槲皮苷对人γδT 细胞增殖及杀伤功能的影响

摘要

目的：探讨槲皮苷（ quercitrin ）对人外周血γδT细胞体外增殖和功能影响及相关机制。方法从健康人外周血中分离单个核细胞（PBMC），加入到含有异戊烯焦磷酸和IL-2的RPMI 1640完全培养基中诱导培养获得γδT细胞。用不同浓度的槲皮苷作用γδT细胞48 h后，用CCK-8法测定不同浓度槲皮苷组γδT细胞增殖和杀伤能力；采用流式细胞术检测各组γδT细胞颗粒酶B和穿孔素的表达；用Western blot法检测p-ERK、p-Akt和Bcl-2蛋白的表达。结果培养10 d后的γδT细胞纯度达到（88．94±2．36）％。槲皮苷浓度在10～80μg/ml时能显著促进γδT细胞的增殖及颗粒酶B、穿孔素的表达和对结肠癌HCT116细胞的杀伤能力。在蛋白水平p-ERK、p-Akt和Bcl-2的表达量均显著高于对照组，而内参GAPDH表达量无变化。结论槲皮苷可体外增强γδT细胞的增殖及杀伤功能，其机制可能通过p-ERK和p-Akt信号通路。%Objective To investigate the in vitro effects of quercitrin on the proliferation and the cytotoxicity of human γδT cells.Methods Peripheral blood mononuclear cells (PBMCs) were isolated from healthy subjects and cultured with isopentenyl pyrophosphate and IL -2 to induce human γδT cells.The hu-manγδT cells were cultured with quercitrin at various concentrations for 48 hours.CCK-8 kits were used to analyze the in vitro proliferation and cytotoxic activities of γδT cells.Flow cytometry was performed to meas-ure the expression of granzyme B and perforin in γδT cells.The expression of p-ERK, p-Akt and Bcl-2 at protein level were detected by Western blot .Results The percentage of human γδT cells in PBMCs was in-creased from (2.96±1.83)%to (88.94±2.36)%after 10 days of culture.The quercitrin at concentrations of 10 to 80 μg/ml could promote the growth of γδT cells and up-regulate the expression of granzyme B , per-forin, p-ERK, p-Akt and Bcl-2 in a dose dependent manner .The cytolytic activities of γδT cells against co-lonic carcinoma cells ( HCT116 ) were enhanced by quercitrin .Conclusion Quercitrin could promote the proliferation of γδT cells and enhance the expression of granzyme B and perforin at certain concentrations in vitro.ERK1/2 and Akt signal transduction systems might be involved in the process .