白藜芦醇增强乳腺癌细胞对γδ T细胞的敏感性及其机制研究

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)%(P0.05)和白藜芦醇+γδT细胞+cFLIP质粒组的(27.9±3.6)%(P0.05)。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。

The effect and mechanism of resveratrol on increasing the sensitivity of breast cancer cells to gd T cells

AIM: To investigate the effect and mechanism of resveratrol on enhancing the cytotoxicity of gd T cells to breast cancer. Methods: Amplifying the gd T cells in vitro. Cell population of gd T was detected by flow cytometry. To perform the following experiments, MDA-MB-231 cells were divided into control group, resveratrol group, gd T cells group, resveratrol + gd T cells group and resveratrol + gd T cells + c-FLIP plasmid group. LDH release assays were performed to evaluate the cytotoxicity of gd T 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 gd T was successfully obtained. Results of LDH release assays showed that the LDH release rate in gd T cells + resveratrol group (68.2±7.1) was significantly higher than the gd T cells group (21.4±3.2, P<0.05) and gd T 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. In addition, results of western blot assays also showed that activation of caspase 8, caspase 9, caspase 3, Bid and release of cytochrome c was significantly higher than that in the gd T cells group and gd T cells + resveratrol + c-FLIP plasmid group. Conclusion: Resveratrol increased the sensitivity of breast cancer cells to gd T cells through inhibition of c-FLIP expression.