水飞蓟宾对γδT细胞杀伤胃癌细胞SGC-7901的影响及其机制初探

The effect of silibinin on γδT cell cytotoxicity against gastric cancer SGC-7901 cells and the mechanism

探讨水飞蓟宾对人γδT细胞杀伤胃癌细胞SGC-7901的影响及其作用机制。分离健康志愿者外周血单个核细胞，体外经多种细胞因子诱导培养为76T细胞；收集培养、扩增7d后的怕T细胞，将其用不同浓度的水飞蓟宾诱导24h、48h及72h，CCK-8法检测76T细胞增殖情况；流式细胞术（FCM）检测78T穿孔素（perforin，PFP）、细胞颗粒酶B（GranzymeB，GranB）及CD107a的表达；western-blot法检测78T细胞P—ERK1／2、Bcl-2、P—AKT、β-catenin的表达；乳酸脱氢酶（LDH）法检测γδT细胞对胃癌细胞SGC-7901的杀伤活性。结果发现浓度在1．6～100μg／ml的水飞蓟宾作用78T细胞72h后，76T细胞增殖率较对照组显著增加（P〈0．05），且在25μg／ml时达到最高峰；将6．25～100μg／ml水飞蓟宾诱导γδT细胞72h后，78T细胞的PFP、GranB及CD107a的表达以及P—ERK1／2、Bcl-2、P—AKT、β-catenin的表达与对照组比较均不同程度增加（P〈0．05），且对胃癌细胞SGC-7901的杀伤活性显著增强（P〈0．05）。以上实验结果提示一定浓度的水飞蓟宾对γδT细胞具有促进增殖作用，其机制可能与激活P—ERK1／2、Bcl-2、P—AKT及G—catenin信号通路有关。一定浓度的水飞蓟宾能够增加78T细胞对胃癌细胞的杀伤活性，其作用机制可能与水飞蓟宾能够增加78T细胞的穿孔素、GranB及CD107a的表达有关。

This study designed to investigate the effect of silihinin on γδT cell cytotoxicity against gastric cancer SGC-7901 ceils and explore the mechanism. γδT cells were generated in vitro by stimulating peripheral blood mononuclear cells of healthy vol unteers with γδT culture medium for 7 days. The cells were then cultured with different concentrations of silibini for 24, 48 and 72 h. The proliferation of the γδT cell was examined by CCK 8 assay. Production of Perforin, granenzyme B and CD107a were determined by flow cytometry. P ERK1/2,Bcl 2,P-AKT,β-catenin were detected by Western blot. Cytotoxic activity against SGC 7901 cell was examined by release of LDH. The results showed that , compared with the control group, γδT cells which have been treated with silibin in the range of concentration from 1.6μg/ml to 100μg/ml for 72 hours, the proliferation rate was increased（P〈0.05） , and the peak value was attained at the concentration of 25μg/ml . When γδT cells were treated with silibin for 72 h at the concentration range of 6.25gg/ml to 100/,g/ml, the expression of PFP,GraB, and CD107a as well as P- ERK1/2,Bcl-2,P-AKT,13-catenin were all increased, compared with those of the control group（P〈0.05 ）. And the cytotoxic activity to SGC cells was significantly elevated. The above evidence suggests that silibin may promote proliferation of γδT cells, the underline mechanism might be related to activation of multiple signaling paths. These findings indicate Silibin at effective concentrations might promote proliferation of γδT cells by enhancing the production of perforin, granzyme B and CD107a.