CN115806935B

CN115806935B - Umbilical cord mesenchymal stem cell culture method

Info

Publication number: CN115806935B
Application number: CN202211032709.0A
Authority: CN
Inventors: 王杰; 肖驰
Original assignee: Xiamen Morsenkell Cell Biotechnology Co ltd
Current assignee: Xiamen Morsenkell Cell Biotechnology Co ltd
Priority date: 2022-08-26
Filing date: 2022-08-26
Publication date: 2023-07-07
Anticipated expiration: 2042-08-26
Also published as: CN115806935A

Abstract

The invention provides a method for culturing umbilical cord mesenchymal stem cells, and in addition, provides a monoclonal antibody specific to NOS2, wherein the antibody can treat cervical cancer by inhibiting the activity of nitric oxide synthase, and particularly can remarkably improve the sensitivity of cervical cancer cells to umbilical cord blood mesenchymal stem cells after the treatment by the NOS2 monoclonal antibody, and the monoclonal antibody can remarkably inhibit the proliferation of cervical cancer cells together, so that the monoclonal antibody has remarkable synergistic effect.

Description

Umbilical cord mesenchymal stem cell culture method

Technical Field

The application relates to the field of biology, in particular to a method for culturing umbilical cord mesenchymal stem cells.

Technical Field

Mesenchymal stem cells (mesenchymal stem cells, MSCs) were originally derived from early mesoderm, were able to self-renew and had the potential for multipotent differentiation, and were isolated from adult bone marrow, dental pulp, adipose tissue, umbilical cord, etc. MSCs are favored in the field of damaged tissue repair and cellular therapy of autoimmune diseases because of their immunomodulatory effects. Among them, mesenchymal stem cells (huchmscs) derived from umbilical cord become an important suitable cell source for cell transplantation in allogeneic environment due to their multipotency, low tumorigenicity, low immunogenicity, high proliferation rate, convenient acquisition and other characteristics, and may become a preferential choice for stem cell therapy. The sources of human umbilical cord mesenchymal stem cells (hUCMSCs) can be roughly classified into 4 types, namely (1) mesenchymal stem cells obtained from human umbilical cord huacorn glue, (2) mesenchymal stem cells derived from human umbilical cord blood (hUCMSCs), (3) mesenchymal stem cells derived from human umbilical cord perivascular tissue (hUCPV-MSCs), and (4) mesenchymal stem cells obtained from amniotic membrane (hAMSCs).

Human mesenchymal stem cells have been demonstrated to exert anti-inflammatory immunomodulatory and repairing effects in various animal models. Bone marrow was found to be the major source of mesenchymal stem cells in most surveys, but harvesting mesenchymal stem cells in bone marrow is invasive and the number of mesenchymal stem cells increases and decreases in marrow age, which significantly limits the clinical use of mesenchymal stem cells. In 2004, studies have demonstrated that human umbilical cord blood is rich in mesenchymal stem cells and is able to induce 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 on the relationship between umbilical cord blood mesenchymal stem cells and cancer metastasis. Umbilical cord blood mesenchymal stem cells are attractive cancer targeted therapeutic tools, and human umbilical cord blood mesenchymal stem cells have the ability to migrate to tumors, and can be used as targeted carriers for tumor treatment. Several reports have been made to confirm the therapeutic effect of mesenchymal stem cells on cancer. The in vivo and in vitro relation between the umbilical cord blood mesenchymal stem cells and the breast cancer stem cells is studied, and the result of CCK-8 in the experiment determines the formation experiment of soft agar colonies, 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 PI3K/Akt signaling pathway to inhibit PI3K and Akt protein kinase activity. It has also been found in experiments that umbilical cord blood mesenchymal stem cells can inhibit the growth of primary glioblastoma multiforme and cause apoptosis of tumor cells. The promotion of apoptosis of primary glioblastoma multiforme by umbilical cord blood mesenchymal stem cells is caused by tumor necrosis factor-related apoptosis-inducing ligands. Umbilical cord blood mesenchymal stem cells can also inhibit proliferation of breast cancer cells MDA-MB-231 by secreting DKK1 and thereby regulating a Wnt signal pathway. The mesenchymal stem cells are a very promising therapeutic tool, can provide medicines for tumor treatment, and in research, the slow virus transfected umbilical cord blood mesenchymal stem cells can be used for carrier encoding soluble human tumor necrosis factor driven by alpha fetoprotein promoter and related apoptosis inducing ligand genes, and can be used for researching the therapeutic effect of the cells on mice in-situ transplantation liver cancer. Their studies have shown that umbilical cord blood mesenchymal stem cells can be effectively transfected by lentiviruses and migrate into liver cancer tissues in vivo and in vitro, and that these related apoptosis-inducing ligands are expressed at tumor sites and exhibit significant antitumor activity after intravenous injection of stem cells, which effect is enhanced when used in combination with 5-FU. Also, studies have shown that artificial fusion of umbilical cord blood mesenchymal stem cells with esophageal cancer cells can inhibit growth of esophageal cancer cells, increase apoptosis, and inhibit tumor generation. However, there is also evidence that the interaction between umbilical cord blood mesenchymal stem cells and cancer cells may promote the risk of tumor cell metastasis. Mesenchymal stem cells promote metastasis of breast cancer. Chemokine ligand 5 secreted by mesenchymal stem cells and chemokine receptor 5 interactions expressed by cancer cells play an important role in the process of regulating tumor metastasis by 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. Tumor invasion and migration are two steps of tumor metastasis, and in order to know whether umbilical cord blood mesenchymal stem cells can affect tumor cell invasion in vitro, it was tested in experiments whether there was a relationship between the migration of breast cancer cells and umbilical cord blood mesenchymal stem cells, and when umbilical cord blood mesenchymal stem cells were not present in the Transwell lower chamber, the number of breast cancer cells migrated and invaded in each chamber was small, however, the number was significantly increased in the experimental group with umbilical cord blood mesenchymal stem cells, which results indicate that umbilical cord blood mesenchymal stem cells promoted migration of breast cancer cells in vitro due to chemokines secreted by cells. They also exclude the possibility that umbilical cord blood mesenchymal stem cells will proliferate breast cancer cells, thereby interfering with the invasive migration of breast cancer cells. In addition, research has shown that mesenchymal stem cells can differentiate into fibroblasts after entering tumor tissue, and then into myofibroblasts, and play an important role in tumor growth, infiltration and metastasis.

Nitric Oxide Synthase (NOS) is an enzyme that catalyzes the biosynthesis of Nitric Oxide (NO), which is the rate-limiting enzyme for endogenous NO synthesis, and since 1987 it was confirmed that the chemical nature of vascular endothelial cell diastolic factor (EDRF) is NO, NO has become a very active area in modern biological research. The variation in NOS content will necessarily be accompanied by a variation in NO synthesis. eNOS and iNOS are known to be two classes of isozymes of NOS, which exert different roles in the body, iNOS also being called inducible nitric oxide synthase (abbreviated iNOS or NOS 2). Many studies have been reported on expression of NOS in tumor tissue at other sites in the body. For example, it has been found that NOS activity is increased in tumors such as human liver cancer, esophageal cancer, colon adenocarcinoma, bladder cancer and prostate cancer. However, there are few reports on the systematic study of NOS expression 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 a control tissue, which shows that the expression of eNOS and iNOS is related to the occurrence and development of cervical cancer. The weak expression of eNOS and iNOS in normal cervical tissues suggests that the small amount of NO produced by their catalysis may be involved in physiological functions such as protection and secretion of normal cervical epithelium, which is consistent with the results of studies such as Mei Zumin. Abnormal expression of eNOS and iNOS in cervical cancer tissues shows that the partial tissues of cervical cancer have more NO which is synthesized by the catalysis of two NOS, and the high-level NO can not only play the anti-tumor activity, but also promote proliferation and differentiation of cervical cancer cells, thereby promoting the growth of tumor cells and playing the role of tumorigenicity and tumorigenesis. 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 NOS inhibitors can also be utilized for treating cervical cancer.

However, at present, studies on NOS inhibitors are not very much in China, the types of inhibitors provided are not very abundant, and studies on NOS inhibitors combined with other drugs, particularly umbilical cord blood mesenchymal stem cells, are not very much.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides an improved separation and culture method of umbilical cord blood mesenchymal stem cells.

Specifically, the invention provides a method for separating and culturing mesenchymal stem cells of umbilical cord blood, which comprises the steps of obtaining mononuclear cells after collecting umbilical cord blood of an infant by using Ficoll cell separation liquid and gradient centrifugation, wherein the volume ratio of Ficoll separation liquid to umbilical cord blood is 1:1-1:3, centrifugally washing the separated cells for a plurality of times by using an IMDM basal medium, and adjusting the cell density for later use; the isolated cord blood mononuclear cells 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 addedPlacing at 37deg.C, 5% CO ₂ In the saturated humidity incubator, half liquid exchange is carried out after 3 days, and the non-adherent cells are removed, and the half liquid exchange is carried out 1 time every 3 days later, and the obtained cells are obtained by culture, namely the umbilical cord blood mesenchymal stem cells.

Furthermore, the present invention also provides a monoclonal antibody specific to NOS 2.

Specifically, the monoclonal antibody of NOS2 is 1A20, and the variable region sequence of the monoclonal antibody is identified by the variable region sequence, so that the variable region sequence of the light chain is shown as SEQ ID NO:1, the heavy chain variable region sequence of which is shown in SEQ ID NO: 2.

Furthermore, the monoclonal antibodies of the invention may be substituted with appropriate conserved amino acids, which substituted antibodies still retain the corresponding activity.

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

Further, the monoclonal antibody of the invention may be found in the light chain variable region sequence SEQ ID NO:1 maintains 99% sequence identity over the heavy chain variable region sequence of SEQ ID NO:2, and still maintain antibody activity.

The invention also provides a pharmaceutical composition, which contains 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 entity and composition 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 efficacy of the pharmaceutical composition in the usual manner. Specific examples of some substances which may be 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; tragacanth powder; 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; polyols such as propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifying agents, such as Tween; wetting agents, such as sodium lauryl sulfate; a colorant; a flavoring agent; tabletting and stabilizing agent; an antioxidant; a preservative; non-thermal raw water; isotonic saline solution; and phosphate buffer, etc.

The pharmaceutical composition of the present invention can be formulated into various dosage forms as needed, and the dosage beneficial to the patient can be determined by the physician according to the type, age, weight and general disease condition of the patient, the administration mode, etc.

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

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

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

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

In some embodiments, the buffer is a histidine salt buffer. The histidine salt buffer concentration is about 5-30mM, preferably about 10-25mM, more preferably about 10-20mM, and most preferably about 10-15mM. 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 concentration is 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. Wherein the acetate buffer comprises acetate and acetic acid. The "acetate" includes 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, sodium acetate or hydrates thereof; preferably, the acetate is sodium acetate or sodium acetate trihydrate.

In some embodiments, the buffer is a citrate buffer. The citrate buffer concentration is about 3-30mM, preferably about 4.5-30mM, more preferably about 5-20mM, and most preferably about 5-10mM. 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. "citric acid" as used herein includes citric acid itself as well as citric acid hydrates, such as citric acid monohydrate; the "citrate" includes various pharmaceutically acceptable inorganic 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 isotonicity modifier/stabilizer is selected from one or more of sodium chloride, mannitol, sucrose, trehalose, maltose, xylitol; preferably one or more of sodium chloride, mannitol and sucrose; sucrose is most preferred. In some embodiments, the isotonicity modifier/stabilizer is present in an amount of about 4 to 150mg/ml, preferably about 6 to 120mg/ml, more preferably about 40 to 100mg/ml, and most preferably about 60 to 80mg/ml.

In some embodiments, the isotonicity modifier/stabilizer is about 20 to 150mg/ml sucrose, preferably about 40 to 100mg/ml sucrose, more preferably about 60 to 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 in addition, provides a monoclonal antibody specific to NOS2, the antibody can treat cervical cancer by inhibiting the activity of nitric oxide synthase, and particularly can remarkably improve the sensitivity of cervical cancer cells to umbilical cord blood mesenchymal stem cells after the treatment of the NOS2 monoclonal antibody, and the monoclonal antibody can remarkably inhibit the proliferation of cervical cancer cells together, so that the monoclonal antibody has remarkable synergistic effect.

Drawings

FIG. 1A chart of ascites antibody titer results

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

FIG. 3 influence of monoclonal antibodies on Hela cell viability

FIG. 4 influence of the experimental groups on cell growth

Detailed Description

The invention may be understood more readily by reference to the following detailed description of some embodiments of the invention and 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 may, of course, vary. It is also to be understood that the terminology used herein 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

Obtaining mononuclear cells by using Ficoll cell separating liquid with the density of 1.077g/ml after the acquisition of the infant umbilical cord blood at 2500r/min and a density gradient centrifugation for 30min, wherein the volume ratio of the Ficoll separating liquid to the umbilical cord blood is 1:2, centrifugally washing the separated cells for 3 times by using an IMDM basal medium (1200 r/min and 3 min), and adjusting the cell density for standby; separating umbilical cord blood mononuclear cells at a ratio of 2x10 ⁶ The cells were inoculated into 6-well plates at a density of one ml, 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, and the mixture was placed at 37℃with 5% CO ₂ And (3) after 3 days, carrying out half liquid exchange and removing non-adherent cells, carrying out half liquid exchange for 1 time every 3 days later, carrying out passage according to the proportion of 1:2 after the cells are fused to reach 85 percent by using 0.125 percent pancreatin plus 0.02 percent EDTA, carrying out digestion on the cells when the cells are transferred to the third generation (P3), collecting the cells, namely the umbilical cord blood mesenchymal stem cells, and carrying out flow detection. The method comprises the following steps: collecting umbilical cord blood mesenchymal stem cells, and adjusting the cell concentration to 10 ⁷ Mu.l of fluorescent-labeled antibody CD13-PE, CD29-F was added per mlITC, CD44-FITC, CD166-PE, CD34-FITC, CD45-FITC, CD105-FITC, HLA-DR-PE, incubating cells on ice for 30min, washing 2 times with PBS, centrifuging at 1000r/min for 6min, and resuspending cells with 500 μl of PBS to give a cell concentration of 10 ⁶ About one per ml, and then flow-on-machine analysis. The results showed that CD13, CD29, CD105, CD166 and CD44 were expressed positively, while CD34, CD45 and HLA-DR were expressed negatively, indicating that the cultured cells were 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 GEL01ST polymer adjuvant is mixed in equal volume, BALB/c mice are immunized by subcutaneous multipoint injection, and the total immunization is 3 times, wherein the immunization interval is 2 weeks, and the dosage is 100 mug/mouse. The spleen cells of the No. 2 mice with the highest titer are taken for fusion after 6d of the three-phase immunization by the impact of NOS2 protein without adjuvant.

SP2/0 cells and spleen cells are fused under the action of PEG1500, and after fusion, HAT and HT are added to the mixture by using DMEM culture medium for liquid exchange. After the hybridoma grows to about 1/3 of the bottom area, the supernatant is sucked, and the antibody titer is measured by an indirect ELISA method. The wells determined to be strongly positive were selected for subcloning and screening to obtain two hybridoma cell lines with highest titers against NOS2 protein, designated 1A20 and 5G23.

The BALB/c mice were sensitized with paraffin in the abdominal cavity, and ascites were prepared by injecting two isolated hybridoma cells after 7 d. The prepared ascites monoclonal antibody is used for measuring the antibody titer by an indirect ELISA method, 2 mug recombinant NOS2 protein is used as a coating antigen, and the ascites monoclonal antibody is serially diluted from 1:10 after being blocked for measuring the antibody titer. The results are shown in FIG. 1.

From the results shown in FIG. 1, the titers of the two monoclonal antibodies are both greater than 1:512000, and the monoclonal antibodies have good titer effects.

The prepared ascites is subjected to crude extraction by an ammonium sulfate salting-out method, then a protein G 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 have obvious two bands at about 25kD and 56kD, namely a monoclonal antibody light chain and a monoclonal antibody heavy chain, and the purer monoclonal antibody is proved to be obtained.

Monoclonal antibody subtype identification was performed on ascites fluid prepared as described above using a mouse Ig class/mab subclass detection kit. The types of 1A20 and 5G23 monoclonal antibodies are both IgG1 type, and the light chains are both kappa chains.

Example 3 determination of affinity of NOS2 monoclonal antibody 1A20

The ELISA plate is coated with NOS2 recombinant protein at a concentration of 1 mug/mL by an indirect ELISA method, the ELISA plate is closed, purified monoclonal antibody diluted by a double ratio is added for incubation, goat anti-mouse IgG marked by HRP is used as a secondary antibody, and an OD450nm absorbance value is read by an ELISA reader. The OD450nm readings of several dilutions in succession are regarded as 100% binding of antigen-antibody when no longer increased, the ordinate is the absorbance at OD450nm, and the ordinate is the scatter plot, and the 50% binding of antigen-antibody when half of the maximum value of the readings is used to generate a logarithmic trend line and formula. Half of the OD450nm maximum was substituted into the formula, and the concentration of the antibody at this time was determined as affinity dissociation constant (Kd). The results showed that Kd value of 1A20 mab was 12.53nM.

EXAMPLE 4 identification of monoclonal antibody 1A20 to NOS2

HeLa cell lines were inoculated into 96-well plates, the same number of cells was maintained in each well, and when the cells grew to 80%, negative control groups and positive control groups were set up to 1400W groups (concentration: 50. Mu.g/L), and 6 multiplex wells were set up for each of the monoclonal antibody groups (concentration: 1, 10, 50, 100. Mu.g/mL, respectively). After 6h of culture, the activity of the cell-free cell is detected according to the specification of a nitric oxide synthase detection kit, a hole without cells is used as a blank control, the excitation wavelength is 495nm, the emission wavelength is 515nm, and the 96-well plate is taken and detected by a fluorescence enzyme-labeled instrument, and the relative activity is calculated. The results are shown in FIG. 2.

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

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

According to a density of 5X 10 ⁴ HeLa cells were seeded in 96-well plates per mL. The control group and the drug groups with different concentrations are set, namely a positive control 1400W group (50 mug/mL) and a monoclonal antibody drug group (the concentrations are respectively 1, 10, 50 and 100 mug/mL). After 48h incubation, 10 μLMTT (5 mg/mL) was added to each well and incubation was continued for 4h, 100 μLDMSO was added to each well, shaking was performed at low speed for 10min, and absorbance at 490nm was measured with an ELISA. Cell viability of each group was calculated as formula cell inhibition = 1- (drug group absorbance/control group absorbance) ×100% with reference to 100% of control group cell viability. The results are shown in FIG. 3.

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

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

HeLa cells with good growth state in the logarithmic phase are paved into 96-well plates, and 10000 HeLa cells are inoculated into each well. The experimental groupings were as follows:

control group: without treatment, only Hela cells;

stem cell co-culture group: placing a Transwell chamber with a pore diameter of 3.0 μm into a 96-well culture plate, and inoculating 2 ten thousand stem cells isolated in example 1 with good log phase growth state into each well;

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

mab co-culture group: adding 100 mug/mL of 1A20 monoclonal antibody;

stem cells in combination with positive control co-culture group: a Transwell chamber with a 3.0 μm aperture was placed in a 96-well plate, and each well was inoculated with 2 ten thousand stem cells isolated in example 1, which were well grown in log phase, while 1400W (INOS inhibitor) was added at 100. Mu.g/mL;

DMEM complete medium make-up system was added to each well of the blank.

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

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

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention 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 invention.

And (3) a sequence table:

Claims

1. a monoclonal antibody 1a20 of NOS2, characterized by a light chain variable region sequence as set forth in SEQ ID NO:1, the heavy chain variable region sequence of which is shown in SEQ ID NO: 2.

2. Use of monoclonal antibody 1a20 of NOS2 for preparing a pharmaceutical composition for inhibiting proliferation of cervical cancer cells, wherein the light chain variable region sequence of the antibody is as shown in SEQ ID NO:1, the heavy chain variable region sequence of which is shown in SEQ ID NO: 2.

3. Use of monoclonal antibody 1a20 of NOS2 and umbilical cord mesenchymal stem cells in the preparation of a pharmaceutical composition for inhibiting proliferation of cervical cancer cells; wherein, the light chain variable region sequence of the antibody is shown as SEQ ID NO:1, the heavy chain variable region sequence of which is shown in SEQ ID NO:2 is shown in the figure; the umbilical cord mesenchymal stem cells are prepared by the following method: obtaining mononuclear cells by using Ficoll cell separating liquid with the density of 1.077g/ml at 2500r/min and density gradient centrifugation for 30min after the acquisition of the infant umbilical cord blood, wherein the volume ratio of the Ficoll separating liquid to the umbilical cord blood is 1:2, and centrifugally washing the separated cells for 3 times by using an IMDM basal medium at 1200r/min for 3min to adjust the cell density for standby; separating umbilical cord blood mononuclear cells at a ratio of 2x10 ⁶ The cells were inoculated into 6-well plates at a density of one ml, IMDM medium containing 10% FBS+315. Mu.g/L, GM-CSF 5. Mu.g/L, G-CSF 5. Mu.g/L was added, and the mixture was placed at 37℃with 5% CO ₂ And (3) after 3 days, carrying out half liquid exchange and removing non-adherent cells, and after every 3 days, carrying out half liquid exchange for 1 time, digesting with 0.125% pancreatin and 0.02% EDTA until the cell fusion reaches 85%, carrying out passage according to the proportion of 1:2, and carrying out cell digestion when the cell is transferred to the third generation, wherein the collected cells are umbilical cord blood mesenchymal stem cells.

4. A use according to any one of claims 2 to 3, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

5. The use according to claim 4, wherein the pharmaceutical composition comprises an antibody and at least one or more 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.