| Malignant tumors have always been the killer of human health.The treatment of tumors still uses surgery and radiotherapy and chemotherapy.The adverse reactions and drug resistance caused by radiotherapy and chemotherapy drugs have seriously reduced the quality of life and survival rate of patients.Targeted drugs for tumors are the current research hotspots.The use of bacterial targeting to treat tumors is a new type of treatment strategy.Attenuated Salmonella is an intracellular parasite with anti-tumor activity and can carry exogenous genes to the tumor site for targeted treatment of tumors.Gastric cancer is a common tumor of the digestive tract and ranks among the top in global malignancies.Because the incidence is relatively hidden,when patients with gastric cancer are diagnosed,they are generally in the advanced stage,missed the best treatment time,and some patients even lost the opportunity to undergo surgical resection.Gastric cell infiltration and metastasis are the main causes of treatment failure and death in patients with gastric cancer.In recent years,a large number of studies have been carried out to improve the prognosis of patients with gastric cancer,and neoadjuvant chemotherapy,radiation therapy and molecular targeted therapy have become effective methods.The pathogenesis and pathological mechanism of gastric cancer are complex,and controlling the recurrence and metastasis of gastric cancer is an important strategy for the treatment of gastric cancer.Melanoma is one of the tumors that endanger human health,and the incidence and mortality are relatively high in the death-related diseases caused by tumors.Although some patients have better drug treatment,more than half of the patients are still not sensitive to drugs.Although the clinical use of immunotherapy drugs can also achieve the effect of treating tumors,but the price is expensive,the drug sensitivity rate is low.The research and development of low-cost,high-specificity,high sensitivity and easy-to-use anti-tumor drugs has become a hot topic in today’s research.Melittin is the main active substance of bee venom and has anti-inflammatory activity.It is mainly used in the treatment of rheumatoid arthritis and analgesic and anti-inflammatory.Melittin also has anti-tumor activity and immunomodulatory activity.Melittin enhances the body’s anti-tumor ability by up-regulating Thl cells and releasing IFN-y,which affects the secretion of CD4+T cells in vivo.The previous experimental study of the research group found that melittin can inhibit the invasion and metastasis of gastric cancer and colon cancer in vitro and in vivo,and can induce apoptosis of tumor cells.This study intends to investigate the mechanism of inhibition of gastric cancer and melanoma by recombinant attenuated Salmonella carrying the melittin gene through in vitro and in vivo experiments,and provide a theoretical basis for the treatment of gastric cancer and melanoma.The research content is divided into two parts.Part I.Mechanism of recombinant attenuated Salmonella inhibiting gastric cancer and melanoma cells in vitroObjective:Recombinant attenuated salmonella carrying melittin gene was injected into gastric cancer SGC7901 and mouse melanoma B16F10 cells to investigate the invasion,migration and wound healing and the effects of molecular mechanisms of apoptosis of gastric cancer SGC7901 and mouse melanoma B16F10 cells by recombinant attenuated Salmonella carrying the melittin gene.Methods:Cell migration and invasion assays were performed to detect the invasion,migration and wound healing of gastric cancer SGC7901 and mouse melanoma B16F10 cells of recombinant attenuated Salmonella infection after 24 hours.Flow cytometry was used to detect the apoptosis of SGC7901 and B16F10 cells infected with recombinant attenuated Salmonella.Western Blot was used to detect the expression levels of PRMT5,P-GSK-3β,Vimentin,Bcl2 and Bax protein in SGC7901 and B16F10 cells infected with recombinant attenuated Salmonella.Results:Cell invasion and migration experiments show that recombinant attenuated Salmonella could significantly inhibit the invasion and migration of gastric cancer and melanoma cells compared with the blank control group,X4550 group,GFP group,KLT and 5-FU group;the results of wound healing experiments showed that recombination attenuated Salmonella group can significantly delay the healing rate of gastric cancer and melanoma cells.The results of apoptosis by flow cytometry showed that the recombinant attenuated Salmonella group could significantly induce apoptosis compared with the GFP control group,and the difference was significant(P<0.05).Western Blot results showed that the protein expression of PRMT5,P-GSK-3β,Vimentin and Bcl2 was decreased and the expression of Bax protein was increased after 24 h of SGC7901 and B16F10 cells were infected by recombinant attenuated Salmonella.Conclusion:Recombinant attenuated Salmonella carrying the melittin gene can inhibit tumor invasion,migration and delay wound healing;it can induce apoptosis,which may be related to apoptosis-related proteins Bcl2 and Bax,and promote apoptosis by down-regulating Bcl2.Up-regulation of Bax inhibits apoptosis and down-regulates PRMT5,P-GSK-3β,and Vimentin protein levels.Part Ⅱ.Experimental study on inhibition of tumor growth by recombinant attenuated SalmonellaObjective:The melanoma B16F10 mouse model was treated with recombinant attenuated Salmonella carrying the melittin gene to investigate the mechanism of recombinant attenuated Salmonella inhibiting tumor growth in vivoMethods:Fifty males C57BL/6 mice were purchased and adapted for one week.44 mice were randomly selected for B16F10 melanoma model:B16F10(1×105 cells/cell)cells were injected subcutaneously into the back of the mice,and the remaining 6 mice were blank control group.The size of the posterior tumor site was examined daily to determine the successful establishment of the model.Thirty-six successful mice were randomly divided into 6 groups(model control group,X4550 group,GFP group,MEL group,KLT Chinese medicine group,5-FU positive drug group).The corresponding bacteria were adjusted to 1 × 1 0 6 CFU/mice,100 μl/mice in the KLT group,and 0.091 mg/g in the 5-FU group.After the tumor formation,the X4550 group,the GFP group,and the MEL group were administered once a week on the first day,and the KLT group and the 5-FU group were administered three times for five days each,and the subcutaneous injection was performed on the back.The cycle is 4 weeks and 28 days.On the first day of administration,changes in body weight and tumor volume of the mice were measured every 5 days and recorded.After the mice were sacrificed on the 21 st day,the tumor tissues were excised,and the tumor mass and the tumor volume was measured.On the 3rd and 21st days,peripheral blood was collected from each supernatant,and the number of T cells,B cells and macrophages in peripheral blood was detected by flow cytometry.The expression level of IL-2,IFN-γ,TNF-α,VEGF in serum were detected by ELISA.The mice were sacrificed on the 21st day,and the number of T cells,B cells,and macrophages in the spleen was detected by flow cytometry.Results:The recombinant attenuated Salmonella carrying the melittin gene can significantly inhibit the growth of tumor and the increase of body weights in mice,and the difference is statistically significant compared with the GFP group without melittin,Chinese medicine and western medicine(P<0.05);The expression levels of IL-2,IFN-y,TNF-a and VEGF in peripheral blood of mice were detected by ELISA.The expression levels of IL-2,IFN-y,TNF-a and VEGF in blood were found to be expressed on the 3rd and 21st day in the MEL group.The amount of VEGF factor was significantly lower in the MEL group than the GFP control group on the 21st day,and the difference between the MEL group and the GFP control group was the difference is statistically significant(P<0.05).Flow cytometry was used to detect the number of T cells,B cells and macrophages in peripheral blood and spleen.The number of CD3+CD4+ T cells and CD3+CD8+T cells in peripheral blood and spleen was significantly increased.The number of B cells in peripheral blood was detected on the 21st day,the number of macrophages increased significantly on the third day,there was no significant difference in the number of macrophages between the peripheral blood and the spleen on the 21st day.Conclusion:Attenuated Salmonella carrying the melittin gene can effectively inhibit the growth of mouse melanoma cells without affecting the body weight of mice,and has good safety.The mechanism may be through up-regulation of IL-2,IFN-y,TNF-a related factor expression and down-regulation of VEGF factor levels;tumor-specific CD4+T cells and CD8+T cell immune responses be promoted and together with B cells and macrophages to regulate the body’s immune response,play a role in cellular immunity and Humoral immmunity,the immune response biased to Thl type. |
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