Application of umbilical cord mesenchymal stem cells and monoclonal antibody in combined treatment of cancer

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of umbilical cord mesenchymal stem cells and monoclonal antibodies in combined treatment of cancers. The monoclonal antibody is a monoclonal antibody specifically aiming at human PAFAH1B3 protein, the amino acid sequence of a heavy chain variable region of the monoclonal antibody is as shown in SEQ ID NO: 1, and the amino acid sequence of a light chain variable region of the monoclonal antibody is as shown in SEQ ID NO: 2. The PAFAH1B3 protein monoclonal antibody can significantly inhibit the proliferation capacity of pancreatic cancer cells, and after the umbilical cord mesenchymal stem cells and the PAFAH1B3 protein monoclonal antibody are combined for use, the inhibition effect on the proliferation capacity of the pancreatic cancer cells is better, and the umbilical cord mesenchymal stem cells can be used for preparing anti-cancer drugs and have good application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of umbilical cord mesenchymal stem cells and monoclonal antibodies in the combined treatment of cancer. Background Art

[0002] Pancreatic cancer is a tumor that originates from the pancreatic ductal epithelium and is one of the most deadly malignant tumors in humans. Pancreatic cancer is a tumor whose pathogenesis is still unknown, and the age of onset is mostly between 40 and 65. Currently, the treatments for pancreatic cancer mainly include chemotherapy, radiotherapy, and targeted therapy. However, it has a high chance of recurrence and strong side effects. Therefore, we need to study a safe biological agent that can resist the growth of pancreatic cancer cells and has targeted therapy.

[0003] Mesenchymal stem cells include mesenchymal stem cells from bone marrow, umbilical cord, and fat. They have a certain self-renewal ability, can be amplified in large quantities, and can also age and die. Mesenchymal stem cells can differentiate into different types of cells under specific culture conditions. Human umbilical cord stem cells are a type of stem cell isolated from human umbilical cord tissue. They have strong differentiation potential and can differentiate into various tissue cells such as nerves, muscles, liver, and myocardium under specific induction conditions. At present, they have been widely used in the fields of tissue engineering and regenerative medicine.

[0004] The uncontrolled proliferation, invasion, and migration of pancreatic cancer cells are the root cause of death in pancreatic cancer patients. Monoclonal antibodies are a very important direction for the treatment of pancreatic cancer. Through the body's autoimmune mechanism, the treatment of pancreatic cancer can improve patient acceptability and tolerance and further reduce the recurrence of the disease. Related studies have shown that the PAFAH1B3 gene is closely related to the proliferation, invasion, and migration of pancreatic cancer cells. Therefore, there is an urgent need to develop safe biological agents targeting the PAFAH1B3 protein or PAFAH1B3 upstream and downstream related molecules to improve the treatment effect of cancer and provide patients with new medication options. Summary of the invention

[0005] In order to fill the gaps in the prior art, the first purpose of the present invention is to provide an umbilical cord mesenchymal stem cell and monoclonal antibody composition, which can effectively inhibit the proliferation of pancreatic cancer cells and provide a new idea for the treatment of pancreatic cancer.

[0006] The second objective of the present invention is to provide an application of umbilical cord mesenchymal stem cells and monoclonal antibodies in the combined treatment of cancer.

[0007] One of the purposes of the present invention is achieved by the following technical solution:

[0008] An umbilical cord mesenchymal stem cell and monoclonal antibody composition, wherein the monoclonal antibody is a monoclonal antibody specific to human PAFAH1B3 protein, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO:2.

[0009] Furthermore, the subtype of the monoclonal antibody is IgG1.

[0010] Furthermore, the umbilical cord mesenchymal stem cells are P2-P5 generation umbilical cord mesenchymal stem cells.

[0011] Furthermore, the method for preparing P2-P5 generation umbilical cord mesenchymal stem cells comprises the following steps:

[0012] (1) Wharton's jelly was removed from the umbilical cord, and the Wharton's jelly was cut into pieces, mixed with culture medium, and cultured in a cell culture incubator at 37°C and 5% CO2;

[0013] (2) When umbilical cord mesenchymal stem cells appear around the tissue block, the medium is completely replaced and recorded as P0;

[0014] (3) When the cell confluence reaches 80-85%, the umbilical cord tissue pieces and culture medium are discarded, 0.25% trypsin is added for digestion, the digestion solution is removed by centrifugation, and the culture medium is added for resuspending, and subculture is performed to obtain P2-P5 umbilical cord mesenchymal stem cells.

[0015] Furthermore, the subculture ratio in step (3) is 1:2.

[0016] The second object of the present invention is achieved by adopting the following technical solution:

[0017] Application of the above-mentioned umbilical cord mesenchymal stem cells and monoclonal antibody composition in combined treatment of cancer.

[0018] Furthermore, the cancer is pancreatic cancer.

[0019] Compared with the prior art, the beneficial effects of the present invention are mainly that the PAFAH1B3 protein monoclonal antibody of the present invention can specifically bind to the human PAFAH1B3 protein and can significantly inhibit the proliferation ability of pancreatic cancer cells that highly express the PAFAH1B3 protein. When the PAFAH1B3 protein monoclonal antibody is used in combination with umbilical cord mesenchymal stem cells, the proliferation and invasion ability of pancreatic cancer cells is effectively reduced, and the invention can be used to prepare anticancer drugs, which has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the morphology of P3 generation umbilical cord mesenchymal stem cells;

[0021] Figure 2 This is a diagram showing the specific identification results of the PAFAH1B3 protein monoclonal antibody;

[0022] Figure 3 is the inhibition rate of PAFAH1B3 protein monoclonal antibody on pancreatic cancer cell proliferation;

[0023] Figure 4 The effects of PAFAH1B3 protein monoclonal antibody and umbilical cord mesenchymal stem cells on the proliferation of pancreatic cancer;

[0024] Figure 5 The effects of PAFAH1B3 protein monoclonal antibody and umbilical cord mesenchymal stem cells on pancreatic cancer tumor size;

[0025] Figure 6 The effects of PAFAH1B3 protein monoclonal antibody and umbilical cord mesenchymal stem cells on pancreatic cancer tumor weight. DETAILED DESCRIPTION

[0026] The technical scheme of the present invention is further described below in conjunction with specific embodiments. However, it should be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the present invention. The specific conditions not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, unless otherwise specified, are conventional products obtained through common purchasing channels.

[0027] Example 1

[0028] Preparation of umbilical cord mesenchymal stem cells:

[0029] (1) Take the umbilical cord of a healthy full-term newborn and wash it with PBS in a clean bench to remove residual blood. Remove the Wharton's jelly from the umbilical cord, cut the Wharton's jelly into tissue blocks, wash it three times with normal saline, and culture the tissue blocks in DMEM medium supplemented with 10% FBS, penicillin (100 U / mL) and streptomycin (100 μg / mL) at 37°C and 5% CO2 in a cell culture incubator, and change half of the medium every 2 days;

[0030] (2) When umbilical cord mesenchymal stem cells appear around the tissue block, the medium is completely replaced and recorded as P0;

[0031] (3) When the cell confluence reaches 80%, the umbilical cord tissue pieces and culture medium are discarded, 0.25% trypsin is added for digestion, the digestion solution is removed by centrifugation, and DMEM medium is added for resuspending, and subculture is performed to obtain P3 umbilical cord mesenchymal stem cells (such as Figure 1 shown).

[0032] Example 2

[0033] Preparation of monoclonal antibody against PAFAH1B3 protein:

[0034] S1. Immunization of experimental animals: 6 healthy female mice of about 11 weeks old with good growth status were selected for immunization, and 1 non-immunized healthy female mouse was used as a negative control. Recombinant human PAFAH1B3 protein (immunogen) was injected into the peritoneal cavity of the mice. The dose of intraperitoneal injection was 50 μg / mouse. The injection method was subcutaneous multi-point injection. Immunization was performed once every two weeks, for a total of 3 times. The recombinant human HPAFAH1B3 protein was mixed with Freund's complete adjuvant for the first immunization, and mixed with Freund's incomplete adjuvant for the 2nd-3rd time. The titer of mouse antibodies was determined by indirect ELISA, and the mice with the highest antibody titer were selected for subsequent related experiments.

[0035] S2. Fusion of hybridoma cells: Take myeloma cells in good growth state and logarithmic phase, and prepare them into myeloma cell suspension. Take the female mouse with the highest antibody titer in step S1, dislocate the neck and then remove the spleen to prepare a spleen cell suspension. Myeloma cells and spleen cells are fused at a ratio of 10:1 under the action of 50% polyethylene glycol. Change the HAT selective medium for culture 5 days after cell fusion. Collect the cell culture medium supernatant after 7 days, and screen the positive hybridoma cells by indirect ELISA.

[0036] S3. Subcloning and screening: Subclone the positive hybridoma cells by limiting dilution method, and select the monoclonal stable cell line with the highest positive value for expansion culture and cryopreservation.

[0037] S4. Preparation of mouse ascites: Six healthy female mice of about 11 weeks old with good growth conditions were intraperitoneally injected with 0.5 mL of liquid paraffin. One week later, the hybridoma cells obtained in step S3 were injected into the mice at an inoculum of 5 × 10 5 The state of the mouse abdomen was observed. When the mouse abdomen was obviously bulging, the mouse ascites was collected. The collected ascites was centrifuged at 4000 rpm for 30 minutes, the supernatant was taken and diluted with buffered saline, silica powder was added to the ascites, and incubated at room temperature for half an hour with shaking during the incubation, and centrifuged for 30 minutes to obtain the supernatant containing PAFAH1B3 antibody.

[0038] S5, affinity chromatography of PAFAH1B3 antibody supernatant: balance-loading-washing-elution. Balance: Use buffer to balance the affinity chromatography column. Loading: Load the anti-PAFAH1B3 monoclonal antibody supernatant onto the affinity chromatography column. Washing: After loading, wash twice with buffer B1 and buffer B2 until the UV absorption value is stable, then stop washing. Elution: After washing, use buffer B3 to elute the affinity chromatography fluid. When the UV absorption value reaches 100mAU, collect the protein solution for later use. Determine the protein concentration using the BCA method, adjust the antibody concentration to 5 mg / mL with PBS buffer, aliquot the purified antibody, freeze-dry it, and store it at -20°C.

[0039] S6. Analysis of monoclonal antibody sequence: The PAFAH1B3 protein monoclonal antibody was identified by sequencing. The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO: 2.

[0040] Heavy chain variable region (SEQ ID NO: 1):

[0041] EVQLVESGAELAQPGASVKVSCAATGYIFSAYDIHWVKQAPGHGLELIDINNP DNGDLGWKEIDTGFTILTSDTSKNTAYMQLSRSESEDTAVYYEARTYFRFAYLAWL AYVTVQGTLVT;

[0042] Light chain variable region (SEQ ID NO: 2):

[0043] AYQMTQSPSSVPKPGTVTVTISCRVSQSLYGMSANWQKWYLISPPQRGRTSPK DNGNRFARFSGRTINGDKLTIFFTLMEAEDVRYEDLFFYYCFFGSHVPYTFGLGTKL ELK.

[0044] Test Example 1

[0045] Monoclonal antibody subtype detection:

[0046] According to the instructions of the monoclonal antibody typing multifunctional detection kit, identify the subtype of PAFAH1B3 protein monoclonal antibody. The operation is as follows: take 100ng of PAFAH1B3 antigen and add it to the ELISA plate, and coat it overnight at 4°C. Add PBST buffer, incubate at 37°C for 3h, and then block the ELISA plate. Dilute the PAFAH1B3 protein monoclonal antibody prepared in Example 2 to a concentration of 1μg / mL, add 100μL / well to the ELISA plate, and incubate at 37°C for 1 hour. Add 100μL of 1μg / mL HRP-labeled sheep anti-mouse IgA-HRP, IgM-HRP, IgG1-HRP, IgG2a-HRP, IgG2b-HRP, IgG3-HRP, κ-HRP, λ-HRP secondary antibodies to different wells, mix gently, cover with a sealing film, and incubate at 37°C for 1h. Wash the plate with PBST buffer to remove free substances, develop with TMB colorimetric solution for 10 min in dark conditions, and add 100 μL sulfuric acid to each well to terminate the reaction. Detect the OD value at 450 nm. The well with the darkest color or the highest OD value corresponds to the PAFAH1B3 protein monoclonal antibody subtype.

[0047] Table 1 Subtype detection results

[0048]

[0049] From the results in Table 1, it can be seen that the monoclonal antibody against PAFAH1B3 protein is of IgG1 subtype.

[0050] Test Example 2

[0051] Specificity Characterization of PAFAH1B3 Monoclonal Antibody:

[0052] PAFAH1B3 recombinant protein, BSA protein and loading buffer (2×) containing SDS were mixed at a volume ratio of 1:1, and human pancreatic cancer cell SW1990 was made into human pancreatic cancer cell SW1990 lysate for SDS-PAGE protein electrophoresis. After the electrophoresis, blocking solution was added for 2 hours. PAFAH1B3 monoclonal antibody was used as the primary antibody, incubated overnight at 4°C to react with membrane protein, washed 3 times with PBST buffer, washed once with PBS, and washed away unbound antibodies and other impurities. Alkaline phosphatase-labeled anti-mouse secondary antibody was added and incubated at room temperature for 1 hour, with slight shaking during incubation, washed 3 times with PBST buffer, washed once with PBS washing solution, and scanned with an infrared imaging system to determine whether the monoclonal antibody has the ability to specifically recognize PAFAH1B3 protein.

[0053] The results are as follows Figure 2As shown in the figure, lane 1, lane 2, and lane 3 correspond to human pancreatic cancer cell SW 1 9 9 0 lysate, PAFAH1B3 recombinant protein, and BSA protein samples, respectively. The results show that human pancreatic cancer cell SW 1 9 9 0 lysate and PAFAH1B3 recombinant protein can form specific bands with monoclonal antibody PAFAH1B3, while the BSA protein in lane 3 cannot form specific bands with the antibody. This indicates that the monoclonal antibody PAFAH1B3 can specifically bind to PAFAH1B3 in cancer cells and can also bind to recombinant PAFAH1B3 protein, but has no obvious binding characteristics with BSA protein, indicating that the PAFAH1B3 protein monoclonal antibody has good specificity.

[0054] Test Example 3

[0055] Functional verification of PAFAH1B3 protein monoclonal antibody:

[0056] A negative control group, 100 μg / mL, 200 μg / mL, 300 μg / mL PAFAH1B3 protein monoclonal antibody groups, and 200 μg / mL cetuximab positive control group were set up, with 6 replicates for each concentration. Human pancreatic cancer cells SW 1 9 9 0 in the logarithmic growth phase were obtained, digested with trypsin, and the cell suspension concentration was adjusted to 1×10 6 / mL, 100 μL was inoculated into a 96-well culture plate, and cultured in a DMEM medium containing 10% FBS, 1% penicillin and streptomycin sulfate. After the cells adhered to the wall, the above drugs were added respectively. After 48 hours of culture, the culture medium was discarded, and 100 μL of DMEM medium containing MTT was added to each well for further incubation for 4 hours. The culture medium was discarded and 100 μL of DMSO was added to each well. After shaking, the absorbance value of each well at a wavelength of 490 nm was measured with an ELISA instrument to calculate the cell growth inhibition rate, which was (1-absorbance value of the experimental group / absorbance value of the control group) × 100%. The effect of PAFAH1B3 protein monoclonal antibody on the proliferation of pancreatic cancer cells was analyzed based on absorbance and inhibition rate, and compared with the positive control group cetuximab.

[0057] The results are as follows Figure 3 As shown in the figure, PAFAH1B3 protein monoclonal antibody can significantly inhibit the proliferation of human pancreatic cancer cell SW 1 9 90, and the inhibitory effect is best when the concentration of PAFAH1B3 protein monoclonal antibody is 200μg / mL, so 200μg / mL of PAFAH1B3 protein monoclonal antibody is the optimal concentration. When the concentration of PAFAH1B3 protein monoclonal antibody is 200μg / mL, the cell growth inhibition rate reaches (92.12±1.67)%, which is higher than the positive control group cetuximab (91.26±1.83)%.

[0058] Test Example 4

[0059] Effects of monoclonal antibodies and umbilical cord mesenchymal stem cells on the proliferation ability of pancreatic cancer:

[0060] The frozen human pancreatic cancer cells SW1990 were quickly dissolved in a 37°C water bath and cultured in DMEM medium containing 10% FBS, penicillin (100 U / mL) and streptomycin (100 μg / mL). Human pancreatic cancer cells SW1990 in the logarithmic growth phase were used for subsequent experiments. The cells were digested with trypsin and the cell suspension density was adjusted to 1×10 6 100 μL of cell suspension was inoculated into 96-well plates for culture. Six parallel controls were set up in each group to reduce errors. After 24 h of culture, 0.9% saline (negative control group), 1×10 umbilical cord mesenchymal stem cells, and 1×10 5 / mL (mesenchymal stem cell group), 200μg / mL PAFAH1B3 monoclonal antibody (monoclonal antibody group), umbilical cord mesenchymal stem cells 1×10 5 / mL + 200μg / mL PAFAH1B3 monoclonal antibody (combination group), 200μg / mL cetuximab (positive control group). The cell absorbance values ​​at 24h, 48h, and 72h were detected by an ELISA instrument to verify the effects of umbilical cord mesenchymal stem cells, monoclonal antibodies, and the combination of umbilical cord mesenchymal stem cells and monoclonal antibodies, and cetuximab on the proliferation ability of pancreatic cancer cells SW1990.

[0061] The results are as follows Figure 4 As shown in the figure, at 72h, the OD value of the negative control group was 1.51, the OD value of the positive control group was 0.14, and the OD value of the combined group was 0.06. The results showed that the combination of umbilical cord mesenchymal stem cells and PAFAH1B3 protein monoclonal antibodies can significantly inhibit the proliferation of human pancreatic cancer cells SW1990, and the ability to inhibit the proliferation of pancreatic cancer cells is slightly stronger than that of the positive control group. At 72h, the OD value of the umbilical cord mesenchymal stem cell group was 0.38, and the OD value of the monoclonal antibody group was 0.12. Although the effect of the umbilical cord mesenchymal stem cell group on inhibiting the proliferation of human pancreatic cancer cells SW1990 was not as significant as that of the monoclonal antibody group, it also had a good inhibitory effect compared with the negative control group. Overall, the combined use of umbilical cord mesenchymal stem cells and monoclonal antibodies has a good synergistic effect and can significantly inhibit the proliferation of human pancreatic cancer cells SW1990.

[0062] Test Example 5

[0063] Effects of umbilical cord mesenchymal stem cells and monoclonal antibodies on tumor size and weight:

[0064] Human pancreatic cancer cells in the logarithmic growth phase were prepared into a cell suspension and 1×10 5 / mouse, a subcutaneous pancreatic cancer tumor model was constructed in female nude mice. The mice with successful tumor transplantation were divided into 5 groups, 8 mice in each group. They were injected with 0.9% saline (negative control group), 2mg / kg monoclonal antibody (monoclonal antibody group), 1×10 6 / kg umbilical cord mesenchymal stem cells (mesenchymal stem cell group), 2mg / kg monoclonal antibody and 1×10 6 The mice were given umbilical cord mesenchymal stem cells (combination group) and cetuximab (positive control group) at a dose of 2 mg / kg. The growth status of the tumors in different groups was observed by inoculating different drugs. The nude mice were weighed, and the size of the tumors was measured using a vernier card, the long diameter L and the short diameter W of the tumor, and the formula V = 0.5LW was used. 2 Estimated tumor volume (mm 3 ), data processing was used to draw a tumor growth curve. After 32 days, the mice were killed, the tumors were skinned and separated, and the weight and volume of the tumors were measured to comprehensively investigate and evaluate the therapeutic effect.

[0065] The results are as follows Figure 5 As shown in the figure, at 32 days, compared with the negative control group, the monoclonal antibody group, umbilical cord mesenchymal stem cell group, combined group, and positive control group were able to inhibit the size of the tumor. The combined group was able to significantly inhibit the size of the tumor, and its inhibitory ability was slightly stronger than that of the positive control group. Although the effect of the umbilical cord mesenchymal stem cell group was not as significant as that of the monoclonal antibody group, it also had a good inhibitory effect compared with the negative control group. The experimental results are shown in the figure. Figure 6 As shown in the figure, the tumor weights of the combined group and the negative control group were 0.31±0.23g and 0.82±0.15g, respectively. The combined group could significantly inhibit the tumor weight, and its ability to inhibit tumor weight was the strongest, slightly stronger than the positive control group. In summary, the combination of monoclonal antibodies and umbilical cord mesenchymal stem cells has a good synergistic effect and can inhibit tumor volume and tumor weight.

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them. The basic principles and main features of the present invention have been described above with specific implementation schemes. On the basis of the present invention, some modifications or replacements may be made thereto, but these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of protection claimed by the present invention.

Claims

1. An umbilical cord mesenchymal stem cell and monoclonal antibody composition, characterized in that: The monoclonal antibody is a monoclonal antibody specific to human PAFAH1B3 protein, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO:

2.

2. The umbilical cord mesenchymal stem cell and monoclonal antibody composition according to claim 1, characterized in that: The subtype of the monoclonal antibody is IgG1.

3. The umbilical cord mesenchymal stem cell and monoclonal antibody composition according to claim 1, characterized in that: The umbilical cord mesenchymal stem cells are P2-P5 generation umbilical cord mesenchymal stem cells.

4. The umbilical cord mesenchymal stem cell and monoclonal antibody composition according to claim 3, characterized in that: The method for preparing P2-P5 generation umbilical cord mesenchymal stem cells comprises the following steps: (1) Wharton's jelly was removed from the umbilical cord, and the Wharton's jelly was cut into pieces, mixed with culture medium, and cultured in a cell culture incubator at 37°C and 5% CO2; (2) When umbilical cord mesenchymal stem cells appear around the tissue block, the medium is completely replaced and recorded as P0; (3) When the cell confluence reaches 80-85%, the umbilical cord tissue pieces and culture medium are discarded, 0.25% trypsin is added for digestion, the digestion solution is removed by centrifugation, and the culture medium is added for resuspending, and subculture is performed to obtain P2-P5 umbilical cord mesenchymal stem cells.

5. The umbilical cord mesenchymal stem cell and monoclonal antibody composition according to claim 4, characterized in that: The subculture ratio in step (3) is 1:

2.

6. Use of the umbilical cord mesenchymal stem cell and monoclonal antibody composition according to any one of claims 1 to 5 in the combined treatment of cancer.

7. The use of the umbilical cord mesenchymal stem cells and monoclonal antibody composition in the combined treatment of cancer according to claim 6, characterized in that: The cancer is pancreatic cancer.

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