heating pad until they woke up. Infection was prevented during the operation, and the mice were manually assisted to urinate until they urinate spontaneously after surgery ^[1] . Administration was started 30 minutes after surgery. The mice in the plasminogen group were given plasminogen by tail vein injection at 1mg/0.1ml/mouse/day, and the mice in the vehicle PBS control group were given the same volume of PBS in the same way. Start administration on the 1st day and continue administration for 7 days. On the 8th day after administration, the mice were dissected and the spinal cord T7-T9 segments were fixed in 4% paraformaldehyde. Fixed tissue samples were dehydrated through alcohol gradients and cleared in xylene before being embedded in paraffin. The thickness of the cross-sectioned spinal cord tissue was 4 μm. After deparaffinization to water, LFB staining was performed with myelin staining solution. Dehydrate with graded alcohol, clear with xylene, and mount with neutral gum. Observe and take pictures under an optical microscope.

The results showed that the myelin sheath of the PBS control group (Figure 5A) showed obvious large-scale disintegration (triangle), lighter blue, and myelin sheath rupture (arrow), compared with the myelin sheath structure of the fibronectin group (Figure 5B) which was obviously more complete, continuous, and darker blue. This shows that fibronectin can significantly promote the regeneration of myelin sheath in the spinal cord compression nerve injury model Plg-/- mice.

Embodiment 6 Plasminogen promotes pain recovery in spinal cord compression injury model mice

Thirty-eight 7-week-old C57BL/6J female mice were taken and weighed the day before modeling. They were randomly divided into 2 groups according to the weight results, 7 in the sham operation group and 31 in the model group. All mice were anesthetized by intraperitoneal injection of pentobarbital sodium solution (50 mg/kg), the midline skin of the back was incised, the back T5-T8 muscles were separated, and the T6-T7 laminae were removed. In the model group, a microvascular clamp (30g, Kent Scientific Corporation) was clamped on the T6-T7 spinal cord for 1 minute. The forceps should be inserted into the vertebral channel with the tip at the top of the lamina, forming a 90° angle ^[5] ; in the sham operation group, only Remove the lamina to avoid dural damage. All mice were sutured immediately after surgery, intramuscularly injected with Tolfedine (0.1 mg/kg) and penicillin potassium (40,000 units/kg), and then placed on a heating pad until they woke up. Infection was prevented during the operation. After the operation, the mice were manually assisted to urinate until they urinate spontaneously, and the mice were observed for 7 days. Seven days after surgery, all mice were evaluated for body weight and BMS score (two people performed blind scoring respectively) ^[5] . According to the body weight and BMS score, the mice in the model group were randomly divided into three groups: Group A and Group B. group and vehicle control group and started administration, which was designated as the first day of administration. The mice in group A were injected with plasminogen solution into the tail vein at 1 mg/100 μl/mouse, and the mice in group B were administered at 0.5 mg/100 μl. Plasminogen solution was injected into the tail vein of each mouse, and mice in the vehicle control group were injected with 100 μl of vehicle solution into the tail vein for 6 days. On the 7th day, the Von-Frey mechanical tenderness test was performed on the left leg of the mice.

The results showed that the mechanical threshold of mice in the vehicle group was significantly higher than that of mice in the sham surgery group, indicating that the pain sensation of mice was lost after the establishment of the spinal cord compression injury model; the pain threshold of mice in the drug administration group A (1 mg) and drug administration group B (0.5 mg) was significantly lower than that in the vehicle group, and the statistical difference was significant (* indicates P < 0.05, ** indicates P < 0.01) (Figure 6). This result shows that fibronectin can promote pain recovery in mice with spinal cord compression injury model.

Embodiment 7 Plasminogen promotes neurofilament protein expression in the spinal cord of paraplegic mouse models

Thirty-two C57 female mice aged 8-10 weeks were weighed before modeling. All mice were randomly divided into two groups according to their weight, 8 mice in the sham operation group and 24 mice in the model group. The model group mice were lightly anesthetized with 50% chlorpheniramine before surgery, and then the mice were completely anesthetized using respiratory anesthesia and placed in a prone position on a respiratory anesthesia machine with 2.5% isoflurane turned on. The skin and muscles were opened during surgery to expose the T9 and T10 segments, and the laminectomy was performed. After the spinal cord was exposed, the right half of the spinal cord between T9 and T10 was transversely cut along the coronal plane to create a semi-transverse injury, and the wound was sutured after completion. The mice in the sham operation group were anesthetized in the same way, and the laminectomy was performed, followed by direct suture. All mice were placed on a heating pad after surgery until they woke up. Postoperative care included daily injections of the analgesic drug Tolfedine and penicillin potassium (40,000 units/kg) for 4 days; mice were ensured to have access to food and water, and urinated until the bladder's autonomous urination function was restored ^[6] . One week after the surgical modeling was completed, the postoperative mice were tested for behavioral behavior and scored. Based on the test results, the model group mice were randomly divided into two groups, a vehicle group and a drug group, with 12 mice in each group; the sham surgery group mice were not grouped. After the grouping was completed, the vehicle group and the sham surgery group mice were injected with the vehicle intravenously at a dose of 0.1 ml/mouse/day, and the drug group mice were injected with 1 mg/mouse/day of fibronectin intravenously. This was recorded as the first day of drug administration, and the drug administration was continued for 28 days. On the 29th day, the mice were sacrificed, and the spinal cords were fixed in 4% paraformaldehyde. The fixed tissue samples were dehydrated with alcohol gradient and transparentized with xylene before being embedded in paraffin. The thickness of the tissue sections was 3 μm, and the sections were dewaxed and rehydrated and then washed once. The sections were fixed with citric acid for 30 minutes, cooled at room temperature for 10 minutes, and then gently rinsed with water. The sections were incubated with 3% hydrogen peroxide for 15 minutes, and the tissues were circled with a PAP pen. The sections were sealed with 10% sheep serum (Vector laboratories, Inc., USA) for 1 hour; after the time was up, the sheep serum was discarded. The rabbit anti-neurofilament protein (NFP) antibody (Abcam, ab207176) was incubated overnight at 4°C and washed twice with PBS for 5 minutes each time. The goat anti-rabbit IgG (HRP) antibody (Abcam) was incubated with secondary antibody at room temperature for 1 hour, and then washed twice with PBS for 5 minutes each time. The color was developed according to the DAB reagent kit (Vector laboratories, Inc., USA), washed three times with water, counterstained with hematoxylin for 30 seconds, and returned to blue in running water for 5 minutes, and then washed once with PBS. Gradient dehydration was performed to make it transparent and the slides were mounted, and the sections were observed under a 400x optical microscope.

The results showed that the spinal cord of the sham surgery group (Figure 7A) expressed a certain level of NFP (indicated by arrows), the expression of NFP in the spinal cord of the vehicle group (Figure 7B) was significantly lower than that of the sham surgery group, and the expression of NFP in the spinal cord of the drug group (Figure 7C) was significantly higher than that of the vehicle group, and the statistical difference was significant (* indicates P < 0.05) (Figure 7D). This suggests that fibronectin can promote the expression of NFP in the spinal cord of mice with spinal cord transection model.

Embodiment 8 Plasminogen improves hindlimb motor function in paraplegic model mice

Forty-two C57 female mice aged 8 to 10 weeks were weighed before modeling. All mice were randomly divided into 2 groups according to their weight, with 14 mice in the sham operation group and 28 mice in the model group. Take the experimental mice, remove the fur on the back of the mice after pre-anesthesia, and then use respiratory anesthesia to completely anesthetize the mice. Place them in a prone position on a respiratory anesthesia machine with 2.5% isoflurane turned on. The skin and muscles are opened surgically, the T9 and T10 segments are exposed, and laminectomy is performed. After the spinal cord is exposed, the right half of the spinal cord between T9 and T10 is transected along the coronal plane to cause a semi-transverse injury. After completion, the wound is sutured. After the mice in the sham operation group were anesthetized in the same way, the skin and muscles were opened surgically, the T9 and T10 segments were exposed, and then sutured directly. The analgesic Tolfedine and penicillin potassium (200,000 units/kg, 80,000 units/ml) were administered before surgery and 24 and 72 hours after surgery. All mice after surgery should be placed on a heating pad. The neck should be kept level and the neck should not be bent until the mouse wakes up. Postoperative care: Normal animals require about 40-80ml/kg of fluid per day. After waking up from anesthesia, if the mouse is found unable to eat or drink, it must be given intraperitoneal injection of normal saline to replenish body fluids. Help mice excrete urine to restore the bladder's autonomous urination function ^[6] . In order to prevent the animal's wound from being contaminated by feces, urine, and bedding on the day after surgery, a clean wiping paper needs to be placed on the bedding in the PC box. Three days after the completion of the surgical modeling, the open field behavior of the postoperative mice was tested and scored. According to the test results, the 28 mice in the model group were randomly divided into two groups, the vehicle group and the drug administration group. Each group had 14 mice; mice in the sham operation group will not be grouped. After the grouping was completed, the mice in the vehicle group and the sham operation group were injected with vehicle into the tail vein at 0.1ml/mouse/day, and the mice in the medication group were injected with plasminogen into the tail vein at 1mg/mouse/day, which was recorded as the first day of administration. , continued administration for 7 days. On the 8th day, perform the Basso Mouse Scale (BMS) test according to Table 1.

The BMS score is specifically used to evaluate the changes in hindlimb function of mice with spinal cord injury, including the main and secondary scoring systems: the main score is scored after observing the mouse's hindlimb ankle joint mobility, coordination, paw posture, trunk stability and tail posture. The secondary score is a supplementary correction to the main score based on coordination, stability, etc. ^[7]

BMS (Basso Mouse Scale) score 0 marks No joint movement 1 point slight joint movement 2 minutes Large range of motion 3 points The soles of the paws are on the ground or there is no weight support, or occasionally, frequently, or always relying on the back to move, but not walking 4 points Occasionally walk on the soles of your feet 5 points Frequent or always walking on the soles of the feet, poor coordination; Frequent or always walking on the soles of the feet, with some coordination, and the paws can rotate when the soles of the feet first come into contact 6 points Frequent or constant walking with a certain degree of coordination, the soles of the feet are parallel when making the first contact; constant walking with good coordination, the claws can rotate when the soles of the feet make the first contact 7 points Frequent or constant walking on the soles of the feet, good coordination, and the soles of the feet are parallel; Frequent or consistent walking on the soles of the feet, good coordination, and the soles of the feet are parallel, and severe limb balance 8 points Frequently or always walking on the soles of the feet, with good coordination, the soles of the feet are parallel, and a slight limb balance; Frequently or always walking on the soles of the feet, with good coordination, the soles of the feet are parallel, with a slight limb balance, normal limb balance and tail direction down or both up or down 9 points Frequent or constant walking on soles, good coordination, soles of feet parallel, slight limb balance, tail always up

The results showed that the BMS score of the mice in the prosthetic hand group was 9 points. The BMS score of the mice in the vehicle group was 6.75±1.92 points, which was significantly lower than that of the mice in the sham operation group. The BMS score of the mice in the drug administration group was 8.29±0.39 points, which was significantly higher. mice in the vehicle group, and the statistical difference was significant (* indicates P < 0.05, *** indicates P < 0.001) (Figure 8). It is suggested that plasminogen can promote the recovery of hindlimb motor function in paraplegic model mice.

Example 9 Fibrinolysin promotes the transcription of fibrolysin gene in the spinal cord of paralysis model mice

Fifteen 8-week-old female C57 mice were weighed before modeling. All mice were randomly divided into two groups according to their weight, a blank control group of 5 mice and a model group of 10 mice. The experimental mice were taken, and the hair on the back of the mice was removed after pre-anesthesia. After that, the mice were completely anesthetized using respiratory anesthesia and placed in a prone position on a respiratory anesthesia machine with 2.5% isoflurane turned on. The skin and muscles were opened surgically to expose the T9 and T10 segments, and the laminectomy was performed. After the spinal cord was exposed, the right half of the spinal cord between T9 and T10 was transversely cut along the coronal plane to create a hemisection injury, and the wound was sutured after completion. Mice in the blank control group were anesthetized in the same way. The skin and muscles were opened surgically to expose the T9 and T10 segments, and then sutured directly ^[6]. Analgesics Tolfedine and penicillin potassium (200,000 units/kg and 80,000 units/ml) were given before surgery and 24 and 72 hours after surgery. All mice were placed on a heating pad after surgery. The necks were kept horizontal and not bent until the mice woke up. Postoperative care: Normal animals require about 40-80 ml/kg of fluid per day. After awakening from anesthesia, if the mice are unable to eat or drink water, they must be given physiological saline intraperitoneally to replenish body fluids. Help the mice to urinate until the bladder's autonomous urination function is restored. To prevent the wound of the animal from being contaminated by feces, urine, and padding on the day after surgery, a clean wipe must be placed on the padding inside the PC box. Three days after the completion of the surgical modeling, the postoperative mice were tested for behavior and scored. According to the test results, the 10 mice in the model group were randomly divided into two groups, 5 mice in the vehicle group and 5 mice in the drug group; the blank control group was not grouped. After the grouping was completed, all experimental mice were injected with a dose of 50 mg/kg/day through the tail vein. The drug group was injected with 10 mg/ml of fibrolysin solution, and the blank control group and the vehicle group were injected with the solvent. Six hours after drug administration, the mice were sacrificed, and the spinal cord homogenate at the modeling site was obtained for RT-PCR detection of Plg gene transcription mRNA, and the CT value was recorded. The primer information is shown in Table 1 below. Calculate the CT value of Plg gene transcription mRNA in 200 ng total RNA.

Target Primer name Primers Length Plg Plg-F (sequence 15) GACCAGTCAGATTCCTCAGTTC 117 Plg-R (sequence 16) CTTCTTCCCTGTGATGGTAGTG

CT value: indicates the number of cycles that each PCR reaction tube goes through when the fluorescent signal reaches the set threshold. Studies have shown that there is a linear relationship between the CT value of each template and the logarithm of the initial copy number of the template. The more the initial copy number, the smaller the CT value. And vice versa.

The results showed that the spinal cord of mice in the blank control group had a certain level of Plg mRNA, and the spinal cord Plg mRNA level of mice in the drug-treated group was significantly higher than that in the vehicle group, and the statistical difference was close to significant (P=0.051) (Figure 9). This suggests that fibronectin can promote the transcription of fibronectin gene in the spinal cord of paralysis model mice.

Example 10 Fibrolysin promotes the expression of synaptophysin in the injured spinal cord of paralysis mice

42 C57 female mice aged 8-10 weeks were weighed before modeling. All mice were randomly divided into two groups according to their weight, 14 mice in the sham operation group and 28 mice in the model group. The experimental mice were taken, and the hair on the back of the mice was removed after pre-anesthesia. Then, the mice were completely anesthetized using respiratory anesthesia and placed in a prone position on a respiratory anesthesia machine with 2.5% isoflurane turned on. The skin and muscles were opened surgically to expose the T9 and T10 segments, and the laminectomy was performed. After the spinal cord was exposed, the right half of the spinal cord between T9 and T10 was transversely cut along the coronal plane to create a hemisection injury, and the wound was sutured after completion. The mice in the sham operation group were anesthetized in the same way, and the skin and muscles were opened surgically to expose the T9 and T10 segments, and then sutured directly. Tolfedine and potassium penicillin (200,000 units/kg and 80,000 units/ml) were administered before surgery and 24 and 72 hours after surgery. All mice were placed on a heating pad after surgery. The necks were kept horizontal and not bent until the mice woke up. Postoperative care: Normal animals require about 40-80 ml/kg of fluid per day. After awakening from anesthesia, if the mice are unable to eat or drink, they must be given saline intraperitoneally to replenish body fluids. Help the mice to urinate until the bladder's autonomous urination function is restored [6]. To prevent the wound of the animal from being contaminated by feces, urine, and padding on the day after surgery, a clean wipe must be placed on the padding inside the PC box. Three days after the surgical modeling, the postoperative mice were tested for behavior and scored. According to the test results, the 28 mice in the model group were randomly divided into two groups, the vehicle group and the drug group, with 14 mice in each group; the mice in the sham group were not grouped. After the grouping was completed, the mice in the vehicle group and the sham group were injected with the solvent through the tail vein at 0.1ml/mouse/day, and the mice in the drug group were injected with 1mg/0.1ml/mouse/day of fibrolysinogen through the tail vein, which was recorded as the first day of drug administration, and the drug administration was continued for 28 days. On the 30th day, the mice were sacrificed and the spinal cords at the injured site were fixed in 10% formaldehyde solution. The fixed tissue samples were dehydrated with alcohol gradient and transparentized with xylene before being embedded in paraffin. The thickness of the tissue sections was 3 μm. The sections were dewaxed and washed once. The sections were fixed with citric acid for 30 minutes, cooled at room temperature for 10 minutes, and then gently rinsed with water. The sections were incubated with 3% hydrogen peroxide for 15 minutes, and the tissues were circled with a PAP pen. The sections were sealed with 10% goat serum (Vector laboratories, Inc., USA) for 1 hour; after the time was up, the goat serum was discarded. The rabbit anti-synaptophysin protein antibody (Wuhan Sanying, 10842-1-AP) was incubated at 4°C overnight, and washed twice with PBS for 5 minutes each time. The goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody was incubated at room temperature for 1 hour, and washed twice with PBS for 5 minutes each time. The cells were stained with DAB reagent kit (Vector laboratories, Inc., USA), washed 3 times with water, counterstained with hematoxylin for 30 seconds, washed with running water to blue for 5 minutes, and then washed once with PBS. The cells were dehydrated to make them transparent and sealed, and the sections were observed under a 200x optical microscope.

Synaptophysin is also known as synaptic vesicle protein, synaptophysin, synaptosome, etc. It is a calcium-binding protein with a molecular weight of 38KDa located on the synaptic vesicle membrane. It is a membrane protein closely related to synaptic structure and function. It is widely present in all nerve terminals of the body and is specifically distributed on the presynaptic vesicle membrane. It participates in calcium-dependent neurotransmitter release and the circulation of synaptic vesicles and is considered to be an important marker of synapse occurrence and synaptic remodeling [8].

The results showed that the spinal cord of the mice in the sham operation group (Figure 10A) expressed a certain level of synaptophysin (marked by the arrow), and the level of synaptophysin in the spinal cord of the mice in the vehicle group (Figure 10B) at the modeling site was significantly lower than that of the mice in the sham operation group. The statistical difference between the two groups was extremely significant (*** indicates P<0.001). The level of synaptophysin in the spinal cord of the mice in the drug administration group (Figure 10C) was significantly higher than that in the vehicle group, and the statistical difference was significant (* indicates P<0.05) (Figure 10D). This shows that plasminogen can promote the expression of synaptophysin in the injured spinal cord of paraplegic model mice and promote the repair of spinal cord injury.

Example 11 Fibrolysinogen promotes the recovery of spinal cord neuron function in paralysis model mice

42 C57 female mice aged 8-10 weeks were weighed before modeling. All mice were randomly divided into two groups according to their weight, 14 mice in the sham operation group and 28 mice in the model group. The experimental mice were taken, and the hair on the back of the mice was removed after pre-anesthesia. Then, the mice were completely anesthetized using respiratory anesthesia and placed in a prone position on a respiratory anesthesia machine with 2.5% isoflurane turned on. The skin and muscles were opened surgically to expose the T9 and T10 segments, and the laminectomy was performed. After the spinal cord was exposed, the right half of the spinal cord between T9 and T10 was transversely cut along the coronal plane to create a hemisection injury, and the wound was sutured after completion. The mice in the sham operation group were anesthetized in the same way, and the skin and muscles were opened surgically to expose the T9 and T10 segments, and then sutured directly. Tolfedine and potassium penicillin (200,000 units/kg and 80,000 units/ml) were administered before surgery and 24 and 72 hours after surgery. All mice were placed on a heating pad after surgery. The necks were kept horizontal and not bent until the mice woke up. Postoperative care: Normal animals require about 40-80 ml/kg of fluid per day. After awakening from anesthesia, if the mice are unable to eat or drink, they must be given saline intraperitoneally to replenish body fluids. Help the mice to urinate until the bladder's autonomous urination function is restored [6]. To prevent the wound of the animal from being contaminated by feces, urine, and padding on the day after surgery, a clean wipe must be placed on the padding inside the PC box. Three days after the completion of the surgical modeling, the postoperative mice were tested for behavioral tests and scored. According to the test results, the 28 mice in the model group were randomly divided into two groups, the vehicle group and the drug group, with 14 mice in each group; the mice in the sham group were not grouped. After the grouping was completed, the mice in the vehicle group and the sham group were injected with the solvent by the tail vein at 0.1ml/mouse/day, and the mice in the drug group were injected with 1mg/mouse/day of fibrolysinogen by the tail vein, which was recorded as the first day of drug administration, and the drug administration was continued for 28 days. The fixed striatal tissue was dehydrated with alcohol gradient and transparentized with xylene before being embedded in paraffin. The thickness of the tissue section was 3μm. After dewaxing to water, it was stained with 0.4% tar violet stain (pH=3). Dehydrate with gradient alcohol, make transparent with xylene, and seal with neutral resin. The sections were observed and photographed under a 400x optical microscope.

Nissl bodies, also known as chromatin, are structures unique to nerve cells. They are composed of many parallel-arranged rough endoplasmic reticulum and free ribosomes during distribution. They have the function of synthesizing proteins. Their number and distribution are similar to those of neurons. Functional status is closely related and is regarded as a marker of nerve cell activity ^[9] .

The results showed that the spinal cord of mice in the sham operation group (Figure 11A) had a certain level of Nissl bodies (marked by arrows). The level of Nissl bodies in the spinal cord of the mice in the vehicle group (Figure 11B) was significantly higher than that of the mice in the sham operation group. The statistical difference between groups is extremely significant (*** indicates P<0.001). The level of Nissl bodies in the spinal cord of mice in the drug administration group (Figure 11C) was significantly lower than that in the vehicle group, and the statistical difference was significant (** indicates P<0.01) (Figure 11D). It shows that plasminogen can promote the recovery of Nissl body levels in the injured spinal cord of paraplegic model mice and promote the repair of spinal cord injury.

Sequence list:

Sequence 1:

gagcctctggatgactatgtgaatacccagggggcttcactgttcagtgtcactaagaagcagctgggagcaggaagtatagaagaatgtgcagcaaaatgtgaggaggacgaagaattcacctgcagggcattccaatatcacagtaaagagcaacaatgtgtgataatggctgaaaacaggaagtcctccataatcattaggatgagagatg tagttttatttgaaaagaaagtgtatctctcagagtgcaagactgggaatggaaagaactacagagggacgatgtccaaaacaaaaaatggcatcacctgtcaaaaatggagttccacttctccccacagacctagattctcacctgctacacacccctcagagggactggaggagaactactgcaggaatccagacaacgatccgcaggggccctggtg ctatactactgatccagaaaagagatatgactactgcgacattcttgagtgtgaagaggaatgtatgcattgcagtggagaaaactatgacggcaaaatttccaagaccatgtctggactggaatgccaggcctgggactctcagagcccacacgctcatggatacattccttccaaatttccaaacaagaacctgaagaagaattactgtcgtaaccccga tagggagctgcggccttggtgtttcaccaccgaccccaacaagcgctgggaactttgtgacatcccccgctgcacaacacctccaccatcttctggtcccacctaccagtgtctgaagggaacaggtgaaaactatcgcgggaatgtggctgttaccgtgtccgggcacacctgtcagcactggagtgcacagacccctcacacacata acaggacaccagaaaacttcccctgcaaaaatttggatgaaaactgccgcaatcctgacggaaaaagggccccatggtgccatacaaccaacagccaagtgcggtgggagtactgtaagataccgtcctgtgactcctccccagtatccacggaacaattggctcccacagcaccacctgagctaacccctgtggtccaggactgctaccat ggtgatggacagagctaccgaggcacatcctccaccaccaccacaggaaagaagtgtcagtcttggtcatctatgacaccacaccggcaccagaagaccccagaaaactacccaaatgctggcctgacaatgaactactgcaggaatccagatgccgataaaggcccctggtgttttaccacagaccccagcgtcaggtgggagtactgcaacctgaaaaa atgctcaggaacagaagcgagtgttgtagcacctccgcctgttgtcctgcttccagatgtagagactccttccgaagaagactgtatgtttgggaatgggaaaggataccgaggcaagagggcgaccactgttatgggacgccatgccaggactgggctgcccaggagccccatagacacagcattttcactccagagacaaatccacgggcg ggtctggaaaaaattactgccgtaaccctgatggtgatgtaggtggtccctggtgctacacgacaaatccaagaaaactttacgactactgtgatgtccctcagtgtgcggccccttcatttgattgtgggaagcctcaagtggagccgaagaaatgtcctggaagggttgtaggggggtgtgtggcccacccacattcct ggccctggcaagtcagtcttagaacaaggtttggaatgcacttctgtggaggcaccttgatatccccagagtgggtgttgactgctgcccactgcttggagaagtccccaaggccttcatcctacaaggtcatcctgggtgcaccaagaagtgaatctcgaaccgcatgttcaggaaatagaagtgtctaggctgttct tggagcccacgaaaagatattgccttgctaaagctaagcagtcctgccgtcatcactgacaaagtaatcccagcttgtctgccatccccaaattatgtggtcgctgaccggaccgaatgtttcatcactggctggggagaaacccaaggtacttttggagctggccttctcaaggaagcccagctccctgtgattgagaataaagt gtgcaatcgctatgagtttctgaatggaagagtccaatccaccgaactctgtgctgggcatttggccggaggcactgacagttgccagggtgacagtggaggtcctctggtttgcttcgagaaggacaaatacattttacaaggagtcacttcttggggtcttggctgtgcacgccccaataagcctggtgtctatgtt cgtgtttcaaggtttgttatacttggattgagggagtgatgagaaataattaa

Sequence 2:

EPLDDYVNTQGASLFSVTKKQLGAGSIEECAAKCEEDEEFTCRAFQYHSKEQQCVIMAENRKSSIIIRMRDVVLFEKKVYLSECKTGNGKNYRGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEGLEENYCRNPDNDPQGPWCYTTDPEKRYDYCDILECEEECMHCSGENYDGKISKTMSGLECQAWDSQSPHAHGYIPSKFPNKNLKKNYCRNP DRELRPWCFTTDPNKRWELCDIPRCTTPPPSSGPTYQCLKGTGENYRGNVAVTVSGHTCQHWSAQTPHTHNRTPENFPCKNLDENYCRNPDGKRAPWCHTTNSQVRWEYCKIPSCDSSPVSTEQLAPTAPPELTPVVQDCYHGDGQSYRGTSSTTTTGKKCQSWSSMTPHRHQKTPENYPNAGLTMNYCRNPDADKGPWCFTTDPSVRWEYCNLKKCSGTEASVV APPPVVLLPDVETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVI PACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN

Sequence 3:

atggaacataaggaagtggttcttctacttcttttatttctgaaatcaggtcaaggagagcctctggatgactatgtgaatacccagggggcttcactgttcagtgtcactaagaagcagctgggagcaggaagtatagaagaatgtgcagcaaaatgtgaggaggacgaagaattcacctgcagggcattccaatatcacagtaaagagcaacaatgtgtgataatggctgaaaacaggaagtcctccataatcattaggatgagagatgtagttttatttgaaaagaaagtgtatctctcag agtgcaagactgggaatggaaagaactacagagggacgatgtccaaaacaaaaaatggcatcacctgtcaaaaatggagttccacttctccccacagacctagattctcacctgctacacacccctcagagggactggaggagaactactgcaggaatccagacaacgatccgcaggggccctggtgctatactactgatccagaaaagagatatgactactgcgacattcttgagtgtgaagaggaatgtatgcattgcagtggagaaaactatgacggcaaaatttccaagaccatgtctgg actggaatgccaggcctgggactctcagagcccacacgctcatggatacattccttccaaatttccaaacaagaacctgaagaagaattactgtcgtaaccccgatagggagctgcggccttggtgtttcaccaccgaccccaacaagcgctgggaactttgtgacatcccccgctgcacaacacctccaccatcttctggtcccacctaccagtgtctgaagggaacaggtgaaaactatcgcgggaatgtggctgttaccgtgtccgggcacacctgtcagcactggagtgcacagacccct cacacacataacaggacaccagaaaacttcccctgcaaaaatttggatgaaaactactgccgcaatcctgacggaaaaagggccccatggtgccatacaaccaacagccaagtgcggtgggagtactgtaagataccgtcctgtgactcctccccagtatccacggaacaattggctcccacagcaccacctgagctaacccctgtggtccaggactgctaccatggtgatggacagagctaccgaggcacatcctccaccaccaccacaggaaagaagtgtcagtcttggtcatctatgacac cacaccggcaccagaagaccccagaaaactacccaaatgctggcctgacaatgaactactgcaggaatccagatgccgataaaggcccctggtgttttaccacagaccccagcgtcaggtgggagtactgcaacctgaaaaaatgctcaggaacagaagcgagtgttgtagcacctccgcctgttgtcctgcttccagatgtagagactccttccgaagaagactgtatgttttgggaatgggaaaggataccgaggcaagagggcgaccactgttactgggacgccatgccaggactgggctgc ccaggagccccatagacacagcattttcactccagagacaaatccacgggcgggtctggaaaaaaattactgccgtaaccctgatggtgatgtaggtggtccctggtgctacacgacaaatccaagaaaactttacgactactgtgatgtccctcagtgtgcggccccttcatttgattgtgggaagcctcaagtggagccgaagaaatgtcctggaagggttgtaggggggtgtgtggcccacccacattcctggccctggcaagtcagtcttagaacaaggtttggaatgcacttctgtgga ggcaccttgatatccccagagtgggtgttgactgctgcccactgcttggagaagtccccaaggccttcatcctacaaggtcatcctgggtgcacaccaagaagtgaatctcgaaccgcatgttcaggaaatagaagtgtctaggctgttcttggagcccacacgaaaagatattgccttgctaaagctaagcagtcctgccgtcatcactgacaaagtaatcccagcttgtctgccatccccaaattatgtggtcgctgaccggaccgaatgtttcatcactggctggggagaaacccaaggta cttttggagctggccttctcaaggaagcccagctccctgtgattgagaataaagtgtgcaatcgctatgagtttctgaatggaagagtccaatccaccgaactctgtgctgggcatttggccggaggcactgacagttgccagggtgacagtggaggtcctctggtttgcttcgagaaggacaaatacattttacaaggagtcacttcttggggtcttggctgtgcacgccccaataagcctggtgtctatgttcgtgtttcaaggtttgttacttggattgagggagtgatgagaaataattaa

Sequence 4:

MEHKEVVLLLLLFLKSGQGEPLDDYVNTQGASLFSVTKKQLGAGSIEECAAKCEEDEEFTCRAFQYHSKEQQCVIMAENRKSSIIIRMRDVVLFEKKVYLSECKTGNGKNYRGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEGLEENYCRNPDNDPQGPWCYTTDPEKRYDYCDILECEEECMHCSGENYDGKISKTMSGLECQAWDSQSPHA HGYIPSKFPNKNLKKNYCRNPDRELRPWCFTTDPNKRWELCDIPRCTTPPPSSGPTYQCLKGTGENYRGNVAVSGHTCQHWSAQTPHTHNRTPENFPCKNLDENYCRNPDGKRAPWCHTTNSQVRWEYCKIPSCDSSPVSTEQLAPTAPPELTPVVQDCYHGDGQSYRGTSSTTTTGKKCQSWSSMTPHRHQKTPENYPNAGLTMNYCRNPDADKGPWCFTTDPS VRWEYCNLKKCSGTEASVVAPPPVVLLPDVETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTR KDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN

Sequence 5:

aaagtgtatctctcagagtgcaagactgggaatggaaagaactacagagggacgatgtccaaaacaaaaaatggcatcacctgtcaaaaatggagttccacttctccccacagacctagattctcacctgctacacacccctcagagggactggaggagaactactgcaggaatccagacaacgatccgcaggggccctggtgctatactactgatccagaaaagagatatgactactgcgacattcttgagtgtgaagaggaatgta tgcattgcagtggagaaaactatgacggcaaaatttccaagaccatgtctggactggaatgccaggcctgggactctcagagcccacacgctcatggatacattccttccaaatttccaaacaagaacctgaagaagaattactgtcgtaaccccgatagggagctgcggccttggtgtttcaccaccgaccccaacaagcgctgggaactttgtgacatccccgctgcacaacacctccaccatcttctggtcccacctaccagtg tctgaagggaacaggtgaaaactatcgcgggaatgtggctgttaccgtgtccgggcacacctgtcagcactggagtgcacagacccctcacacacataacaggacaccagaaaacttcccctgcaaaaatttggatgaaaactactgccgcaatcctgacggaaaaagggccccatggtgccatacaaccaacagccaagtgcggtgggagtactgtaagataccgtcctgtgactcctccccagtatccacggaacaattggctccc acagcaccacctgagctaacccctgtggtccaggactgctaccatggtgatggacagagctaccgaggcacatcctccaccaccaccacaggaaagaagtgtcagtcttggtcatctatgacaccacaccggcaccagaagaccccagaaaactacccaaatgctggcctgacaatgaactactgcaggaatccagatgccgataaaggcccctggtgttttaccacagaccccagcgtcaggtgggagtactgcaacctgaaaaaat gctcaggaacagaagcgagtgttgtagcacctccgcctgttgtcctgcttccagatgtagagactccttccgaagaagactgtatgtttgggaatgggaaaggataccgaggcaagagggcgaccactgttactgggacgccatgccaggactgggctgcccaggagccccatagacacagcattttcactccagagacaaatccacgggcgggtctggaaaaaaattactgccgtaaccctgatggtgatgtaggtggtccctggtg ctacacgacaaatccaagaaaactttacgactactgtgatgtccctcagtgtgcggccccttcatttgattgtgggaagcctcaagtggagccgaagaaatgtcctggaagggttgtaggggggtgtgtggcccacccacattcctggccctggcaagtcagtcttagaacaaggtttggaatgcacttctgtggaggcaccttgatatccccagagtgggtgttgactgctgcccactgcttggagaagtccccaaggccttcatcc tacaaggtcatcctgggtgcacaccaagaagtgaatctcgaaccgcatgttcaggaaatagaagtgtctaggctgttcttggagcccacacgaaaagatattgccttgctaaagctaagcagtcctgccgtcatcactgacaaagtaatcccagcttgtctgccatccccaaattatgtggtcgctgaccggaccgaatgtttcatcactggctggggagaaacccaaggtacttttggagctggccttctcaaggaagcccagctcc ctgtgattgagaataaagtgtgcaatcgctatgagtttctgaatggaagagtccaatccaccgaactctgtgctgggcatttggccggaggcactgacagttgccagggtgacagtggaggtcctctggtttgcttcgagaaggacaaatacattttacaaggagtcacttcttggggtcttggctgtgcacgccccaataagcctggtgtctatgttcgtgtttcaaggtttgttacttggattgagggagtgatgagaaataattaa

Sequence 6:

KVYLSECKTGNGKNYRGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEGLEENYCRNPDNDPQGPWCYTTDPEKRYDYCDILECEEECMHCSGENYDGKISKTMSGLECQAWDSQSPHAHGYIPSKFPNKNLKKNYCRNPDRELRPWCFTTDPNKRWELCDIPRCTTPPPSSGPTYQCLKGTGENYRGNVAVTVSGHTCQHWSAQTPHTHNRTPENFPCKNLDENYCRNPDGKRAPWCHTTNSQVRWEYCKIPSCDSSPVSTEQLAPTAPPELTPVVQDCYHGDGQSYRGTSSTTTTGKKCQSWSSMTPHRHQKTPENYPNAGLTMNYCRNPDADKGPWCFTTDPSVRWEYCNLKK CSGTEASVVAPPPVVLLPDVETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN

Sequence 7:

gagcctctggatgactatgtgaatacccagggggcttcactgttcagtgtcactaagaagcagctgggagcaggaagtatagaagaatgtgcagcaaaatgtgaggaggacgaagaattcacctgcagggcattccaatatcacagtaaagagcaacaatgtgtgataatggctgaaaacaggaagtcctccataatcattaggatgagagatgtagttttatttgaaaagaaagtgtatctctcagagtgcaagactgggaatggaaagaactacagagggacgatgtccaaaacaaaaaatggcatcac ctgtcaaaaatggagttccacttctccccacagacctagattctcacctgctacacacccctcagagggactggaggagaactactgcaggaatccagacaacgatccgcaggggccctggtgctatactactgatccagaaaagagatatgactactgcgacattcttgagtgtgaagaggcggccccttcatttgattgtgggaagcctcaagtggagccgaagaaatgtcctggaagggttgtaggggggtgtgtggcccacccacattcctggccctggcaagtcagtcttagaacaaggtttggaa tgcacttctgtggaggcaccttgatatccccagagtgggtgttgactgctgcccactgcttggagaagtccccaaggccttcatcctacaaggtcatcctgggtgcacaccaagaagtgaatctcgaaccgcatgttcaggaaatagaagtgtctaggctgttcttggagcccacacgaaaagatattgccttgctaaagctaagcagtcctgccgtcatcactgacaaagtaatcccagcttgtctgccatccccaaattatgtggtcgctgaccggaccgaatgtttcatcactggctggggagaaacc caaggtacttttggagctggccttctcaaggaagcccagctccctgtgattgagaataaagtgtgcaatcgctatgagtttctgaatggaagagtccaatccaccgaactctgtgctgggcatttggccggaggcactgacagttgccagggtgacagtggaggtcctctggtttgcttcgagaaggacaaatacattttacaaggagtcacttcttggggtcttggctgtgcacgccccaataagcctggtgtctatgttcgtgtttcaaggtttgttacttggattgagggagtgatgagaaataattaa

Sequence 8:

EPLDDYVNTQGASLFSVTKKQLGAGSIEECAAKCEEDEEFTCRAFQYHSKEQQCVIMAENRKSSIIIRMRDVVLFEKKVYLSECKTGNGKNYRGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEGLEENYCRNPDNDPQGPWCYTTDPEKRYDYCDILECEEAAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN

Sequence 9:

gtcaggtgggagtactgcaacctgaaaaaatgctcaggaacagaagcgagtgttgtagcacctccgcctgttgtcctgcttccagatgtagagactccttccgaagaagactgtatgtttgggaatgggaaaggataccgaggcaagagggcgaccactgttatgggacgccatgccaggactgggctgcccaggagccccatagaca cagcattttcactccagagacaaatccacgggcgggtctggaaaaaaattactgccgtaaccctgatggtgatgtaggtggtccctggtgctacacgacaaatccaagaaaactttacgactactgtgatgtccctcagtgtgcggccccttcatttgattgtgggaagcctcaagtggagccgaagaaatgtcctggaagggt tgtaggggggtgtgtggcccacccacattcctggccctggcaagtcagtcttagaacaaggtttggaatgcacttctgtggaggcaccttgatatccccagagtgggtgttgactgctgcccactgcttggagaagtccccaaggccttcatcctacaaggtcatcctgggtgcaccaagaagaagtgaatctcgaaccgcatg ttcaggaaatagaagtgtctaggctgttcttggagcccacacgaaaagatattgccttgctaaaagctaagcagtcctgccgtcatcactgacaaagtaatcccagcttgtctgccatccccaaattatgtggtcgctgaccggaccgaatgtttcatcactggctggggagaaacccaaggtacttttggagctggccttct caaggaagcccagctccctgtgattgagaataaagtgtgcaatcgctatgagtttctgaatggaagagtccaatccaccgaactctgtgctgggcatttggccggaggcactgacagttgccagggtgacagtggaggtcctctggtttgcttcgagaaggacaaatacattttacaaggagtcacttcttggggtcttggctgt gcacgccccaataagcctggtgtctatgttcgtgtttcaaggtttgttatacttggattgagggagtgatgagaaataattaa

Sequence 10:

VRWEYCNLKKCSGTEASVVAPPPVVLLPDVETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEP TRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN

Sequence 11:

gccccttcatttgattgtgggaagcctcaagtggagccgaagaaatgtcctggaagggttgtaggggggtgtgtgtggcccacccacattcctggccctggcaagtcagtcttagaacaaggtttggaatgcacttctgtggaggcaccttgatatccccagagtgggtgttgactgctgcccactgcttggagaagtccccaaggccttcatcctacaaggtcatcctgggtgcacaccaagaagtgaatctcgaaccgcatgttcaggaaatagaagtgtctaggctgttcttggagcccacacgaaaagatattgccttgctaaagctaagcagtcctgccgtcatcactgacaaagtaatcccagcttgtctg ccatccccaaattatgtggtcgctgaccggaccgaatgtttcatcactggctggggagaaacccaaggtacttttggagctggccttctcaaggaagcccagctccctgtgattgagaataaagtgtgcaatcgctatgagtttctgaatggaagagtccaatccaccgaactctgtgctgggcatttggccggaggcactgacagttgccagggtgacagtggaggtcctctggtttgcttcgagaaggacaaatacattttacaaggagtcacttcttggggtcttggctgtgcacgccccaataagcctggtgtctatgttcgtgtttcaaggtttgttacttggattgagggagtgatgagaaataattaa

Sequence 12:

APSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN

Sequence 13:

gttgtaggggggtgtgtgtggcccacccacattcctggccctggcaagtcagtcttagaacaaggtttggaatgcacttctgtggaggcaccttgatatccccagagtgggtgttgactgctgcccactgcttggagaagtccccaaggccttcatcctacaaggtcatcctgggtgcacaccaagaagtgaatctcgaaccgcatgttcaggaaatagaagtgtctaggctgttcttggagcccacacgaaaagatattgccttgctaaagctaagcagtcctgccgtcatcactgacaaagtaatcccagcttgtctgccatccccaaattatgtggtcgct gaccggaccgaatgtttcatcactggctggggagaaacccaaggtacttttggagctggccttctcaaggaagcccagctccctgtgattgagaataaagtgtgcaatcgctatgagtttctgaatggaagagtccaatccaccgaactctgtgctgggcatttggccggaggcactgacagttgccagggtgacagtggaggtcctctggtttgcttcgagaaggacaaatacattttacaaggagtcacttcttggggtcttggctgtgcacgccccaataagcctggtgtctatgttcgtgtttcaaggtttgttacttggattgagggagtgatgaga

Sequence 14:

VVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRF VTWIEGVMR

Sequence 15:

gaccagtcagattcctcagttc

Sequence 16:

cttcttccctgtgatggtagtg

References

[1] Suelen Adriani Marques. A simple, inexpensive and easily reproducible model of spinal cord injury in mice: Morphological and functional assessment. Journal of Neuroscience Methods 177 (2009) 183–193.

[2] Sun SW, Liang HF, Trinkaus K et al. Noninvasive detection of cuprizone induced axonal damage and demyelination in the mouse corpus callosum. Magn Reson Med. 2006 Feb;55(2):302-8.

[3]Vallières N1, Berard JL, David S et al. Systemic injections of lipopolysaccharide accelerates myelin phagocytosis during Wallerian degeneration in the injured mouse spinal cord. Glia. 2006 Jan 1;53(1):103-13.

[4]Gotow T. Neurofilaments in health and disease. Med Electron Microsc. 2000; 33 (4):173-99.

[5]Joshi M. Development and characterization of a graded, in vivo, compressive, murine model of spinal cord injury [J]. American Journal of Mathematics, 2000, 97(2):308-311.

[6] Wen-Ge Ding, Spinal Cord Injury Causes More Damage to Fracture Healing of Later Phase than Ovariectomy in Young Mice, Connective Tissue Research, 53(2): 142–148, (2012)

[7] RV Ung, NP Lapointe, Spontaneous recovery of hindlimb movement in completely spinal cord transected mice: a comparison of assessment methods and conditions, Spinal Cord (2007) 45, 367–379.

[8] Li GL, Farooque M, Isaksson J, Olsson Y. Changes in synapses and axons demonstrated by synaptophysin immunohistochemistry following spinal cord compression trauma in the rat and mouse. Biomed Environ Sci. 2004 Sep;17(3):281-90.

[9] HEBERT AE, DASH PK. Nonredundant roles for hippocampal and entorhinal cortical plasticity in spatial memory storage [J]. Pharmacology Biochemistry and Behavior, 2004, 79(1) :143-153.

without

Figure 1A-C Representative pictures of H&E staining of spinal cord in spinal cord compression injury model mice after administration of plasminogen for 7 days. A is the sham operation group, B is the control group given the vehicle PBS, and C is the plasminogen given group. The results showed that the neurons and nerve fibers in the sham operation group were in normal shape and the cytoplasm was red-stained; the control group given vehicle PBS and the group given plasminogen were collected from the junction of normal nerves and damaged nerves, and the control group given vehicle PBS were collected from the junction of normal nerves and damaged nerves. Large injury cavities (triangle marks) can be seen in the area, and the degree of nerve repair at the injured site in the plasminogen-administered group is significantly better than that in the vehicle PBS-administered control group, with no obvious cavities. This shows that plasminogen can significantly promote the repair of spinal cord injured nerves in mice with spinal cord compression injury.

Figure 2A-C Representative images of LFB staining of the spinal cord of mice with spinal cord compression injury model given fibrinolysin for 7 days. A is the sham surgery group, B is the vehicle PBS control group, and C is the fibrinolysin group. The results showed that the myelin sheath structure of the sham surgery group (Figure 2A) was intact and continuous; the myelin sheath of the vehicle PBS control group (Figure 2B) showed obvious large-scale disintegration (arrows), lighter blue, and myelin sheath rupture; compared with the vehicle PBS control group, the myelin sheath structure of the fibrinolysin group (Figure 2C) was significantly more intact, and the blue color was darker. This shows that fibrinolysin can promote the regeneration of myelin sheaths in mice with spinal cord compression model.

Figure 3A-C Representative images of spinal cord neurofilament protein (NFP) staining in mice with spinal cord compression injury model given fibrinolysin for 7 days. A is the sham surgery group, B is the vehicle PBS control group, and C is the fibrinolysin group. The results showed that the expression of NFP (indicated by arrows) in the fibrinolysin group was significantly higher than that in the vehicle PBS control group, and the NFP expression was closer to that of the sham surgery group. This shows that fibrinolysin can promote NFP expression and promote spinal cord neurofiber regeneration in mice with spinal cord compression injury model.

Figure 4 A-B Representative pictures of spinal H&E staining of spinal cord compression injury model Plg-/- mice given plasminogen for 7 days. A is the control group given PBS as vehicle, and B is the group given plasminogen. The results showed that the control group given vehicle PBS and the group given plasminogen were collected from the junction of normal nerves and injured nerves. It can be seen that the normal neurons and nerve fiber structures at the injured site disappeared and were replaced by repaired fibers and cells. The plasma was lightly stained and infiltrated by inflammatory cells. However, in the control group given vehicle PBS, larger damage cavities (triangular marks) could be seen in the junction area, while the degree of repair of the damaged area in the group given plasminogen was significantly higher than that in the control group given vehicle PBS. Good, no obvious cavities were seen, and inflammatory cell infiltration was significantly reduced. This shows that plasminogen can significantly promote the repair of spinal cord injury in the spinal cord compression nerve injury model Plg-/- mice.

Figure 5A-B Representative pictures of spinal cord LFB staining in spinal cord compression injury model Plg-/- mice given plasminogen for 7 days. A is the control group given PBS as vehicle, and B is the group given plasminogen. The results showed that the nerve myelin sheath in the control group given vehicle PBS showed obvious and larger area of disintegration (triangle mark), lighter blue, and myelin sheath breakage (arrow mark). In contrast, the myelin sheath in the plasminogen group was The sheath structure is obviously more complete and continuous, and the blue color is darker. This shows that plasminogen can significantly promote the repair of myelin damage in the spinal cord compression nerve injury model Plg-/- mice.

Figure 6 Results of Von-frey mechanical pain threshold test of C57 mice with spinal cord compression injury model given fibronectin for 6 days. The results showed that the mechanical threshold of mice in the vehicle group was significantly increased compared with that in the sham group, indicating that the pain sensation of mice was lost after the establishment of the spinal cord compression model; the pain threshold of mice in group A (1 mg) and group B (0.5 mg) was significantly lower than that in the vehicle group, and the statistical difference was significant (* indicates P < 0.05, ** indicates P < 0.01). This result shows that fibronectin can promote pain recovery in mice with spinal cord compression model.

Figure 7A-D shows immunohistochemical staining results of NFP in the spinal cord of spinal cord transection model mice given plasminogen for 28 days. A is the sham operation group, B is the vehicle PBS control group (also called the vehicle group, the same below), C is the plasminogen group (also called the medication group, the same below), and D is the average optical density quantitative analysis results. . The results show that the spinal cord of mice in the sham operation group expresses a certain level of NFP (arrow mark), the expression of NFP in the spinal cord of the mice in the vehicle group) is significantly less than that of the mice in the sham operation group, and the expression of NFP in the spinal cord of the mice in the drug group is significantly less than that of the mice in the sham operation group. The expression of was significantly higher than that of the vehicle group, and the statistical difference was significant (* indicates P<0.05). It is suggested that plasminogen can promote the expression of NFP in the spinal cord of spinal cord transection model mice.

Figure 8 BMS scoring results of spinal cord transection model mice. The results showed that the BMS score of the mice in the prosthetic hand group was 9 points. The BMS score of the mice in the vehicle group was 6.75±1.92 points, which was significantly lower than that of the mice in the sham operation group. The BMS score of the mice in the drug administration group was 8.29±0.39 points, which was significantly higher. to the mice in the vehicle group, and the statistical difference was significant (* indicates P＜0.05, *** indicates P＜0.001). It is suggested that plasminogen can promote the recovery of hindlimb motor function in paraplegic model mice.

Figure 9 RT-PCR detection results of plasminogen mRNA in the spinal cord of injured spinal cord transection model mice 6 hours after administration of plasminogen. The results showed that the spinal cord of mice in the blank control group had a certain level of plasminogen mRNA, and the level of plasminogen mRNA in the spinal cord of mice in the administration group was significantly higher than that in the vehicle group, and the statistical difference was close to significant (P=0.051). It is suggested that plasminogen can promote the transcription of plasminogen gene in the spinal cord of paraplegic model mice.

Figure 10A-D Results of immunohistochemical staining of synaptophysin in the spinal cord of mice with spinal cord transection model after 28 days of administration of fibrinolysinogen. A is the sham group, B is the vehicle group, C is the drug group, and D is the average optical density quantitative analysis result. The results showed that the spinal cord of mice in the sham group expressed a certain level of synaptophysin (indicated by arrows), and the level of synaptophysin in the spinal cord of mice in the vehicle group was significantly lower than that in the sham group, and the statistical difference between the two groups was extremely significant (*** indicates P < 0.001). The level of synaptophysin in the spinal cord of mice in the drug group was significantly higher than that in the vehicle group, and the statistical difference was significant (* indicates P < 0.05). This shows that fibrinolysinogen can promote the expression of synaptophysin in the injured spinal cord of mice with paralysis model and promote the repair of spinal cord injury.

Figure 11A-D shows the results of Nissl staining of the spinal cord of mice with spinal cord transection model after 28 days of administration of fibrinolysin. The results showed that the spinal cord of mice in the sham surgery group had a certain level of Nissl bodies (indicated by arrows), and the level of Nissl bodies in the spinal cord of mice in the vehicle group was significantly higher than that in the sham surgery group, and the statistical difference between the two groups was extremely significant (*** indicates P < 0.001). The level of Nissl bodies in the spinal cord of mice in the drug group was significantly lower than that in the vehicle group, and the statistical difference was extremely significant (** indicates P < 0.01). This shows that fibrinolysin can promote the recovery of Nissl bodies in the injured spinal cord of mice with paralysis model and promote the repair of spinal cord injury.

without

Claims (15)

A method of treating nerve damage, comprising administering to a subject a therapeutically effective amount of one or more compounds selected from the group consisting of components of the plasminogen activation pathway, capable of activating plasminogen directly or by activating fibrin. Compounds that indirectly activate plasminogen by upstream components of the lysinogen activation pathway, compounds that mimic the activity of plasminogen or plasmin, and compounds that can upregulate plasminogen or plasminogen activators Expressed compounds, plasminogen analogs, plasmin analogs, tPA or uPA analogs and antagonists of fibrinolysis inhibitors. The method according to claim 1, wherein the components of the plasminogen activation pathway are selected from plasminogen, recombinant human plasmin, Lys-plasminogen, Glu-fibrinogen Zymogens, plasmin, plasminogen and plasmin variants and analogs containing one or more kringle domains and protease domains of plasminogen and plasmin, small fibers Mini-plasminogen, mini-plasmin, micro-plasminogen, micro-plasmin, delta-plasminogen, delta -Plasmin (delta-plasmin), plasminogen activator, tPA and uPA. As described in claim 1, the nerve damage is central nerve damage or peripheral nerve damage. The method of any one of claims 1-3, wherein the compound promotes repair of damaged nerves. A method as described in any one of claims 1-4, wherein the compound promotes the regeneration of nerve myelin sheath. A method as described in any one of claims 1 to 5, wherein the compound promotes the expression of spinal cord neurofilament protein and nerve fiber regeneration. A method as described in any one of claims 1 to 6, wherein the compound promotes the recovery of sensory nerve function or motor nerve function. The method of claim 7, wherein the compound promotes restoration of pain sensation. The method of any one of claims 1-8, wherein the compound promotes the expression of synaptophysin. A method as described in any one of claims 1-9, wherein the compound promotes the recovery of Nissl body levels in damaged nerve cells. The method of any one of claims 1-10, wherein the compound is fibrolytic enzyme. The method of any one of claims 1-11, wherein the plasminogen and sequence 2 have at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity and proteolytic activity of plasminogen. A method as described in claim 12, wherein the fibronectin is a conservatively substituted variant of the fibronectin described in sequence 2. The method according to any one of claims 1-13, wherein the plasminogen comprises at least 75%, 80%, 85%, 90%, 95%, An amino acid sequence that has 96%, 97%, 98% or 99% sequence identity and has proteolytic activity to plasminogen. The method according to any one of claims 1 to 14, wherein the fibrinolysinogen is natural or synthetic human full-length fibrinolysinogen.