甲胎蛋白腺病毒载体诱导的树突状细胞对人肝癌细胞HepG2的免疫治疗作用

摘要

目的 研究重组甲胎蛋白(AFP)特异性T细胞的体外抗肝细胞癌免疫活性及其机制.方法 实验分为:空白对照HepG2组;CD8+T+AFP空载组;CD8+T+AFP过表达组;CD8+T+AFP空载+ JAK2抑制剂组;CD8+T+AFP过表达+JAK2抑制剂组.分离、鉴定DC细胞,构建AFP腺病毒并转染树突状细胞(dendritic cells,DC),活化AFP特异性的CD8+T细胞,检测CD8+T细胞对人肝癌细胞HepG2的影响.采用CCK8法检测细胞增殖情况;流式细胞术检测细胞周期和凋亡情况;ELISA法检测IL-2、IL-6、IL-6、IL-10、IFN-γ(人干扰素γ)、TNF-α、PFP(穿孔素)、granzyme B(颗粒酶)表达情况;蛋白印迹检测p-STAT3/STAT3、p-JAK2/JAK2、Bax/Bcl-2、Fas/Fasl表达情况.结果 实验显示,AFP特异性CD8+T细胞体外能有效抑制HepG2细胞增殖,上调IL-2、IFN-γ、TNF-α、PFP、granzyme B、Bax/Bcl-2的表达水平,显著抑制T细胞活化的负调控因子IL-10及p-STAT3/STAT3、p-JAK2/JAK2表达.结论 活化的AFP特异性T细胞能显著抑制肝细胞癌增殖.其抗肿瘤机制可能是重组AFP腺病毒活化、促进T细胞增殖,通过升高Bax/bcl-2和促进穿孔素/颗粒酶通路并特异性杀伤HepG2.JAK2抑制剂在该机制中起到一定调控作用.%Objective To investigate the immunoreactivity of the recombinant AFP-specific T cells in hepatocellular carcinoma in vitro and explore its potential mechanism.Methods To isolate and identify dendritic cells (DC),AFP adenovirus were constructed and transfected into DC to activate CD8 + T cells.There were 5 groups based on different treatments:HepG2 control Group,CD8 + T mock Group,CD8 + T Ad-AFP/DCs Group,CD8+ T mock + JAK2 inhibitor Group,and CD8+ T Ad-AFP/DCs + JAK2 inhibitor Group.To detect the cytotoxic effect of CD8 + T cells on HepG2 cells,CCK-8 assay was used to detect cell proliferation,and cell cycle and apoptosis were examined by flow cytometry.The expression of IL-2,IL-6,IL-6,IL-10,IFN-γ,TNF-α,perforin (PFP) and granzyme B were tested by ELISA assay,and western blot was used to detect the protein expression of p-STAT3/STAT3,p-JAK2/JAK2,Bax/Bcl-2 and Fas/Fasl.Results The results showed that CD8 + T cells can inhibit the proliferation of HepG2 cells,up-regulate the expression level of IL-2,IFN-γ,TNF-α,PFP,granzyme B and Bax/Bcl-2,but significantly inhibit expression of T cell activation negative regulator as IL-10,p-STAT3/STAT3 and p-JAK2/JAK2.Conclusions The activated AFP-specific T cells can dramatically suppress the proliferation of hepatocellular carcinoma,possibly by increasing Bax/bcl-2,promoting perforin/granzyme pathway and specifically killing HepG2.Besides,JAK2 inhibitors may play a regulatory role in the process.