IL2-TNFα基因诱导大鼠C6胶质瘤细胞凋亡

Induction of Apoptosis by IL2-TNFα in C6 Rat Glioma

目的 为深入进行胶质瘤细胞凋亡的分子生物学研究及为提高胶质瘤辅助免疫治疗打下基础。方法 通过光镜、电镜、荧光显微镜分析 ,DNA断裂分析及流式细胞仪分析 ,进行通过IL2 TNFα诱导C6胶质瘤细胞凋亡研究。结果 本研究用脂质体将pLXSN IL2 TNFα基因导入病毒包装细胞PA31 7,经G41 8抗性筛选 ,用NIH3T3细胞测得病毒滴度为 5×1 0 5CFU ml的细胞克隆 ,利用病毒上清感染C6细胞 ,测得IL2的生物活性为 2 0～ 8 0U 1 0 6 cells 2 4h ,测得TNFα生物活性为 3～ 1 1 0U 1 0 6 cells 2 4h ,实验证实在作用 72h后 ,C6细胞出现细胞凋亡 ;光镜和电镜可见细胞形态学上出现细胞皱缩 ,染色质浓集贴边 ;流式细胞仪结果显示有凋亡峰出现 ,凋亡细胞占细胞总数 1 4 0 %± 1 3% ;荧光显微镜观察出现染色质浓集、断裂 ;DNA电泳表现出DNA呈梯状带断裂。结论 上述结果提示用IL2 TNFα成功诱导大鼠C6胶质瘤细胞发生凋亡。

Objective\ To explore the molecular mechanism of glioma cell apoptosis and improve the effect of chemotherapy for glioma.Methods\ pLXSN-IL2-TNFα was introduced into packaging cell line PA317 by lipofectmine transfection method.Several G418-resistant colonies were obtained and the virus titer in supernatant of the colonies was determined by NIH3T3 cells with the highest one of 5×10\+5CFU/ml.C6 rat glioma cell line was exposed in vitro to pLXSN-IL2-TNFα.The biological activity of IL2 in culture supernatant was monitored in the range of 2.0～8.0U/10\+6 cells/24 h while that of TNFα was in 23～110U/10\+6 cells/24 h.Cell apoptosis was ascertained by (1) cell morphology;(2)DNA fragment analysis;(3)fluoroscopy;(4)flow cytometric study.Results\ When C6 glioma cells were treated with IL2-TNFα for 72 hours,cell apoptosis occurred.Typical apoptotic morphological features of cell shrinkage and condensation and margination of nuclear chromatin were showed by light and electron microscopy.Condensed nuclear chromatin and the fragments of nuclei were demonstrated by fluoroscopy.Apoptotic peak was identified by blow cytometric study.The apoptotic cells accounted for (14.5±1.3)% of the cell population.DNA ladder was not showed by DNA fragment analysis.Conclusion\ The apoptosis of C6 glioma cell was successsfully induced by IL2-TNFα.\;