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作 者:刘扬[1] 王跃如[2] 丁光辉[1] 杨庭松[1] 姚乐[1] 钱明平[1] 张柏和[2] 吴孟超[3] LisaH.Butterfield
机构地区:[1]同济大学附属第十人民医院肝胆外科,上海200072 [2]上海交通大学附属上海市第一人民医院内科 [3]上海东方肝胆外科医院肿瘤综合治疗科 [4]美国匹兹堡大学肿瘤研究所外科及免疫学组
出 处:《中华肝胆外科杂志》2015年第7期486-489,共4页Chinese Journal of Hepatobiliary Surgery
基 金:国家自然科学基金(81372461)
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+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.
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