CN116836276B

CN116836276B - Stem cell preparation for repairing knee joint injury and preparation process thereof

Info

Publication number: CN116836276B
Application number: CN202310940735.1A
Authority: CN
Inventors: 蔡俊; 喻长杰; 贾鹏飞
Original assignee: Aiketaike Zhejiang Holding Co ltd
Current assignee: Aiketaike Zhejiang Holding Co ltd
Priority date: 2023-07-28
Filing date: 2023-07-28
Publication date: 2023-12-08
Anticipated expiration: 2043-07-28
Also published as: CN116836276A

Abstract

The application relates to a stem cell preparation for repairing knee joint injury and a preparation process thereof. The application provides a monoclonal antibody specific to NGF, which is a murine monoclonal antibody. The antibody can be used for effectively inhibiting the generation of inflammatory factors in osteoarthritis by singly or jointly umbilical cord mesenchymal stem cells, can effectively promote the healing of osteoarthritis, and has better pharmaceutical application.

Description

Stem cell preparation for repairing knee joint injury and preparation process thereof

Technical Field

The application relates to the field of biological treatment, in particular to a stem cell preparation for repairing knee joint injury and a preparation process thereof.

Background

The knee joint is the biggest bending joint of the whole body, and meanwhile, the ligament structure of the knee joint plays a great role in maintaining the normal function and stability of the knee joint because the appearance of the knee joint also determines that the knee joint is not a very stable joint. The knee joint is a bending joint, but can slightly grind and rotate when bending the knee. The main functions of the knee joint are loading, transferring load and participating in sports to provide a couple for calf movement. The knee joint is not as flexible as the hip joint, mainly performs bending and stretching movements, but is positioned in the middle of the lower limb and between two largest lever arms of the body, bears larger force, and is easy to cause sprain and fracture. Especially in sporting activities, injuries to ligaments and menisci are most common. Knee osteoarthritis is a disorder based on degenerative pathological changes. Symptoms are usually manifested as red and swelling pain of the knee, and joint deformity can be caused if the treatment is not carried out in time. Joint diseases such as gonarthromeningitis, ligament injury, meniscus injury and the like are also frequently caused at knee joint parts. The occurrence of knee osteoarthritis is generally caused by degenerative changes of the knee joint, trauma, excessive strain, and the like. In addition, incorrect walking posture, cold catching of the knee joint, and cold catching are also causes of osteoarthritis of the knee joint.

Clinically, there is no effective control measure for the disease progress of knee osteoarthritis. Although the treatment methods for early knee osteoarthritis are various, including oral medication, injection of medicines into the joint cavity, wearing of braces, physical therapy, changing of lifestyle, etc., the treatment effects are poor. Cartilage repair or transplantation under arthroscope can be adopted after the failure of non-operative treatment, or operative treatment such as osteotomy correction, unicondylar and total knee replacement can be carried out. Currently, with the penetration of stem cell research and the widespread use of bioengineering techniques, more and more researchers are exploring the feasibility of these techniques for application in the field of osteoarthritis treatment.

The research shows that MSCs can be continuously differentiated into chondrocytes, and can be used for treating osteoarthritis. MSCs can be cultured and amplified without losing the multi-directional differentiation potential; MSCs have the potential for cartilage differentiation, whether cultured in vivo or ex vivo. MSCs are widely distributed, and can be isolated from bone marrow, periosteum, trabecular bone, adipose pad tissue, synovium, skeletal muscle, deciduous teeth, and other tissues. Regardless of the tissue source from which the cells are derived, have the ability to differentiate into a variety of cell lines, including connective tissue cells, such as bone, fat, cartilage, and muscle. Based on this recognition, more and more methods for treating knee osteoarthritis by cell technology have been developed.

MSCs are relatively easy to separate, have good in vitro expansion capacity, can keep the stem cell characteristics in the expansion process, and promote the proliferation of chondrocytes, so that MSCs are another option for the sources of articular chondrocytes. MSCs were initially isolated from bone marrow and recognized for their properties, and subsequently isolated from other connective tissue, including periosteum, synovium, and adipose tissue. Animal experiment research shows that the MSCs after amplification culture can repair cartilage and subchondral bone and control the progress of secondary osteoarthritis. Using rabbits as subjects, collagen gel with suspended bone marrow or periosteum MSCs was injected into the medial side of the fully injured lateral femoral condyle, and it was observed that glass-like repair tissue was formed in the joint soon, while collagen without MSCs was injected to form fiber-like tissue. Although the glassy repair tissue becomes progressively thinner and degenerates over time, the biomechanical properties are inferior to those of the original cartilage tissue and the cartilage surface is more rough and uneven, it is still superior to the fibrous tissue. In recent years, there have been continuous studies on the treatment of osteoarthritis with MSCs into the clinical trial phase. Clinical trials have shown that autologous MSCs are injected into the body after in vitro expansion culture, with the result that regenerated cartilage appears in the damaged area, and this therapy is therefore considered safe and viable. The local application of MSCs has a plurality of advantages, not only can strengthen joint repair, but also can slow down degeneration caused by osteoarthritis, and is the simplest and easy method for treating osteoarthritis. MSCs obtained and cultured from fibroblast growth factor receptor positive rats were transplanted into the femoral space of a fibroblast osteoarthritis mouse, and this method was found to have therapeutic effects, and the transplanted cells were able to differentiate into chondrocytes. It was found that the therapeutic effect was also observed by transplanting human bone marrow MSCs to osteoarthritis goats, and therefore it is thought that local cell therapy could stimulate meniscus tissue regeneration and mitigate further damage to the damaged area. The implanted autologous MSCs were found to stimulate cartilage growth and alleviate pain symptoms of the degenerated joints by 24-week MRI follow-up monitoring. According to the research, MSCs have certain effect of treating bone diseases, but the methods of current research are not enough, and particularly the effect of single treatment is not good enough, so that development of combined therapeutic pharmaceutical compositions and further improvement of the therapeutic effect are further development directions.

Furthermore, nerve Growth Factor (NGF) belongs to secreted proteins associated with peripheral nervous system and brain cell development. In addition to play an important role in neuronal growth, differentiation, maturation, and lesion repair, clinical study data also indicate that significant increases in NGF levels are detected in synovial fluid of patients with rheumatoid arthritis and other types of arthritis (including KOA). Whereas abnormal NGF expression levels and chronic inflammatory responses at the site of inflammation or degenerated joints are considered to be the primary causes of pain in patients. Animal models and clinical studies show that inhibiting NGF can reduce pain and hyperalgesia, and NGF antibody L148M inhibits subchondral bone abnormal angiogenesis by downregulating VEGFA and Ang-1 protein levels, thereby alleviating joint inflammatory pain response; and reduces the pathological damage of cartilage and subchondral bone by inhibiting IL-1 beta, TNF-alpha and MMP expression. Development of specific NGF antibodies for the treatment of osteoarthritis is also an important research direction.

Disclosure of Invention

The application provides an umbilical mesenchymal stem cell combined with NGF antibody for treating osteoarthritis.

In particular, in one aspect, the application provides a monoclonal antibody specific for NGF, which is a murine monoclonal antibody.

Further, the monoclonal antibody is named as 3N12, and the light chain variable region sequence of the monoclonal antibody is identified and obtained by sequencing as shown in SEQ ID NO:1, the heavy chain variable region sequence of which is shown in SEQ ID NO: 2.

In some embodiments, the light chain variable region of the application comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID No. 1; or alternatively

(i) Comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO. 1: or alternatively

(ii) Comprising or consisting of an amino acid sequence having 1 or more (preferably NO more than 10, more preferably NO more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence selected from SEQ ID NO. 1, preferably, the amino acid changes do not occur in the CDR regions.

In some embodiments, the heavy chain variable regions of the application

i) Comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID No. 2; or alternatively

ii) comprises or consists of an amino acid sequence selected from SEQ ID NO. 2; or alternatively

(ii) Comprising or consisting of an amino acid sequence having 1 or more (preferably NO more than 10, more preferably NO more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence selected from SEQ ID NO. 2, preferably, the amino acid changes do not occur in the CDR regions.

Fragments and derivatives of the antibodies of the application (as used in the present application, which are encompassed by the term "antibody" unless otherwise indicated or clearly contradicted by context) are preferably produced by techniques known in the art. An "immunoreactive fragment" includes a portion of an intact antibody, typically an antigen-binding site or variable region. Examples of antibody fragments include: fab, fab '-SH, F (ab') 2, and Fv fragments; a bifunctional antibody; any antibody fragment, which is a polypeptide having a primary structure, wherein the primary structure consists of one uninterrupted sequence of contiguous amino acid residues (referred to herein as a "single chain antibody fragment" or "single chain polypeptide"), including but not limited to (1) single chain Fv (scFv) molecules (2) single chain polypeptides comprising only one light chain variable domain, or fragments thereof comprising three CDRs of a light chain variable domain without an associated heavy chain portion, and (3) single chain polypeptides comprising only one heavy chain variable region, or fragments thereof comprising three CDRs of a heavy chain variable region without an associated light chain portion; and multispecific antibodies formed from antibody fragments. For example, fab or F (ab') 2 fragments can be produced by protease digestion of the isolated antibodies according to conventional techniques. It should be appreciated that the immunoreactive fragments may be modified using known methods.

Furthermore, the application also provides application of the monoclonal antibody 3N12 of NGF in preparing a pharmaceutical composition for treating osteoarthritis.

Furthermore, the application also provides application of the monoclonal antibody 3N12 of NGF in combination with umbilical cord mesenchymal stem cells in preparing a pharmaceutical composition for treating osteoarthritis.

In particular, any suitable carrier known to those of ordinary skill in the art may be used in the pharmaceutical compositions of the present application, and the type of carrier will vary with the manner of administration. For parenteral administration (e.g., subcutaneous injection), the preferred carrier contains water, saline, alcohol, fat, wax, or a buffer. For oral administration, any of the carriers or solid carriers described above, such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be used. Biodegradable centers (e.g., polylactic acid salts, polyglycolic acid salts) may also be used as carriers for the pharmaceutical compositions of the present application.

Pharmaceutically acceptable carriers that may be used in these compositions include, but are not limited to: ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and lanolin.

The compositions of the application may be used in methods of enhancing the repair of osteoarthritis in a patient. The method comprises the step of contacting said patient or biological sample with said composition. Such a method would be useful for diagnostic and therapeutic purposes. For use with biological samples, the antibody composition may be administered by simple mixing with the sample or directly applied to the sample, depending on the nature of the sample (liquid or solid). The biological sample may be in direct contact with the antibodies in any suitable device (plate, pouch, vial, etc.). For use with a patient, the composition must be formulated for administration to the patient. The composition of the present application may be administered by: oral, topical, through an implanted reservoir. As used herein, the term "parenteral" includes subcutaneous, intralesional injection or infusion techniques.

In particular, umbilical cord mesenchymal stem cells involved in the pharmaceutical composition of the present application may be commercially purchased or may be isolated from umbilical cord. The separation and preparation method is a method known in the art, and the concentration of the extracted cells can be appropriately adjusted, which is a conventional technique in the art.

Advantageous effects

The application provides a monoclonal antibody specific to NGF, which is a murine monoclonal antibody. The antibody can be used for effectively inhibiting the generation of inflammatory factors in osteoarthritis by singly or jointly umbilical cord mesenchymal stem cells, can effectively promote the healing of osteoarthritis, and has better pharmaceutical application.

Drawings

FIG. 1 shows a diagram of the results of specific identification of NGF monoclonal antibodies

FIG. 2 graph of results of articular cartilage scoring for each treatment group

Detailed Description

Specific embodiments of the present application will be described in more detail below with reference to the accompanying drawings. While specific embodiments of the application are shown in the drawings, it should be understood that the application may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the application to those skilled in the art.

Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the presently described subject matter belongs.

EXAMPLE 1 preparation of NGF monoclonal antibodies

Recombinant human NGF protein product No. 519648 New York organism was used as immunogen. Immunization was performed using the following method:

immunization 1: 3 Balb/c female mice with the weight of 18-22 g are selected, NGF protein and Freund's complete adjuvant are mixed and emulsified in equal volume, and then primary immunization is carried out by adopting an abdominal subcutaneous multipoint injection mode, wherein the dosage is 80 mug/mouse. Immunization 2: the time and the 1 st immunization are separated by 1 month, NGF protein and Freund's incomplete adjuvant are mixed and emulsified in equal volume, transferred into a 1mL syringe, and subjected to subcutaneous multipoint injection in the abdomen for immunization, wherein the immunization dose is 70 mug/dose. After 1 month of the second immunization, the 3 rd immunization was performed by the same method, and the immunization dose was 50. Mu.g/dose. ELISA (enzyme-Linked immuno sorbent assay) respectively measures the polyclonal antibody titer of mice after 3 times of immunization, and selects the mouse with the highest titer No. 2 to carry out hybridoma cell fusion, and carries out intraperitoneal boosting immunization on the mice 3d before fusion, wherein the immune dose is 50 mug/mouse.

The cell number ratio of spleen cells of immunized mice to myeloma cells SP2/0 of the mice is 10:1, the fusion time is 2min, and the fusion reagent is 50% polyethylene glycol; observing the form of SP2/0 through a microscope before cell fusion, continuously culturing after 5d of half liquid replacement, culturing until 7d of full liquid replacement, observing the growth state of cells in the half liquid replacement and full liquid replacement by microscopic examination, collecting cell plate culture solution supernatant after the fusion for about 7-10 d, screening positive cell plate holes by adopting indirect ELISA, and subcloning positive cells for 4 times by adopting a limiting dilution method to obtain 4 positive monoclonal strains, wherein the clone with the strongest positive reaction is named as 3N12, and then performing bottle expansion and amplification culture.

Taking about 8-week-old Balb/c mice, injecting 0.5mL liquid paraffin into the abdominal cavity, and injecting 5X 10 into the abdominal cavity after 7d ⁵ And 3N12 hybridoma cells, and collecting ascites after the abdomen of the mouse swells. Purifying ascites by octanoic acid-ammonium sulfate method, dialyzing with PBS, measuring protein concentration by BCA method, adjusting protein concentration to 1mg/mL, and storing at-20deg.C.

EXAMPLE 2 specific identification of NGF monoclonal antibodies

NGF recombinant protein, BSA protein and loading buffer solution are mixed in a ratio of 3:1, boiled at 100 ℃ for 3-5 min for denaturation treatment, and then SDS-PAGE electrophoresis is carried out. And (3) starting electrophoresis at a constant voltage of 50V until the voltage is adjusted to 100V after the sample solution enters the separation gel, and ending electrophoresis. The gel was immersed in TF buffer for 30min, transferred to nitrocellulose membrane with a semi-dry gel transfer instrument, and transferred at constant voltage of 18V for 30min at 0.8mA/cm2 nitrocellulose membrane. Blocking the blocking solution at 37 ℃ for 60min, washing with TBST for 1 time, discarding the liquid, adding the monoclonal antibody for reaction for 1h, washing with TBST for 1 time, discarding the liquid, adding the alkaline phosphatase-labeled anti-mouse secondary antibody, and shaking for reaction at 37 ℃ for 40min. The TBST buffer was washed 5 times for 8min each. The mixed BCIP/NBT color development solution was added for reaction for 5min, and the results were observed as shown in FIG. 1.

As can be seen from the results of the monoclonal antibody hybridization analysis of FIG. 1, the 3N12 monoclonal antibody can specifically and specifically band NGF recombinant protein at about 27KD of lane 2, and the monoclonal antibody has no band in the control protein lane 1, thus showing better specificity.

EXAMPLE 3 affinity and potency assays for NGF monoclonal antibodies

The affinity of the NGF monoclonal antibody is measured by adopting a biological film interferometer, and the specific method comprises the following steps: NGF protein was adsorbed to a biosensor [ Anti-Penta-HIS (HIS 1K) Biosensors ], and the purified 3N12 monoclonal antibody was added thereto, and affinity constant measurement was performed in a ForteBioBlitz biomolecular interaction analysis system.

The titer of the monoclonal antibody was determined by indirect ELISA, 1. Mu.g/mL NGF protein was added to the wells of the enzyme-labeled plate at 100. Mu.L/well, coated overnight at 4℃and blocked with 5% skim milk after washing the plate with PBST. The liquid in the wells is discarded, the PBST is washed for 3 times, the monoclonal antibody is taken and diluted by PBS gradient and then added into the enzyme-labeled wells of the plate for reaction for 45min, and the HRP enzyme-labeled goat anti-mouse IgG antibody (1:5000) is added after the plate washing for incubation for 45min. After the liquid in the hole is discarded and the PBST plate is washed, 100 mu L/hole of TMB color development liquid is added, the reaction is stopped by adding 2mol/L sulfuric acid after 7min of light-proof color development, and the reaction is put in an enzyme-labeled instrument and the OD value is measured at the wavelength of 450 nm. The results are shown in Table 1.

TABLE 1 3N12 monoclonal antibody titers and affinity assay results

Antibody name	Valency of	Affinity for
			3N12 monoclonal antibodies	1:2048000	1.437±0.054×10 ^-9 mol/L

As can be seen from Table 1, the 3N12 monoclonal antibody of the application has better potency and affinity and better activity.

Example 3 isolation and preparation of umbilical cord mesenchymal Stem cells

Isolation and culture of HUCMSC: fresh term caesarean section is taken to produce neonatal umbilical cord, and the umbilical cord is repeatedly washed by PBS buffer solution containing penicillin/streptomycin double antibody to remove residual blood. The umbilical cord tissue was cut to 0.5mm ³ About the size of the fragments, transferring to a petri dish, culturing in high sugar DMEM medium containing 10% of exosome-removed fetal bovine serum and 1% of double antibody (100U/mL penicillin, 100mg/mL streptomycin), and standing at 37deg.C and 5% CO ₂ And (3) adding 5mL of culture medium into the incubator for 20 hours, changing the liquid for 1 time every 3 days, observing the growth state of the cells after 7 days by using an inverted microscope, and digesting with 0.25% EDTA pancreatin when the confluence degree reaches 80% -90%, and carrying out 1:3 passage. P3 generation cells were subjected to surface antigen detection, and logarithmic phase cells were collected, and labeled fluorescent antibodies CD29-PE, CD166-PE, CD73-PE, CD14-PE, CD144-PE, CD34-FITC, CD45-FITC, HLA-ABC, HLA-DR, CD44-FITC, CD105-FITC, and flow cytometry detection were carried out, which revealed that umbilical cord mesenchymal stem cells strongly expressed CD29 (99.5%), HLA-ABC (98.2%), CD166 (95.3%), CD105 (90.5%), CD73 (98.9%), CD44 (98.2%), and hardly expressed CD34, CD14, HLA-DR, CD45, CD31, and CD144. Umbilical cord mesenchymal stem cells are prepared. Stem cells were adjusted to 5X 10 ⁷ And (5) after concentration, standby.

EXAMPLE 4NGF monoclonal antibody and/or umbilical cord mesenchymal Stem cell function validation

C57BL/6 mice were randomly divided into 7 groups of 10:

a negative control group, in which 50. Mu.L of sterile physiological saline is injected into the right knee joint cavity after the mice are anesthetized;

the mice were anesthetized with 1% pentobarbital sodium in each group below, and the right knee surgical site was conventionally sterilized with iodophor, with the right knee flexed 90 degrees, and 27G needle was pierced perpendicular to the patellar ligament until there was a feeling of empty. According to different groupings, 50 μl of sterile saline or MIA solution was slowly injected into the knee joint cavity. After the needle is pulled out, the knee joint is pressed for 10 seconds by gauze and then bent and stretched for 5 times. After the end, the mice were placed in a warm place and returned to the animal room after the mice wake up, allowing free feeding.

Wherein, in the model control group, 50 mu L of 20mg/mL MIA solution is injected into the cavity of the right knee joint after the anesthesia of the mice, and sterile physiological saline (10 mg/kg) is injected into the abdominal cavity 2 weeks after the operation;

in the positive control group, 50 mu L of 20mg/mL MIA solution is injected into the cavity of the right knee joint after the anesthesia of the mice, and L148M (10 mg/kg) is injected into the abdominal cavity 2 weeks after the operation;

the low dose 3N12 monoclonal antibody group, 50 mu L of 20mg/mL MIA solution is injected into the right knee joint cavity after the anesthesia of the mice, and the NGF monoclonal antibody (1 mg/kg) is injected into the abdominal cavity 2 weeks after the operation;

high dose 3N12 monoclonal antibody group, 50 mu L of 20mg/mL MIA solution is injected into the right knee joint cavity after the anesthesia of the mice, and the NGF monoclonal antibody (10 mg/kg) is injected into the abdominal cavity 2 weeks after the operation;

stem cell treatment group, mice were anesthetized, and 50 μL of 20mg/mL MIA solution was injected into the right knee joint cavity, and umbilical mesenchymal stem cells 1×10 prepared in example 3 were injected into the joint cavity 2 weeks after the operation ⁶ ；

Stem cell combined with 3N12 monoclonal antibody treatment group, injecting 50 mu L of 20mg/mL MIA solution into right knee joint cavity after anesthesia of mice, injecting umbilical cord mesenchymal stem cells 1×10 prepared in example 3 into joint cavity 2 weeks after operation ⁶ Simultaneously, the NGF monoclonal antibody (10 mg/kg) is injected intraperitoneally;

at week 4 after MIA modelling (week 2 after drug intervention), 5 mice were randomly withdrawn from each group, anesthetized with chloral hydrate by intraperitoneal injection, and after cervical dislocation, the right knee joints of the mice were removed by rongeur and scalpel separation, and the soft tissues and muscles around the knee joints were carefully cleaned. HE staining, OARSI joint soft pathology scoring method for articular cartilage scoring. The results are shown in FIG. 2.

OARSI scoring results showed that model group mice had significantly higher OARSI scores (8.27±0.43) than negative control group (P < 0.05), whereas high-dose mab group mice had significantly lower OARSI scores (3.03±0.23) than model group (P < 0.05), and also had better effect than positive control group. In particular, after stem cells are combined with monoclonal antibodies, the knee joint of the mouse has more complete cartilage and subchondral bone structure and more remarkable treatment effect compared with a control group.

A portion of the cartilage portion of each of the remaining groups of mice described above was extracted with RIPA lysis buffer containing 1% PMSF to give total protein of knee cartilage cells, and the cartilage tissue was subjected to ultrasonic disruption on ice and centrifuged at 12000 Xg 4℃for 30min. Protein concentration was detected using BCA protein assay kit. 50 μg of total protein was loaded onto a 10% SDS-PAGE gel and then transferred to PVDF membrane at 300mA constant current. The membranes were incubated in Tris buffer containing 0.1% Tween-20 and 5% nonfat milk powder for 2h at room temperature and washed 3 times with TBS-T. Membranes were incubated overnight with anti-INOS (1:1000), COX-2 (1:1000), NGF (1:1000) primary antibody at 4 ℃. The membrane was then washed 3 times with TBS-T. Then incubated with the secondary antibody for 2h at room temperature. After washing with TBS-T, detection was performed using an enhanced chemiluminescence kit, and quantitative analysis was performed by Quantitone, with the amount of GAPDH protein expressed as a relative expression basis. The results are shown in Table 2.

Table 2 analysis results of the expression level of the related proteins in each group

* Indicating that the differences between treatment groups were extremely significant compared to the model group (P < 0.01).

The Westernblotting detection result shows that compared with a control group, the expression level of the INOS and COX-2 NGF proteins of the mice in the model group is increased (P < 0.05); the positive control, the strain of mab, and the strain of mab combined with stem cell treatment group had reduced levels of NGF, INOS, and COX-2 expression (P < 0.01) compared to the model group. The experimental result of the research shows that the 3N12 monoclonal antibody can obviously inhibit the expression level of mouse chondrocyte INOS, COX-2 and NGF protein, COX-2 is a key effector of arthritis pain and inflammatory reaction, and can effectively protect articular cartilage structure and reduce pain and inhibit inflammation by inhibiting NGF expression and then COX-2 and INOS expression. In addition, the monoclonal antibody has better effect than single antibody treatment after being combined with stem cells.

The foregoing is merely a preferred embodiment of the present application and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present application, which are intended to be comprehended within the scope of the present application. Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present application, and not for limiting the same; although the application has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the application.

Claims

1. An NGF monoclonal antibody, which is characterized in that the variable region sequence of the light chain of the antibody is shown in SEQ ID NO:1, the heavy chain variable region sequence of which is shown in SEQ ID NO: 2.

2. A pharmaceutical composition for repairing knee joint injury, which consists of umbilical mesenchymal stem cells and the NGF monoclonal antibody of claim 1, wherein the knee joint injury corresponds to knee osteoarthritis.

3. Use of umbilical cord mesenchymal stem cells and NGF monoclonal antibodies in the preparation of a pharmaceutical composition for treating knee osteoarthritis, wherein the NGF monoclonal antibodies have light chain variable region sequences as set forth in SEQ ID NOs: 1, the heavy chain variable region sequence of which is shown in SEQ ID NO: 2.

4. The use according to claim 3, wherein the pharmaceutical composition further comprises an excipient.