CN115806935A

CN115806935A - Umbilical cord mesenchymal stem cell culture method

Info

Publication number: CN115806935A
Application number: CN202211032709.0A
Authority: CN
Inventors: 王杰; 肖驰
Original assignee: Guangzhou Lianda Technology Co ltd
Current assignee: Xiamen Morsenkell Cell Biotechnology Co ltd
Priority date: 2022-08-26
Filing date: 2022-08-26
Publication date: 2023-03-17
Anticipated expiration: 2042-08-26
Also published as: CN115806935B

Abstract

The invention provides a culture method of umbilical cord mesenchymal stem cells, and also provides a monoclonal antibody specifically aiming at NOS2, wherein the antibody can treat cervical cancer by inhibiting the activity of nitric oxide synthase, particularly can obviously improve the sensitivity of cervical cancer cells to umbilical cord blood mesenchymal stem cells after the treatment of the NOS2 monoclonal antibody, and can obviously inhibit the proliferation of the cervical cancer cells together, so that the antibody has an obvious synergistic effect.

Description

Umbilical cord mesenchymal stem cell culture method

Technical Field

The application relates to the field of biology, in particular to a culture method of umbilical cord mesenchymal stem cells. Technical Field

Mesenchymal Stem Cells (MSCs) were first originated from early mesoderm, capable of self-renewal and have the potential to differentiate in multiple directions, and can be isolated from adult bone marrow, dental pulp, adipose tissue, umbilical cord, and other tissues. Because of their immunomodulatory role, MSCs are gaining favor in the areas of damaged tissue repair and cell therapy for autoimmune diseases. Among them, mesenchymal stem cells (hucsmcs) derived from umbilical cord have the characteristics of pluripotency, low tumorigenicity, low immunogenicity, high proliferation rate, convenient acquisition and the like, and thus become an important applicable cell source for cell transplantation in allogeneic environment, and they may also become a preferred choice for stem cell therapy. The sources of human umbilical cord mesenchymal stem cells (hUCMSCs) can be roughly divided into 4 types, namely, (1) mesenchymal stem cells derived from human umbilical cord Wharton jelly, (2) mesenchymal stem cells derived from human umbilical cord blood (hUCMSCs), (3) mesenchymal stem cells derived from human umbilical cord blood vessel peripheral tissues (hUCPV-MSCs), and (4) mesenchymal stem cells derived from amnion (hAMSCs).

Human mesenchymal stem cells have been shown to play an anti-inflammatory immunomodulatory and reparative role in various animal models. Bone marrow was found to be the main source of mesenchymal stem cells in most surveys, but the collection of mesenchymal stem cells in bone marrow is invasive and the number of mesenchymal stem cells increases and decreases with age, which significantly limits the clinical application of mesenchymal stem cells. In 2004, there was a study that demonstrated that human cord blood is rich in mesenchymal stem cells and capable of inducing differentiation of its surface markers, and is similar to mesenchymal stem cells extracted from various tissues, such as bone marrow. Therefore, umbilical cord blood-derived mesenchymal stem cells have been widely used in various experimental models. The focus of recent research is the relationship between umbilical cord blood mesenchymal stem cells and cancer metastasis. The umbilical cord blood mesenchymal stem cells are an attractive tool for targeted therapy of cancers, and the human umbilical cord blood mesenchymal stem cells have the capacity of migrating to tumors and can be used as a targeting vector for tumor therapy. There are several reports that confirm the efficacy of mesenchymal stem cells in cancer. The relation between the umbilical cord blood mesenchymal stem cells and the breast cancer stem cells in vivo and in vitro is researched, and the result of CCK-8 in the experiment determines the formation experiment of the soft agar colony, which shows that the human umbilical cord blood mesenchymal stem cells can inhibit the growth of human breast cancer cells. They speculate that umbilical cord blood mesenchymal stem cells may inhibit tumor cell proliferation by blocking the activity of PI3K and Akt protein kinases through a PI3K/Akt signal pathway. It has also been found in experiments that umbilical cord blood mesenchymal stem cells can inhibit the growth of primary glioblastoma multiforme and cause tumor cell apoptosis. The promotion of the apoptosis of the primary glioblastoma multiforme by the umbilical cord blood mesenchymal stem cells is caused by a tumor necrosis factor-related apoptosis-inducing ligand. The umbilical cord blood mesenchymal stem cells can also inhibit the proliferation of the breast cancer cell MDA-MB-231 cells by secreting DKK1 so as to regulate a Wnt signal pathway. The mesenchymal stem cells are a promising therapeutic tool, can provide medicaments for treating tumors, and in the research, the mesenchymal stem cells of umbilical cord blood transfected by lentiviruses can carry a carrier to code a soluble human tumor necrosis factor and a related apoptosis inducing ligand gene driven by an alpha-fetoprotein promoter, and can be used for researching the therapeutic effect of the cells on mouse in-situ transplantation liver cancer. Their studies indicate that umbilical cord blood mesenchymal stem cells can be efficiently transfected by lentiviruses and can migrate into liver cancer tissues in vivo and in vitro, and after intravenous injection of stem cells, these relevant apoptosis-inducing ligands are expressed at the tumor site and exhibit significant anti-tumor activity, which will be stronger when used in combination with 5-FU. Research also shows that the artificial fusion of the umbilical cord blood mesenchymal stem cells and the esophageal cancer cells can inhibit the growth of the esophageal cancer cells, increase apoptosis and inhibit the occurrence of tumors. However, there is also evidence for the risk that the interaction between mesenchymal stem cells of umbilical cord blood and cancer cells may contribute to the metastasis of tumor cells. Mesenchymal stem cells promote metastasis of breast cancer. The interaction of chemokine ligand 5 secreted by the mesenchymal stem cells and chemokine receptor 5 expressed by cancer cells plays an important role in the process of regulating tumor metastasis by the mesenchymal stem cells. Subsequently, the mesenchymal stem cells promote cancer cell metastasis by secreting cell-derived factor-1, interleukin 6 and vascular endothelial growth factor. Invasion and migration of tumors are two steps of tumor metastasis, in order to know whether umbilical cord blood mesenchymal stem cells can influence tumor cell invasion in vitro, whether the relations between the migration of breast cancer cells and the umbilical cord blood mesenchymal stem cells exist is tested in an experiment, when umbilical cord blood mesenchymal stem cells do not exist in a Transwell lower chamber, the number of the breast cancer cells migrating and invading in each chamber is small, however, the number of the experiment groups with umbilical cord blood mesenchymal stem cells is obviously increased, and the result shows that the umbilical cord blood mesenchymal stem cells promote the migration of the breast cancer cells in the in vitro experiment, which is caused by chemotactic factors secreted by the cells. Meanwhile, the possibility that the umbilical cord blood mesenchymal stem cells can cause proliferation of the breast cancer cells so as to interfere invasion and migration of the breast cancer cells is eliminated. In addition, research shows that the mesenchymal stem cells can be differentiated into fibroblasts after entering tumor tissues and then into myofibroblasts, and play an important role in tumor growth, infiltration and metastasis.

Nitric Oxide Synthase (NOS), an enzyme that catalyzes the biosynthesis of Nitric Oxide (NO), is a rate-limiting enzyme of endogenous NO synthesis, and since the 1987 that the chemical nature of vascular endothelial cell relaxing factor (EDRF) is NO, NO has become an active area in modern biological research. Changes in NOS content must be accompanied by changes in NO synthesis. eNOS and iNOS are known as two types of isozymes of NOS, which exert different actions in vivo, and iNOS is also called inducible nitric oxide synthase (abbreviated iNOS or NOS 2). Many studies have been reported on the expression of NOS in tumor tissues at other sites in the body. For example, NOS activity is found to be increased in human tumors such as liver cancer, esophageal cancer, colon adenocarcinoma, bladder cancer, and prostate cancer. However, there are few reports on the studies of systemic expression of NOS in cervical cancer tissues. The existing results show that: the positive expression rate of eNOS and iNOS (two isozymes of NOS) in cervical cancer is obviously higher than that of the control tissues, which indicates that the expression of eNOS and iNOS is related to the generation and development of cervical cancer. The weak expression of eNOS and iNOS in normal cervical tissues indicates that a small amount of NO catalytically generated by the eNOS and the iNOS possibly participates in physiological functions such as protection and secretion of normal cervical epithelium and the like, which is consistent with the research results of Mei Zumin and the like. The abnormal expression of eNOS and iNOS in cervical cancer tissues shows that the amount of NO catalytically induced and synthesized by two NOS in local tissues of the cervical cancer is large, and the high-level NO can not play a role in anti-tumor activity but promotes the proliferation and differentiation of cervical cancer cells, thereby promoting the growth of the tumor cells and playing roles in tumorigenicity and promoting the growth of tumors. The expression characteristics of NOS in cervical tissues are utilized, NOS is used as one of detection indexes for early diagnosis of cervical cancer in the future, and an NOS inhibitor can be utilized for treating the cervical cancer.

However, at present, the research on inhibitors against NOS is not numerous at home, the types of inhibitors provided are not abundant enough, and the research on the combination of NOS inhibitors with other drugs, particularly umbilical cord blood mesenchymal stem cells, is also not numerous.

Disclosure of Invention

The invention provides an improved method for separating and culturing umbilical cord blood mesenchymal stem cells aiming at the defects of the prior art.

Specifically, after the umbilical cord blood of an infant is collected, carrying out density gradient centrifugation on the collected umbilical cord blood by using Ficoll cell separation liquid to obtain mononuclear cells, wherein the volume ratio of the Ficoll separation liquid to the umbilical cord blood is 1:1-1:3, carrying out centrifugal washing on the separated cells for a plurality of times by using an IMDM basic culture medium, and adjusting the cell density for later use; the cord blood mononuclear cells obtained by the isolation were inoculated into a 6-well plate, and IMDM medium containing 10% FBS + IL-3 (15. Mu.g/L) + GM-CSF (5. Mu.g/L) + G-CSF (5. Mu.g/L) was added thereto, and the mixture was set at 37 ℃ and 5% CO ₂ And in the saturated humidity incubator, after 3 days, carrying out half-amount liquid change to remove non-adherent cells, and then, carrying out half-amount liquid change 1 time every 3 days to obtain the cells, namely the umbilical cord blood mesenchymal stem cells.

Furthermore, the present invention also provides monoclonal antibodies specific for NOS 2.

Specifically, the monoclonal antibody of NOS2 is 1a20, and the variable region sequence of the light chain thereof is identified by SEQ ID NO:1, and the heavy chain variable region sequence thereof is shown as SEQ ID NO:2, respectively.

Furthermore, the monoclonal antibodies of the invention may also be subjected to appropriate conservative amino acid substitutions, the substituted antibodies still retaining the corresponding activity.

In addition, the number of amino acids of the monoclonal antibody of the present invention after the modification is preferably within 20 amino acids, more preferably within 10 amino acids, and still more preferably within 5 amino acids (for example, within 3 amino acids, 1 amino acid), that is, in the present specification, the "plurality of amino acids" is preferably 20 amino acids or less, more preferably 10 amino acids or less, still more preferably 5 amino acids or less, and still more preferably 2 amino acids or less. For example, among acidic amino acids (aspartic acid and glutamic acid), basic amino acids (lysine, arginine, histidine), and neutral amino acids, amino acids having a hydrocarbon chain (glycine, alanine, isoleucine, proline), amino acids having a hydroxyl group (serine, threonine), and amino acids containing sulfur (cysteine, methionine) are mentioned.

Further, the monoclonal antibody of the present invention may be represented by the variable region sequence of light chain SEQ ID NO:1, a modification in the heavy chain variable region sequence SEQ ID NO:2, and still retains antibody activity.

The invention also provides a pharmaceutical composition, which comprises the monoclonal antibody of the invention with pharmaceutically effective dose and a pharmaceutically acceptable carrier.

The term "pharmaceutically acceptable" as used herein means that the molecular entities and compositions do not produce adverse, allergic, or other untoward reactions when properly administered to an animal or human.

As used herein, a "pharmaceutically acceptable carrier" should be compatible with, i.e., capable of being blended with, the muteins of the present invention without substantially reducing the effectiveness of the pharmaceutical composition as is often the case. Specific examples of some substances that may serve as pharmaceutically acceptable carriers or components thereof are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and methyl cellulose; powdered gum tragacanth; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyhydric alcohols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as Tween; wetting agents, such as sodium lauryl sulfate; a colorant; a flavoring agent; tabletting agents, stabilizers; an antioxidant; a preservative; pyrogen-free water; isotonic saline solution; and phosphate buffer, and the like.

The pharmaceutical composition of the present invention can be formulated into various dosage forms as required, and can be administered at a dose that is determined by a physician in consideration of the kind, age, weight and general condition of a patient, administration manner, and the like, which are beneficial to the patient.

Furthermore, the invention also provides application of the monoclonal antibody of NOS2 in preparation of a pharmaceutical composition for treating cervical cancer.

Furthermore, the invention also provides application of the NOS2 monoclonal antibody and the umbilical cord blood mesenchymal stem cells in preparing a pharmaceutical composition for treating cervical cancer, wherein the NOS2 monoclonal antibody can be used for treating sensitivity of the cervical cancer cells to the umbilical cord blood mesenchymal stem cells.

The invention aims at providing a highly stable pharmaceutical composition, which is characterized in that: the pharmaceutical composition comprises an antibody and at least one or more of a buffer, an isoosmotic adjusting agent, a stabilizing agent and/or a surfactant. In particular, the pharmaceutical composition comprises 1-150mg/ml anti-NOS 2 monoclonal antibody (mAb), 3-50mM buffer, 2-150mg/ml isotonic regulator/stabilizer and 0.01-0.8mg/ml surfactant, and has a pH of about 4.5-6.8. The preparation can prevent polymer growth of antibody therein, and can maintain biological binding activity of antibody for a long time.

In some embodiments, the buffer is selected from citrate buffer, acetate buffer, histidine buffer, or phosphate buffer; preferably citrate buffer or histidine buffer; more preferably a histidine salt buffer. In some embodiments, the buffer has a concentration of about 4.5 mM to about 50mM, preferably about 5mM to about 25mM, more preferably about 10mM to about 20mM, and most preferably about 10mM to about 15mM.

In some embodiments, the buffer is a histidine salt buffer. The histidine salt buffer is present at a concentration of about 5 to about 30mM, preferably about 10 to about 25mM, more preferably about 10 to about 20mM, and most preferably about 10 to about 15mM. In some embodiments, the histidine salt buffer is about 5mM, about 10mM, about 15mM, about 20mM, about 25mM, or about 30mM. In some embodiments, the histidine salt buffer is about 10mM. In other embodiments, the histidine salt buffer is about 15mM. In other embodiments, the histidine salt buffer is about 20mM. Wherein the histidine salt buffer comprises histidine and hydrochloric acid.

In some embodiments, the buffer is an acetate buffer. The acetate buffer is present at a concentration of about 5mM to about 30mM, preferably about 10mM to about 25mM, more preferably about 10mM to about 20mM, and most preferably about 10mM to about 15mM. Wherein the acetate buffer comprises acetate and acetic acid. The acetate salt comprises various pharmaceutically acceptable inorganic acid salts or organic acid salts of acetic acid or hydrates thereof, including but not limited to potassium acetate or hydrates thereof, and sodium acetate or hydrates thereof; preferably, the acetate salt is sodium acetate or sodium acetate trihydrate.

In some embodiments, the buffer is a citrate buffer. The citrate buffer is present at a concentration of about 3 to about 30mM, preferably about 4.5 to about 30mM, more preferably about 5 to about 20mM, and most preferably about 5 to about 10mM. In some embodiments, the citrate buffer is about 5mM, about 10mM, about 15mM, about 20mM, or about 25mM. In some embodiments, the citrate buffer is about 5mM. In other embodiments, the citrate buffer is about 10mM. In other embodiments, the citrate buffer is about 15mM. The citric acid comprises citric acid per se and hydrate of citric acid, such as monohydrate citric acid; the citrate comprises various pharmaceutically acceptable inorganic acid salts or organic acid salts of citric acid or hydrates thereof, including but not limited to potassium citrate or hydrates thereof, sodium citrate or hydrates thereof; preferably, the citrate is sodium citrate or sodium citrate dihydrate.

In some embodiments, the isotonic adjusting/stabilizing agent is selected from one or more of sodium chloride, mannitol, sucrose, trehalose, maltose, xylitol; one or more of sodium chloride, mannitol and sucrose are preferred; most preferred is sucrose. In some embodiments, the isotonicity adjusting/stabilizing agent is present in an amount of about 4 to about 150mg/ml, preferably about 6 to about 120mg/ml, more preferably about 40 to about 100mg/ml, and most preferably about 60 to about 80mg/ml.

In some embodiments, the isotonicity adjusting/stabilizing agent is about 20-150mg/ml sucrose, preferably about 40-100mg/ml sucrose, more preferably about 60-80mg/ml sucrose, calculated as w/v. In some embodiments, the sucrose is at a concentration of about 40mg/ml, 50mg/ml, 60mg/ml, 70mg/ml, 80mg/ml, 90mg/ml, or 100mg/ml. In some embodiments, the sucrose is at a concentration of about 60mg/ml. In some embodiments, the sucrose is at a concentration of about 70mg/ml. In some embodiments, the sucrose is at a concentration of about 80mg/ml. In some embodiments, the sucrose is at a concentration of about 90mg/ml.

Advantageous effects

The invention provides a separation culture method of umbilical cord mesenchymal stem cells, and also provides a monoclonal antibody specifically aiming at NOS2, wherein the antibody can treat cervical cancer by inhibiting the activity of nitric oxide synthase, particularly can obviously improve the sensitivity of cervical cancer cells to umbilical cord blood mesenchymal stem cells after the treatment of the NOS2 monoclonal antibody, and can obviously inhibit the proliferation of the cervical cancer cells together, so that the monoclonal antibody has an obvious synergistic effect.

Drawings

FIG. 1 ascites antibody titer results chart

FIG. 2 Effect of monoclonal antibodies on nitric oxide synthase Activity

FIG. 3 Effect of monoclonal antibodies on Hela cell survival

FIG. 4 Effect of various groups on cell growth

Detailed Description

The present invention may be understood more readily by reference to the following description of certain embodiments of the invention and the detailed description of the examples included therein. Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such embodiments are necessarily varied. It is also to be understood that the terminology used in the description is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Example 1 isolation and culture of umbilical cord blood mesenchymal Stem cells

Collecting umbilical cord blood of infant, separating with Ficoll cell separating medium with density of 1.077g/ml at 2500r/min,obtaining mononuclear cells after 30min density gradient centrifugation, wherein the volume ratio of Ficoll separating medium to umbilical cord blood is 1:2, centrifugally washing the separated cells for 3 times (1200 r/min,3 min) by using an IMDM basic culture medium, and adjusting the cell density for later use; separating the obtained cord blood mononuclear cells at 2x10 ⁶ Each/ml was inoculated into 6-well plates, and IMDM medium containing 10% FBS IL-3 (15. Mu.g/L) + GM-CSF (5. Mu.g/L) + G-CSF (5. Mu.g/L) was added at 37 ℃ and 5% CO ₂ After 3 days, half of the liquid is changed every 3 days for 1 time, when the cells are fused to 85 percent, the cells are digested by 0.125 percent pancreatin and 0.02 percent EDTA, passaged according to the proportion of 1:2, when the cells are transferred to the third generation (P3), the cells are digested, and the collected cells are the umbilical cord blood mesenchymal stem cells, and the flow detection is carried out. The method comprises the following steps: collecting umbilical cord blood mesenchymal stem cells, and adjusting the cell concentration to 10 ⁷ Mu.l of fluorescence labeled antibodies CD13-PE, CD29-FITC, CD44-FITC, CD166-PE, CD34-FITC, CD45-FITC, CD105-FITC and HLA-DR-PE are added into the cells per ml, the cells are incubated on ice for 30min, washed with PBS for 2 times, centrifuged at 1000r/min for 6min, and resuspended in 500. Mu.l of PBS to a cell concentration of 10 ⁶ About one/ml, and then analyzed on a flow computer. The result shows that CD13, CD29, CD105, CD166 and CD44 are positively expressed, and CD34, CD45 and HLA-DR are negatively expressed, which indicates that the cells obtained by culture are the umbilical cord blood mesenchymal stem cells.

EXAMPLE 2 preparation of NOS2 monoclonal antibody

Recombinant expression of NOS2 protein as immunogen (Hzbscience, cat: ZY837Hu 011), and 5-fold dilution of Montanide ^TM Equal volume mixing of GEL01ST polymer adjuvant, subcutaneous multi-point injection immunization of BALB/c mice, 3 times of total immunization, 2 weeks of each immunization interval, and 100 μ g/mouse. And performing impact immunization by using NOS2 protein without an adjuvant 7d after the three-immunization, and fusing splenocytes of the No. 2 mouse with the highest titer 6d later.

Fusing SP2/0 cells and splenocytes under the action of PEG1500, and adding HAT and HT into DMEM medium after fusion for liquid exchange. When the hybridoma cells grow to about 1/3 of the basal area, the supernatant is sucked up, and the antibody titer is measured by an indirect ELISA method. And selecting the well which is determined to be strong positive for subcloning and screening to obtain two hybridoma cell strains with the highest potency and anti-NOS 2 protein, wherein the two hybridoma cell strains are named as 1A20 and 5G23.

And (3) carrying out abdominal paraffin sensitization on the BALB/c mouse, and respectively injecting the two separated hybridoma cells after 7d to prepare ascites. The prepared ascites monoclonal antibody is used for measuring the antibody titer by an indirect ELISA method, 2 mu g of recombinant NOS2 protein is used as a coating antigen, and after the blocking, the ascites monoclonal antibody is continuously diluted from 1. The results are shown in FIG. 1.

From the results in fig. 1, the potency of both monoclonal antibodies was greater than 1 512000, and the potency effect was better.

The prepared ascites is crudely extracted by an ammonium sulfate salting-out method, then a protein affinity chromatographic column is used for further purification to obtain an IgG antibody with higher purity, and the SDS-PAGE identification result of the purified monoclonal antibody shows that the monoclonal antibodies 1A20 and 5G23 are obviously two bands at about 25kD and 56kD, namely a monoclonal antibody light chain and a monoclonal antibody heavy chain, thereby proving that the purer monoclonal antibody is obtained.

And (3) carrying out monoclonal antibody subtype identification on the prepared ascites by using a mouse Ig class/monoclonal antibody subtype detection kit. The types of 1a20 and 5G23 monoclonal antibodies are both IgG1 types, and the light chains are both kappa chains.

Example 3 determination of affinity of NOS2 monoclonal antibody 1A20

Applying an indirect ELISA method, coating an ELISA plate with NOS2 recombinant protein at the concentration of 1 mu g/mL, sealing, adding a purified monoclonal antibody diluted in a multiple proportion for incubation, taking goat anti-mouse IgG marked by HRP as a secondary antibody, and reading an OD450nm light absorption value by an ELISA reader. The OD450nm readings of several serial dilutions were regarded as 100% binding of antigen and antibody, the antibody concentration (mol/L) was used as abscissa, the OD450nm absorbance was used as ordinate to make scatter plot, the binding rate of antigen and antibody was 50% at half the maximum reading, and a logarithmic trend line and formula were generated. The half of the maximum value at OD450nm was substituted into the formula to determine the antibody concentration at that time, i.e., the affinity dissociation constant (Kd). The result showed that the Kd value of 1A20 monoclonal antibody was 12.53nM.

Example 4 Activity identification of NOS2 monoclonal antibody 1A20

The HeLa cell strain was inoculated into a 96-well plate, the same number of cells in each well was maintained, and when 80% of the cells grew, a negative control group and a positive control group of 1400W (at a concentration of 50. Mu.g/L) were divided, and 6 replicate wells were set for each monoclonal antibody group (at a concentration of 1, 10, 50, 100. Mu.g/mL, respectively). Culturing for 6h, detecting the activity according to the instruction of the nitric oxide synthetase detection kit, taking a hole without cells as a blank control, wherein the excitation wavelength is 495nm and the emission wavelength is 515nm, taking the 96-well plate, detecting by using a fluorescence microplate reader, and calculating the relative activity. The results are shown in FIG. 2.

As can be seen from the results in fig. 2, 1400W and the 1a20 antibody of the present invention both significantly inhibit the activity of nitric oxide synthase, and the difference is significant compared to the negative group, P <0.05. Meanwhile, the antibody group of the 1A20 of the invention has concentration-dependent activity for inhibiting concentration nitric oxide synthase, when the concentration of the 1A20 monoclonal antibody is 50 mu g/mL, the activity is (0.30 +/-0.04) relative to that of a negative control group, and the antibody group has better activity inhibition effect and far better inhibition effect than that of a positive control group.

Example 5 Effect of NOS2 monoclonal antibody 1A20 on Hela cell Activity

According to density 5X 10 ⁴ Hela cells were seeded in 96-well plates per mL. A control group and drug groups with different concentrations are set, wherein the control group comprises a positive control 1400W group (50 mu g/mL) and a monoclonal antibody drug group (the concentrations are 1, 10, 50 and 100 mu g/mL respectively). After 48h incubation, 10 μ LMTT (5 mg/mL) was added to each well and incubation continued for 4h, 100 μ LDMSO was added to each well, shaking at low speed for 10min, and absorbance was measured at 490nm in each well using an ELISA. The cell viability of each group was calculated by setting the cell viability of the control group at 100% and the cell inhibition rate =1- (drug absorbance/control absorbance) × 100% according to the formula. The results are shown in FIG. 3.

From the results in FIG. 3, it was shown that 1400W alone and low concentrations of mAb did not significantly inhibit cell proliferation. Under the condition of the monoclonal antibody with the concentration of 50 mu g/mL, the survival rate of the cells is (70.2 +/-1.8)%, which shows that the monoclonal antibody has certain effect of inhibiting the survival rate of the cancer cells.

Example 6 Effect of NOS2 monoclonal antibody 1A20 monoclonal antibody on HeLa cell Activity in combination with umbilical cord blood mesenchymal Stem cells

HeLa cells with good logarithmic phase growth state were taken and spread in a 96-well plate, and 10000 cells were inoculated per well. The experimental groups were as follows:

control group: no treatment, only Hela cells;

stem cell co-culture group: putting a Transwell chamber with a pore diameter of 3.0 mu m into a 96-well culture plate, and inoculating 2 ten thousand stem cells which are separated in the example 1 and have good growth state in the logarithmic phase in each well;

stem cell and monoclonal antibody co-culture group: placing a Transwell chamber with the aperture of 3.0 mu m into a 96-well culture plate, inoculating 2 ten thousand stem cells separated in the example 1 with good logarithmic phase growth state in each well, and simultaneously adding 100 mu g/mL of 1A20 monoclonal antibody;

monoclonal antibody co-culture group: adding 100 mu g/mL of 1A20 monoclonal antibody;

stem cell in combination with positive control co-culture group: a Transwell chamber with a pore size of 3.0 μm was placed in a 96-well plate, and 2 ten thousand of the stem cells isolated in example 1, which had good growth state in log phase, were seeded per well, while 1400W (INOS inhibitor) was added at 100. Mu.g/mL;

the blank control group was supplemented with DMEM complete medium per well.

3 replicates per group were set and cells were digested after 3 days of culture. After digestion, 100. Mu.l of basal medium and 10. Mu.l of CCK-8 staining solution were added, mixed well and incubated in a constant temperature incubator. After incubation for 2h, 100. Mu.l of the supernatant was pipetted into a new 96-well plate and placed in a microplate reader to determine the D value at a wavelength of 450 nm.

As can be seen from the results in fig. 4, the monoclonal antibody or stem cell co-culture group alone can inhibit cell proliferation and reduce the OD value of the cells, but the effect is not obvious after the monoclonal antibody and stem cell are used in combination, the D value of the HeLa cells in the stem cell and monoclonal antibody co-culture group is significantly lower than that in the control group (P < 0.01), the result is only 0.13 ± 0.02, and the effect is also significant compared with 0.24 ± 0.02 in the stem cell combination positive control group.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and these modifications or substitutions do not depart from the spirit of the corresponding technical solutions of the embodiments of the present invention.

Claims

1. A monoclonal antibody 1A20 of NOS2, characterized in that the light chain variable region sequence is as shown in SEQ ID NO:1, and the heavy chain variable region sequence thereof is shown as SEQ ID NO:2, respectively.

2. The application of a monoclonal antibody 1A20 of NOS2 in the preparation of a pharmaceutical composition for inhibiting the proliferation of cervical cancer cells, wherein the light chain variable region sequence of the antibody is shown as SEQ ID NO:1, and the heavy chain variable region sequence thereof is shown as SEQ ID NO:2, respectively.

3. The application of a monoclonal antibody 1A20 of NOS2 and umbilical cord mesenchymal stem cells in preparing a pharmaceutical composition for inhibiting the proliferation of cervical cancer cells; wherein the light chain variable region sequence of the antibody is shown as SEQ ID NO:1, and the heavy chain variable region sequence thereof is shown as SEQ ID NO:2 is shown in the specification; the umbilical cord mesenchymal stem cells are prepared by adopting the following method: after the collection of the infant umbilical cord blood, carrying out gradient centrifugation on Ficoll cell separation liquid with the density of 1.077g/ml at the density of 2500r/min for 30min to obtain mononuclear cells, wherein the volume ratio of the Ficoll cell separation liquid to the umbilical cord blood is 1:2, the separated cells are centrifugally washed for 3 times by an IMDM basic culture medium of 1200r/min for 3min, and the cell density is adjusted for later use; separating the obtained cord blood mononuclear cells at 2x10 ⁶ One/ml density was inoculated into 6-well plates, IMDM medium containing 10% FBS IL-315. Mu.g/L, GM-CSF 5. Mu.g/L, G-CSF 5. Mu.g/L was added, set at 37 ℃,5% CO + ₂ Changing the liquid for half after 3 days in a saturated humidity incubator, removing non-adherent cells, changing the liquid for half every 3 days for 1 time, until the cells are fused to 85 percent, digesting with 0.125 percent pancreatin and 0.02 percent EDTA, carrying out passage according to the proportion of 1:2, digesting the cells when the cells are transferred to the third generation,collecting the cells to obtain the umbilical cord blood mesenchymal stem cells.

4. Use according to any one of claims 2 to 3, characterised in that the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

5. Use according to claim 4, characterized in that the pharmaceutical composition comprises an antibody and at least one or several of a buffer, an isotonicity adjusting agent, a stabilizer and/or a surfactant.

6. Use according to claim 5, characterized in that the buffer is selected from citrate buffer, acetate buffer, histidine buffer or phosphate buffer.