白藜芦醇增强乳腺癌细胞对γδT细胞的敏感性及其机制研究

Effect and Mechanism of Resveratrol on Increasing the Sensitivity of Breast Cancer Cells to Cells

目的探讨γδT细胞对乳腺癌细胞的杀伤活性,观察白藜芦醇增强γδT细胞的抗肿瘤活性。方法体外扩增培养人γδT细胞并用流式细胞术进行鉴定。实验分为对照组、白藜芦醇组、γδT细胞组、白藜芦醇+γδT细胞组及白藜芦醇+γδT细胞+c-FLIP质粒组。LDH释放实验检测γδT细胞对MDA-MB-231乳腺癌细胞的杀伤活性;western blot实验检测白藜芦醇及γδT细胞处理后MDA-MB-231细胞的细胞Fas-相关性死亡结构域样白介素-1β转换酶抑制蛋白(c-FLIP)的表达水平、活化半胱天冬酶(caspase)8、caspase 9、caspase 3的活化和细胞色素c的释放。结果流式细胞实验结果显示在体外用Br HPP和IL-2培养14天的人单个核细胞、CD3和γδTCR,确定这些定向培养的效应细胞为γδT细胞。LDH释放实验显示γδT细胞+白藜芦醇组对MDA-MB-231的杀伤活性为(68.2±7.1)%,显著高于γδT细胞组的(21.4±3.2)%(P<0.05)和白藜芦醇+γδT细胞+cFLIP质粒组的(27.9±3.6)%(P<0.05)。Western blot实验结果显示白藜芦醇处理能显著降低MDAMB-231细胞中c-FLIP蛋白的表达,γδT细胞+白藜芦醇组MDA-MB-231的活化caspase 8及细胞色素c的释放均显著高于γδT细胞组和白藜芦醇+γδT细胞+c-FLIP质粒组。结论白藜芦醇下调乳腺癌细胞c-FLIP的表达,提高乳腺癌细胞对γδT细胞的敏感性。

Objective To investigate the effect of resveratrol on enhancing the cytotoxicity of cells to breast cancer and the underlying mechanism. Methods The cellswere amplified in vitroand cell population of was detected by flow cytometry. MDA-MB-231 cells were divided into control group, resveratrol group, cell group, resveratrol＋cell group and resveratrol＋cell＋c-FLIP plasmid group. LDH release assays were performed to evaluate the cytotoxicity of cells to MDA-MB-231. Western blot analysis was performed to evaluate the expression of c-FLIP, activation of caspase 8, caspase 9, caspase 3, Bid and release of cytochrome c in MDA-MB-231 cells. Results Flow cytometry analysis showed that Br HPP and IL-2-cultrued peripheral blood mononuclear cells highly expressed CD3 and CR, thereby these cells were cells. Results of LDH release assays showed that the LDH release rate in cells＋resveratrol group（68.2±7.1）% was significantly higher than that in the cells group[（21.4±3.2）%,P〈0.05] and cells＋resveratrol ＋c-FLIP plasmid group [（27.9 ±3.6）%, P〈0.05]. Results of western blot assays showed that resveratrol downregulated the expression of c-FLIP and that activation of caspase 8, caspase 9, caspase 3, Bid and release of cytochrome c was significantly higher than that in the cells group and cells ＋resveratrol ＋c-FLIP plasmid group.Conclusion Resveratrolincreased the sensitivity of breast cancer cells to cells through inhibition of c-FLIP expression.