外源性TRAIL基因转染联合氯喹诱导胶质瘤U251细胞凋亡

目的探讨外源性肿瘤坏死因子凋亡相关诱导配体(TRAIL)联合氯喹对U251细胞凋亡的作用。方法构建稳定表达TRAIL的质粒p EGFP-TRAIL,然后转染到胶质瘤细胞系U251细胞中(TRAIL组),以p EGFP-C1质粒为阴性对照,以不转染质粒为空白对照;将氯喹(50μmol/L)加入到转染p EGFP-TRAIL质粒U251细胞培养基中,作为联合组。共聚焦显微镜检测GFP-TRAIL蛋白表达,MTT法检测细胞抑制率,Annexin V-FITC/PI双染法检测细胞凋亡,免疫印迹法检测GFP-TRAIL和Cleaved caspases-8蛋白表达。结果质粒转染后,共聚焦荧光显微镜检测结果显示,p EGFP-C1在细胞质中成弥散分布;而GFP-TRAIL在细胞质中成聚点分布,且荧光表达可以持续48 h以上;免疫印迹法分析结果显示TRAIL组TRAIL蛋白表达水平明显高于空白对照组和阴性对照组(P0.05)。联合组细胞增殖抑制率(47.22±0.15)%]明显高于阴性对照组(3.21±0.04)%,P0.05]和TRAIL组(23.88±0.22)%,P0.05]。联合组细胞凋亡率(41.62±0.44)%]明显高于空白对照组(2.14±0.09)%,P0.05]、阴性对照组(3.46±0.17)%,P0.05]和TRAIL组(22.48±0.43)%,P0.05]。联合组Cleaved caspases-8蛋白表达水平明显高于阴性对照组、TRAIL组(P0.05)。结论外源性TRAIL基因转染后可以在细胞中稳定表达并诱导细胞凋亡,与氯喹联合应用可以增强TRAIL诱导的细胞凋亡,其机制可能是氯喹抑制细胞自噬。

Effect of exogenous TRAIL gene transfection combined with chloroquine on glioma U251 cells apoptosis

Objective To investigate the effect of the exogenous tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and chloroquine (CQ) on the apoptosis of glioma U251 cells. Methods Human glioma U251 cells were treated with exogenous green fluorescent protein (GFP)-TRAIL and/or CQ. The expression level of GFP-TRAIL in U251 cells was detected by confocal microscope. The cells vitality was detected by MTT assay and U251 cells apoptosis was detected by Annexin V-FITC/PI Apoptosis Detection Kit. The expression levels of proteins related to apoptosis were detected by Western blot. Results GFP-TRAIL was stably expressed in U251 cells and induced the U251 cells apoptosis after the transfection of exgenous TRAIL into U251 cells. The rate of inhibition of U251 cells proliferation (47.22 ± 0.15)%and the U251 cell apoptosis rate (41.62 ± 0.44)%were significantly higher in the glioma U251 cells treated with GFP-TRAIL combined with CQ respectively than those (23.88±0.22)%and (22.48±0.43)%respectively] in the glioma U251 cells only treated with GFP-TRAIL or CQ and control group (P<0.05). Conclusion The GFP-TRAIL protein can be stably expressed in U251 cells transfected with TRAIL gene and induce the apoptosis of U251 cells. GFP-TRAIL combined with CQ can enhance the apoptosis of U251 cells.