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

AFP gene-modified dendritic cells induced immune response against human hepatocellular carcinoma HepG2 cells

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＋T4-AFP过表达＋JAI（2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（dendriticcells，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-1（人干扰素1）、TNF—d、PFP（穿孔素）、granzymeB（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1L-2、IFN-γ、TNF-α、PFP、granzymeB、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 ceils on I-IepG2 cells, CCK-g assay was used to detect ceU proliferation, and cell cycle and apoptosis were examined by flow cytometry. The expression of 1L-2, IL-6, IL-6, IL-10, IFN-3,, TNF-ct, 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 ceils, up-regulate the expression level of IL-2, IFN-3, TNF-α, PFP, granzyme B and Bax/Bcl-2, but significant｜y inhibit expres- sion 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. Be- sides, JAK2 inhibitors may play a regulatory role in the process.