表达VASH1基因的人脑胶质瘤U-87MG细胞对化疗药物敏感性的变化  

Effect of lentiviral vector-mediated VASH1 gene on chemosensitivity of human glioma U-87MG cells

作　　者：付锴[1] 江普查[1] 宫睿[1] 王伟[1] 

机构地区：[1]武汉大学中南医院神经外科,武汉430071

出　　处：《中国临床神经外科杂志》2016年第1期34-37,共4页Chinese Journal of Clinical Neurosurgery

摘　　要：目的探讨表达人血管生成抑制因子1(VASH1)的人脑胶质瘤U-87MG细胞对化疗药物的敏感性变化。方法构建针对VASH1的慢病毒载体p GCL-GFP-VASH1,经测序鉴定后转染293T细胞,筛选出适合浓度的慢病毒转染人脑胶质瘤U-87MG细胞,荧光显微镜下检测转染效率;通过RT-PCR和Western blot分析U-87MG细胞VASH1 m RNA和蛋白表达水平;用CCK-8法检测U-87MG细胞在化疗药物顺铂和替莫唑胺作用下的存活率。流式细胞仪检测U-87MG细胞凋亡。结果成功构建p GCL-GFP-VASH1慢病毒载体,并成功转染U-87MG细胞,转染率达70%以上;RT-PCR和Western blot结果证实转染VASH1慢病毒载体的U-87MG细胞表达VASH1 m RNA和蛋白。在顺铂或替莫唑胺作用下,表达VASH1的U-87MG细胞存活率均较未表达VASH1的U-87MG细胞明显降低(P<0.01),而且U-87MG细胞凋亡率明显增加(P<0.01)。结论 VASH1慢病毒载体转染U-87MG细胞可使其稳定表达VASH1,并提高人脑胶质瘤U-87MG细胞对化疗药物敏感性、增加细胞凋亡率。Objective To construct the lentiviral vector over-expressing VASH1 and study the relationship between the expression of VASH1 and the ehemosensitivity of human glioma U-87MG cells. Methods The lentiviral vector carrying human VASH1 gene （pGCL-GFP-VASH1）, which was constructed and sequenced, was transinfected into 293 T cells. The transinfeetion efficiency was evaluated and then the lentiviral vector was transfeeted into the human glioma U-87MG cells. Transfeetion efficiency was determined by Fluorescence microscopy. The expression of VASH1 mRNA and protein in U87MG cells were detected respectively by RT-PCR and Western blot. The changes in the sensitivity of transfeeted U-87MG cells to the chemotherapeutic drugs including cispatin and temozolomide were determined by cell counting kit-8 （CCK-8）. The apoptosis of U-87MG cells transfeeted with pGCL-GFP-VASH1 was determined by flow eytometry （FCM）. Results The lentiviral vector-mediated VASH1 gene was successfully constructed and transfeq^ted into the U-87 cells. Immunofluorescence assay demonstrated that the transfection efficiency was above 70%. RT-PCR and Western blot analyses demonstrated that pGCL-GFP-VASH1 significantly increased the expressions of VASH1 mRNA and protein by 21.57% and 27.29% respectively in transfected U-87MG cells 96 hours after the transfection compared to the control group（P〈O.O1）; CCK-8 results showed that when exposed to cisplatin or temozolomide, the survival rate of pGCL-GFP-VASHl-transfected U-87 cells were significantly decreased to 23.96 and 17.24% respectively, which were significantly lower than those in the control groups. FCM results showed that the apoptosis rate of U-87MG cells transfected with pGCL-GFP-VASH1 was significantly higher than that in the control groups （P〈0.01）. Conclusion The lentiviral vector-mediated VASH1, which can significantly increase the expression of VASH1 in U-87 MG cells, may enhance chemosensitivity and apoptosis of human glioma U-87MG ceils.

关 键 词：脑胶质瘤 U-87MG细胞 血管生成抑制因子1 慢病毒载体 化疗 药物敏感性 

分 类 号：R739.41[医药卫生—肿瘤] R730.53[医药卫生—临床医学]