The Effect And Mechanism Of Dendritic Cell Tumor Vaccines On The Immune Response Of Esophageal Carcinoma Cells By Inhibiting IL-10 Receptor With Small Interfering RNA

Background and Objective The morbidity of esophageal cancer is seventh in the world for malignant tumors and sixth in mortality.Nearly half of the new cases of esophageal cancer worldwide come from China each year,which is one of the top five major causes of cancer death in China.At present,esophageal cancer is mainly treated by surgery,combined with multi-disciplinary comprehensive treatment of radiotherapy and chemotherapy,but its prognosis is not good,and radiotherapy and chemotherapy may also damage its own immune system,thereby inducing immune tolerance.Therefore,it has profound implication for clinical significance to explore the effective treatment of esophageal cancer with little damage to the body.Current research has found that the occurrence,development and prognosis of esophageal cancer are closely related to dendritic cells.Due to the influence of the tumor microenvironment,the number of dendritic cells in tumor patients are lacking,and most of them are abnormal,thus seriously affecting its own anti-tumor immune function.There are many kinds of immunosuppressive factors in the tumor microenvironment,and IL-10 is the strongest immunosuppressive factor.Therefore,in recent years,research of tumor immunotherapy has focused on finding and optimizing anti-tumor vaccines,as well as reversing the body's immunosuppressive state and overcoming the inhibitory effect of immunosuppressive factors on vaccines.In this study,the dendritic cells were inhibited the expression of IL-10 receptor by small interfering RNA and used in vivo experiments of animal.First,the fluorescent dye CFSE was used to label DCs to observe their migration ability in local lymph nodes;second,TUNEL staining and flow cytometry were used used to detect the in situ apoptosis of tumor cells after injection of different tumor vaccines.Finally,compare the effects of subcutaneous,intralymphatic and intravenous injection of DCs vaccines on the transplanted tumor cell apoptosis,and the difference in Curative effect.The preliminary study of the role and mechanism of IL-10R-si RNA DCs against esophageal cancer immune response provides an experimental and theoretical basis for clinically improving the therapeutic effect of DCs vaccines,and provides new ideas for the clinical returned method of tumor immune vaccines.In short,this study used IL-10 receptors on the surface of dendritic cells as the research target,mainly studying the role and mechanism of IL-10R-si RNA DCs anti-esophageal cancer in immunity response with fluorescent marker tracing,TUNEL staining,flow cytometry and the technology of si RNA gene silencing,experiment were divided into three parts.Part I: Research on the migration ability of IL-10R-si RNA DCs in local lymph nodesMethods 1.constructing a nude transplanted tumor mouse model with the human esophageal cancer cell TE-11 was inoculated subcutaneously on the right armpit of nude mice and randomly divided into 2 groups;2.Two groups of mice were respectively injected with the same amount of CFSE-labeled IL-10R-si RNA DCs and ordinary DCs,and using fluorescent tracing method to observe the ability of DCs to migrate to lymph nodes in animals.Results 1.Inoculate TE-11 cells(human esophageal cancer cells)subcutaneously in nude mice to successfully construct transplanted tumor models;2.The accumulation of IL-10R-si RNA DCs in the lymph nodes of the axillary drainage area was increased,and its migration ability was significantly enhanced.Part 2: The study of the inhibitory effect of IL-10R-si RNA DCs on transplanted tumor in vivo of miceMethods 1.Use TUNEL staining technique to detect in situ apoptosis of tumor cells after injection of different tumor vaccines;2.Using flow cytometry and Expo 32 ADCs software to analyze the apoptosis rate of transplanted tumors;3.Use Multicycle AV software to fit and analyze the DNA cycle changes in the transplanted tumor cell apoptosis process;4.Using flow cytometry to detect Fas and Caspase-3 protein expression in transplanted tumor tissue.Results1.IL-10R-si RNA DCs can significantly reduce the tumor cell proliferation index and increase the tumor cell apoptosis rate;2.IL-10R-si RNA DCs can short the S and M phase of the transplanted tumor cells,inhibit the mitosis of the transplanted tumor cells,and increase the tumor cell apoptosis in situ;3.IL-10R-si RNA DCs can significantly improve the expression of Fas and Caspase-3protein and promote tumor cell apoptosis;4.IL-10R-si RNA DCs have a strong cytotoxic effect on esophageal cancer in vivo.Part III: Research on the optimal reinfusion method of IL-10R-si RNA DCsMethods IL-10R-si RNA DCs were injected by subcutaneous,intralymphatic and intravenous injections to compare the effects of the three reinfusion methods on the apoptosis of transplanted tumor cells and the difference in efficacy.Results 1.Intralymphatic were injected IL-10R-si RNA DCs can significantly reduce the size and weight of the transplanted tumor and increase the tumor suppression rate;2.Transfusion of IL-10R-si RNA DCs into the lymph can obviously reduce the proliferation index of transplanted tumor cells and increase the apoptosis rate;3.Intralymphatic were injected IL-10R-si RNA DCs can significantly increase the expression of tumor apoptosis-related proteins Fas and Caspase-3,and promote apoptosis of cell in situ;4.IL-10R-si RNA DCs injected in Intralymphatic can notablely shorten the S phase and M phase of the DNA cycle in transplanted tumor cell,and the mitosis of the transplanted tumor cell is significantly inhibited;5.Compared with the other two reinfusion routes,the intralymphatic reinfusion vaccine can better exert the vaccine anti-tumor immune response.Conclusion1.IL-10R-si RNA DCs relieve inhibitory effect of IL-10 on DCs,enhance migration ability,and create favorable prerequisites for DCs to effectively exert anti-tumor immune response;2.IL-10R-si RNA DCs enhance the anti-tumor immune response andpromote tumor cell apoptosis of DCs by blocking the IL-10 inhibitory pathway,and provide an experimental basis and theoretical basis for clinically improving the therapeutic effect of DCs vaccines;3.IL-10R-si RNA DCs were reinfused by Intralymphatic route can effectively protect DCs from destruction,and then produce a stronger anti-tumor immune effect,provide new ideas for the clinical reinfusion of tumor immune vaccines.