CN116947976B - Method for treating cartilage injury by mesenchymal stem cells - Google Patents
Method for treating cartilage injury by mesenchymal stem cells Download PDFInfo
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- CN116947976B CN116947976B CN202310932015.0A CN202310932015A CN116947976B CN 116947976 B CN116947976 B CN 116947976B CN 202310932015 A CN202310932015 A CN 202310932015A CN 116947976 B CN116947976 B CN 116947976B
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
- C12N9/0073—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13
- C12N9/0075—Nitric-oxide synthase (1.14.13.39)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
- C12Y114/13—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen (1.14.13)
- C12Y114/13039—Nitric-oxide synthase (NADPH dependent) (1.14.13.39)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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Abstract
The invention relates to a method for treating cartilage injury by mesenchymal stem cells. According to the invention, the polypeptide NOS-S2 which specifically targets and inhibits NOS activity is screened by the phage library, and the polypeptide can effectively inhibit the apoptosis of cartilage cells after cartilage injury so as to promote the repair of cartilage injury. The polypeptide and the bone marrow mesenchymal stem cells can be combined to remarkably improve the treatment effect, and the application prospect is wide.
Description
Technical Field
The application relates to the field of biological treatment, in particular to a method for treating cartilage injury by using mesenchymal stem cells.
Background
Cartilage defects can be caused by trauma, abrasion, neoplasms, congenital deformities, and the like. Trauma is the most common cause of articular cartilage defects. Mainly due to sports or accidents. Shear forces generated by injury can lead to compression fractures of articular cartilage or osteochondral separation. Articular cartilage belongs to hyaline cartilage, and is recovered and replaced by cells and matrixes formed by surrounding tissues after being damaged, but cartilage tissue has poor repair capability, and the repair capability is limited by the histological and biological characteristics of the articular cartilage, so that the articular cartilage cannot recover the original structure and function. Although the synthesis capacity of mature chondrocytes is increased when damaged, it is limited to meet the need for repair.
The damage of the articular cartilage brings great harm to human health, and the burden of social medical treatment is increased. The current surgical treatment methods mainly comprise arthroscopic joint cavity flushing, drilling micro fracture, cartilage cleaning and shaping, and autologous or allogenic bone cartilage transplanting. Although these methods are different, they are important in alleviating pain symptoms associated with injury, delaying joint degeneration, and restoring certain joint function. In recent years, with the development of technology, stem cell therapy brings good news to cartilage repair.
Stem cells can be classified into embryonic stem cells and adult stem cells according to their sources, and ES cells have unlimited proliferation capacity and differentiation totipotency, and have been primarily successful in several tissue construction studies of muscle, vascular endothelial, nerve, etc. However, studies on the directional differentiation of ES cells into chondrocytes remain in the initiation phase. Furthermore, the problems of immunogenicity, tumorigenicity, and renaturability in the application of ES cells have limited the further popularization and application thereof. In contrast, adult stem cells are widely distributed in vivo, have strong proliferation activity and multidirectional differentiation potential, and the directional induced differentiation technology is relatively mature, so that the adult stem cells become hot spots for tissue engineering seed cell research. Mesenchymal stem cells may be derived from various tissues such as bone marrow, fat or cord blood. These cells have the ability to proliferate and differentiate into a range of tissues in vitro, including osteoblasts, chondrocytes, adipocytes, cardiomyocytes and vascular cells. Because of the capability, the mesenchymal stem cells provide a broad prospect for research and clinical application in the field of tissue regeneration.
Mesenchymal Stem Cells (MSCs) have typical stem cell characteristics, are present in bone marrow and other tissue organs throughout the life, are responsible for tissue repair and renewal, and have multipotency differentiation potential, and can differentiate into a number of different tissues such as bone tissue, cartilage tissue, adipose tissue, endothelial tissue, muscle tissue, neural tissue, epithelial tissue, etc. under different induction conditions. Due to the ability of MSCs to self-replicate and multi-differentiate, it has become the seed cells most commonly used in tissue engineering bone construction in recent years. Bone marrow MSCs are convenient to obtain materials, have small wounds, are easy to separate from bone marrow and amplify and purify in vitro, and do not need enzyme digestion pretreatment unlike primary culture of MSCs from synovium, fat and the like. Although bone marrow MSCs only occupy 0.001% -0.01% of the number of nuclear cells, they always maintain the capacity of differentiating into chondrocytes stably, and are cartilage precursor cells which are available for life. Bone marrow MSCs have a chondrogenic potential of CD105 cells that appears to be involved in CD105 expression. Compared with MSCs from synovium, fat and the like, the bone marrow MSCs marker gene has no obvious difference in planar culture, has obvious difference in three-dimensional high-density culture, and can be obviously improved only by cartilage differentiation of bone marrow MSCs, so the bone marrow MSCs marker gene is an ideal seed cell for cartilage tissue engineering research. The differentiation of MSCs cells into chondrocytes can be promoted by isolation and in vitro culture of MSCs cells and control of culture conditions. And bone marrow mesenchymal stem cells and the like can migrate to cartilage injury parts to generate reparative fibrocartilage tissues. Although the repairing tissue is inferior to healthy cartilage in terms of biological and physical properties, durability and the like, the repairing tissue can cover and protect subchondral bones in a defect area and reduce cartilage abrasion and free chip formation.
Compared with mature chondrocytes or old skeletal muscle cells, the mesenchymal stem cells have high proliferation capacity and cartilage differentiation potential, and are more beneficial to cartilage injury repair. In terms of cartilage surface microdamage, unipotent chondrocytes or articular cavity cells may be optimal. Clinically, researchers have a greater propensity to apply primary, untreated bone marrow mesenchymal stem cells to stage I fractures. Indeed, the joint microenvironment is associated with a variety of factors including growth factors, biological materials, proteases and adjacent cartilage and subchondral osseointegrated cells, all of which may be involved in joint reconstructive therapy. In the rabbit model, bone marrow borehole size has a significant impact on the number of fibroblastic clones at the fractured clot. In fact, a 4mm osteochondral joint breach can release 300 cells, from which it is inferred that bone marrow mesenchymal stem cells play an important role in human osteoarthritis repair.
It has been found in studies that NOS inhibitors have the effect of inhibiting chondrocyte apoptosis and thus treating cartilage damage. However, currently, the available NOS inhibitors are not sufficiently wide in variety and are in need of further development. In addition, there are few methods for treating cartilage damage using stem cells, and in particular, there is a need for further research and development of therapeutic effects for improving cartilage damage using other drugs in combination.
Disclosure of Invention
According to the invention, a polypeptide NOS-S2 which specifically targets and inhibits NOS activity is screened by a phage library, and the polypeptide sequence is shown as SEQ ID NO: 2.
The polypeptide NOS-S2 for inhibiting NOS activity can be used for preparing medicines for inhibiting NOS activity.
In particular, the invention provides the use of polypeptide NOS-S2 which inhibits NOS activity in the manufacture of a medicament for the treatment of cartilage damage.
In order to increase the curative effect, the invention further provides application of a combination of the polypeptide NOS-S2 inhibiting NOS activity and bone marrow mesenchymal stem cells in preparing a pharmaceutical composition for treating cartilage injury.
Specifically, the polypeptide is used at a concentration of 1 to 100mg/kg, preferably 1 to 50mg, more preferably 1 to 20mg/kg.
In particular, the stem cells are used at a concentration of 1-9×10 7 Preferably 1 to 5X 10 per mL 7 Per mL, more preferably 1X 10 7 /mL。
Specifically, the administration dose of the pharmaceutical composition in the animal model is that the injury site is in situ injected with BMSCs cell sap (1×10) 7 Per mL) 0.5mL in combination with subcutaneous injection of 10mg/kg NOS-S2 polypeptide.
Still further, the pharmaceutical composition of the present invention further comprises other agents having a beneficial effect on the treatment of cartilage damage.
Furthermore, the polypeptides of the invention may also be made as variants by conservative substitutions. By polypeptide "variant" is meant a biologically active polypeptide having at least about 80% amino acid sequence identity to a native sequence polypeptide after aligning the sequences and introducing gaps (if desired) to achieve the maximum percent sequence identity and not considering any conservative substitutions as part of the sequence identity. Such variants include, for example, polypeptides in which one or more amino acid residues are added, or deleted, at the N-or C-terminus of the polypeptide. In some embodiments, the variant will have at least about 80% amino acid sequence identity. In some embodiments, the variant will have at least about 90% amino acid sequence identity. In some embodiments, the variant will have at least about 95% amino acid sequence identity to the native sequence polypeptide.
Further, the pharmaceutical composition also contains a pharmaceutically acceptable carrier.
By "pharmaceutically acceptable carrier" is meant a nontoxic solid, semisolid or liquid filler, diluent, encapsulating material, formulation aid or carrier conventional in the art that is used in conjunction with therapeutic agents that comprise a "pharmaceutical composition" for administration to a subject. The pharmaceutically acceptable carrier is non-toxic to the recipient at the dosage and concentration used and is compatible with the other ingredients of the formulation. Pharmaceutically acceptable carriers are suitable for the formulation used.
In certain embodiments, the pharmaceutical composition may be in the form of a solid dosage form, preferably a capsule or tablet. Tablets, pills, capsules, troches and the like may contain any of the following components or compounds having similar properties: an adhesive; a diluent; a decomposing agent; a lubricant; a slip agent; a sweetener; and a flavoring agent.
Examples of binders include microcrystalline cellulose, tragacanth, dextrose solution, acacia, plant mucilage, gelatin solution, sucrose and starch paste. Lubricants include talc, starch, magnesium or calcium stearate, lycopodium and stearic acid. Diluents include, for example, lactose, sucrose, starch, kaolin, salts, mannitol and dibasic calcium phosphate. Slip aids include, but are not limited to, colloidal silica. The decomposer comprises croscarmellose sodium, sodium carboxymethyl starch, alginic acid, corn starch, potato starch, bentonite, methylcellulose, agar and carboxymethyl cellulose. Colorants include, for example, any approved legal water-soluble FD and C dyes, mixtures thereof; and water insoluble FD and C dyes suspended on hydrated alumina. The sweetener comprises sucrose, lactose, and mannitol.
Pharmaceutically acceptable carriers for use in parenteral pharmaceutical compositions include aqueous vehicles, anhydrous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, chelating or sequestering agents and other pharmaceutically acceptable substances. Examples of aqueous vehicles include sodium chloride injection, ringer's injection, isotonic dextrose injection, sterile water injection, dextrose and lactate ringer's injection. Anhydrous parenteral vehicles include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil and peanut oil. Antimicrobial agents in concentrations that achieve a bacteriostatic or fungistatic effect must be added to parenteral formulations packaged in multi-dose containers, including phenol or cresol, mercurial, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoates, thimerosal, benzalkonium chloride and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. Buffers include phosphate and citrate. Antioxidants include sodium bisulfate. The local anesthetic comprises lidocaine hydrochloride. Suspending and dispersing agents include sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, and polyvinylpyrrolidone. The emulsifier comprises polysorbate 80. Chelating or sequestering agents for metal ions include EDTA. The pharmaceutical carrier also includes ethanol, polyethylene glycol and propylene glycol as water miscible vehicles and sodium hydroxide, hydrochloric acid, citric acid or lactic acid for pH adjustment.
Further, the pharmaceutical composition of the present invention may be designed for injection for local administration and systemic administration. Typically, a therapeutically effective dose is formulated to contain the active polypeptide at a concentration of at least about 0.1% w/w to up to about 90% w/w, or more, preferably at a concentration of greater than 1% w/w, relative to the tissue being treated. It will be appreciated that the precise dosage and duration of treatment will be related to the tissue being treated and may be determined empirically using known test protocols or inferred from in vivo or in vitro test data. It should be noted that the concentration and dosage values may also vary with the age of the individual receiving the treatment. It should further be understood that the specific dosage regimen for any particular patient should be adjusted over time in accordance with the needs of the individual and the professional judgment of the person performing or supervising the administration of the formulations, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed formulations.
In particular, the polypeptides or bone marrow mesenchymal stem cells provided herein may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, for example, in ampoules or in multi-dose containers, with a preservative added. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form, which is reconstituted with a suitable medium, such as sterile pyrogen-free water or other solvent, prior to use. For example, provided herein are parenteral formulations in stabilized solution or lyophilized form containing an effective amount of a polypeptide and bone marrow mesenchymal stem cells.
The polypeptide and bone marrow mesenchymal stem cells may be suspended in micronized or other suitable form, or may be derivatized to produce a more soluble active product or to produce a prodrug. The form of the resulting mixture depends on many factors, including the intended mode of administration and the solubility of the polypeptide in the carrier or vehicle chosen. The effective concentration is sufficient to ameliorate symptoms of the condition and can be determined empirically.
Sterile lyophilized powders can be prepared by dissolving a solution containing the polypeptide and bone marrow mesenchymal stem cells in a suitable solvent or mixing small aliquots of a solution containing the polypeptide and bone marrow mesenchymal stem cells with a suitable solvent. The solvent may contain excipients or other pharmacological components that improve the stability of the powder or reconstituted solution prepared from the powder. Excipients that may be used include, but are not limited to, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, lactose, or other suitable agents. The solvent may also contain a buffer such as citrate, sodium or potassium phosphate or other such buffers, as would be known to those skilled in the art, typically at about neutral pH. The solution is then sterile filtered and lyophilized under standard conditions known to those skilled in the art to provide a lyophilized formulation. Typically, the solution obtained from sterile filtration is dispensed into vials for lyophilization. Each vial may contain a single dose of polypeptide, such as 10-1000mg or 100-500mg, or multiple doses of polypeptide. Briefly, lyophilized powders may be prepared by dissolving dextrose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, lactose or other suitable agents in an amount of about 1-20% in a suitable buffer such as citrate, sodium or potassium phosphate or other such buffers known to those skilled in the art and bringing the pH to about neutral.
Advantageous effects
According to the invention, the polypeptide NOS-S2 which specifically targets and inhibits NOS activity is screened by the phage library, and the polypeptide can effectively inhibit the apoptosis of cartilage cells after cartilage injury so as to promote the repair of cartilage injury. The polypeptide and the bone marrow mesenchymal stem cells can be combined to remarkably improve the treatment effect, and the application prospect is wide.
Drawings
FIG. 1 binding Properties of different NOS inhibitory peptides
FIG. 2 Effect of different groups on apoptotic cells
Detailed Description
Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention. The methods, apparatus, materials, and so forth in the following examples, unless otherwise indicated, are all conventional in the art and are commercially available.
EXAMPLE 1 screening of NOS inhibitory peptides
Inducible nitric oxide synthase (NOS 2) is purchased from Goybio under accession number GOY-01D340. Phage random 12 peptide library \host E.coli ER2738 were purchased from New England Biolabs company.
Immobilization of inducible nitric oxide synthase recombinant proteins as target proteins in 96 well plates with 10X 10 per well 9 Washing non-specifically bound phage with acidic eluent of 0.2mol/L GIycine-HCI (pH 2.2), 1mg/ml BSA, neutralizing the acidic eluent with 1mol/L Tris-HCI (pH 9.1), and performing 4 rounds of adsorption to obtain good enrichment effect5.8×10 -3 (%) and the following. After the 4 th round of screening, 30 clones were randomly selected and tested by conventional ELISA according to the peptide library kit. The D490 value was measured by using the original phage library and M13 phage without exogenous peptide as controls, with 3 parallel wells per sample, and o-phenylenediamine developed.
As can be seen from the results of FIG. 1, the positive reactions were strongest in 5 clones, designated NOS-S1, NOS-S2, NOS-S3, NOS-S4, NOS-S5, respectively, and ELISA showed that these 5 clones were able to bind specifically to NOS.
Amplifying and purifying positive phage clone, preparing phage single-stranded DNA, and sending to Shanghai biological company for full-automatic sequencing, wherein the sequences are S1: SEQ ID NO: 1. s2: SEQ ID NO:2, seq ID NO:3 wherein S3 and S4 are the same, S5: SEQ ID NO:4.
titer determination was performed on 4 phage clones obtained by screening, and phage titer was 10 11 1. Mu.l of each phage clone of (C) was mixed with inducible nitric oxide synthase (NOS 2), respectively, and the enzyme activity was measured by a microphotometer method. A blank negative control and a normal enzyme control with only enzyme solution were established with the non-screened and non-phage peptide libraries as controls, respectively. Inhibition was calculated as 100% of the enzyme activity of the normal enzyme control tube without phage. The results are shown in Table 1.
Table 1 inhibition of enzyme results by polypeptides
Group of | Inhibition ratio (%) |
Prophage peptide library | 3.57±0.18 |
Blank control | 0 |
NOS-S1 | 27.52±3.46* |
NOS-S2 | 68.73±6.41* |
NOS-S3 | 30.55±3.78* |
NOS-S5 | 35.46±5.21* |
As can be seen from Table 1, the NOS-S1, NOS-S2, NOS-S3, NOS-S5 phage clones all inhibited NOS activity, with the highest inhibition being NOS-S2. S2 polypeptide is synthesized artificially with purity of 98.2 percent for standby.
EXAMPLE 2 isolated preparation of bone marrow mesenchymal Stem cells
1C 57 mouse with the age of 8 weeks is taken, heparinized for 30min, anesthetized with 1% pentobarbital sodium, and the lower limbs of the mouse are aseptically separated, and muscle tissues are removed. The two ends of the long bone were cut off, and the bone marrow cavity was repeatedly flushed with D-Hanks solution containing 10% calf serum by aspiration with a 1ml base syringe. Blowing the flushed bone marrow uniformly, centrifuging, discarding supernatant, adding 0.83% NH 4 Cl red blood cell lysate is lysed for 10min and centrifuged again, and after washing with D-Hanks solution containing 10% calf serum for 1 time, the sediment is resuspended in LG-DMEM containing 10% FBS and blown up uniformly and then cultured in a culture dish, after 48h, the culture dish is gently rocked, and fresh LG-DMEM containing 10% FBS is replaced for 1 time every 3 days to continue culturing mesenchymal stem cells, and the culture is carried out according to 1:3.
Taking 3 rd generation cells, digesting the cells into single cell suspension by pancreatin, washing the single cell suspension for 3 times by PBS, respectively adding fluorescent marked mesenchymal markers CD29 and CD40 and blood-derived markers CD34 and CD45, incubating the mixture at 4 ℃ for 30min, washing the unlabeled antibodies by PBS, fixing 1% paraformaldehyde, and detecting the mixture by using a flow cytometerThe results showed that CD29 (. + -.)%, CD90 (. + -.)%, CD34 (. + -.)%, and CD45 (. + -.)%. From the above results, it can be seen that mesenchymal stem cells were isolated from the preparation. Diluting the P3 generation cells to adjust the concentration to 10 7 And (3) one/mL for later use.
EXAMPLE 3 Effect of NOS-S2 polypeptide on chondrocyte apoptosis after cartilage injury
Taking 10 male C57 mice, injecting 35mg/kg pentobarbital sodium into abdominal cavity to anesthetize the mice, cutting off joint capsules after shaving and sterilizing the parts of right knee joints, cutting off inner meniscus shin ligaments by using ophthalmic surgical scissors, suturing and sterilizing, and injecting 10mg/kg NOS-S2 polypeptide and equivalent normal saline into the experimental group 1 and the control group at 12 hours after the operation after the end of the meniscus injury operation, wherein the injection is further enhanced for 1 time per week and 4 times; experiment group 2: in situ injection of BMSCs cell sap (1×10) 7 Per mL) 0.5mL, re-booster injections 1 time per week for a total of 4 injections; experiment group 3: in situ injection of BMSCs cell sap (1×10) 7 Per mL) 0.5mL combined subcutaneous injection of 10mg/kg NOS-S2 polypeptide; both were re-boosted 1 injection per week for a total of 4 injections; all mice were free to move and eat within the cages. After 6 weeks, according to the histological treatment standard of articular cartilage, the joint heals completely: normal chondrocytes are isolated from the articular cartilage defect and surrounded by matrix, the joint surface is smooth, and the joint surface is tightly combined with surrounding tissues; incomplete healing: the differentiation of the adsorbed tissue is poor, the cell arrangement is irregular, the surface of the new tissue is rough, and the new tissue is partially or completely connected with surrounding adjacent tissues; unhealed: the defect area contains little cartilage tissue, fibrous granulation tissue fills, the surface is depressed, and the treatment effect is scored with or without partial attachment to the surrounding tissue, the results are shown in Table 2.
Table 2 results of therapeutic effects of Polypeptides and BMSCs on cartilage defects
Group of | Complete repair | Incomplete repair | Unrepaired |
Model control group | 0 | 0 | 10 |
Experiment group 1 | 4 | 5 | 1 |
Experiment group 2 | 3 | 5 | 2 |
Experiment group 3 | 6 | 4 | 0 |
The number of cartilage repair cases was significantly increased in each of the treatment groups compared to the model control group, indicating that both the polypeptide and BMSCs alone had better therapeutic effects. And after the polypeptide and BMSCs are combined, the number of complete repair cases is obviously increased (P is less than 0.05), and the curative effect of the cartilage defect is best. This also means that the two have a certain synergistic effect.
The injured cartilage after treatment is taken and sheared after PBS rinsing. Digestion was carried out overnight at 37℃in type IA collagenase (1 mg/ml DMEM), and nylon membranes were washed by filtration. Centrifugation at 2000rpm for 5min, decanting the supernatant, draining, washing 3 times with PBS, and centrifuging. 400. Mu.l of PBS was used to form a cell suspension, 400. Mu.l of rhodamine (10. Mu.g/ml) was added, capped and incubated in a water bath at 37℃for 30 minutes. Centrifugation at 2000rpm for 5min, decanting the supernatant, draining, washing 3 times with PBS, and centrifuging again. 400 μl of 50 μg/ml of propidium iodide was added, 3000 cells were counted by a FACScan flow cytometer, and the percentage of apoptotic cells was automatically calculated. The results are shown in FIG. 2.
As can be seen from fig. 2, the articular chondrocytes of the control group had 50% of the chondrocytes apoptosis after collagenase digestion, and the apoptosis rate was high, while the mesenchymal stem cells were able to inhibit the apoptosis of chondrocytes to some extent, but not to a high extent. And only about 20% of cartilage cells undergo apoptosis after the polypeptide treatment, compared with a control group, the apoptosis cells are obviously reduced (P is less than 0.01), and after the polypeptide is combined with the bone marrow mesenchymal stem cells, the apoptosis of the cartilage cells can be further reduced.
From the above results, it can be seen that the NOS-S2 polypeptide of the present invention can effectively inhibit the apoptosis of chondrocytes after cartilage injury, thereby promoting the repair of cartilage injury. The polypeptide and the bone marrow mesenchymal stem cells can be combined to remarkably improve the treatment effect, and the application prospect is wide.
The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The foregoing embodiments are, therefore, to be considered in all respects illustrative rather than limiting of the present disclosure. The scope of the disclosure is, therefore, indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Claims (7)
1. A polypeptide NOS-S2 which inhibits NOS activity, characterized in that the amino acid sequence of said polypeptide is as set forth in SEQ ID NO: 2.
2. Use of a polypeptide NOS-S2 that inhibits NOS activity in the manufacture of a medicament for treating cartilage damage, wherein the amino acid sequence of the polypeptide is as set forth in SEQ ID NO: 2.
3. Use of a combination of a polypeptide NOS-S2 that inhibits NOS activity and bone marrow mesenchymal stem cells for the preparation of a pharmaceutical composition for treating cartilage damage, wherein the amino acid sequence of the polypeptide is as set forth in SEQ ID NO: 2.
4. The use according to claim 2 or 3, wherein the medicament or pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
5. The use according to claim 4, wherein the carrier is an excipient.
6. The method of claim 4, wherein the carrier comprises any of sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, and polyvinylpyrrolidone.
7. The method according to claim 4, wherein the carrier contains sodium hydroxide, hydrochloric acid, citric acid or lactic acid for adjusting the pH.
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US5759836A (en) * | 1995-03-27 | 1998-06-02 | Hospital For Joint Diseases | Osteoarthritis-associated inducable isoform of nitric oxide synthetase |
WO2000071112A1 (en) * | 1997-11-13 | 2000-11-30 | Cold Spring Harbor Laboratory | Therapeutic uses for nitric oxide inhibitors |
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US5759836A (en) * | 1995-03-27 | 1998-06-02 | Hospital For Joint Diseases | Osteoarthritis-associated inducable isoform of nitric oxide synthetase |
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