外源性TRAIL基因转染联合氯喹诱导胶质瘤U251细胞凋亡  

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

作　　者：冯驰[1] 郭双毅 程龙海[1] 罗杰[1] FENG Chi;GUO Shuang-yi;CHENG Long-hai;LUO Jie(Department of Neurosurgery, Taihe Hospital, Shiyan 44200, China)

机构地区：[1]十堰市太和医院神经外科,湖北442000

出　　处：《中国临床神经外科杂志》2017年第8期563-565,共3页Chinese Journal of Clinical Neurosurgery

摘　　要：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(P<0.05)。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)%,P<0.05]和TRAIL组[(23.88±0.22)%,P<0.05]。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)%,P<0.05]、阴性对照组[(3.46±0.17)%,P<0.05]和TRAIL组[(22.48±0.43)%,P<0.05]。联合组Cleaved caspases-8蛋白表达水平明显高于阴性对照组、TRAIL组(P<0.05)。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。Objective To investigate the effect of the exogenous tumor necrosis factor-related apoptosis-inducing ligand(TRAIL)and chloroquine(CQ)on the apoptosis of glioma U251cells.Methods Human glioma U251cells were treated with exogenous green fluorescent protein(GFP)-TRAIL and/or CQ.The expression level of GFP-TRAIL in U251cells was detected by confocal microscope.The cells vitality was detected by MTT assay and U251cells 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 U251cells and induced the U251cells apoptosis after the transfection of exgenous TRAIL into U251cells.The rate of inhibition of U251cells proliferation(47.22±0.15)%and the U251cell apoptosis rate(41.62±0.44)%were significantly higher in the glioma U251cells treated with GFP-TRAIL combined with CQ respectively than those[(23.88±0.22)%and(22.48±0.43)%respectively]in the glioma U251cells only treated with GFP-TRAIL or CQ and control group(P<0.05).Conclusion The GFP-TRAIL protein can be stably expressed in U251cells transfected with TRAIL gene and induce the apoptosis of U251cells.GFP-TRAIL combined with CQ can enhance the apoptosis of U251cells.

关 键 词：胶质瘤 U251细胞 肿瘤坏死因子凋亡相关诱导配体 氯喹 细胞凋亡 细胞自噬 

分 类 号：R739.41[医药卫生—肿瘤]