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目的 观察单纯疱疹病毒胸苷激酶基因 (HSV - tk) -环丙氧苷 (GCV )系统在胶质瘤基因治疗过程中 ,GCV能否诱导转导有 HSV- tk基因的胶质瘤细胞凋亡。方法 采用光镜、电镜和荧光显微镜观察转导有 HSV- tk基因的大鼠 C6 /tk胶质瘤细胞形态学的改变 ,以未转导 HSV- tk基因的 C6细胞作为对照 ,并以流式细胞术、DNA凝胶电泳分析进一步验证细胞凋亡的发生。结果 在 5μg/ml GCV作用 72 h后 ,C6 /tk细胞呈现凋亡状态。光镜和电镜下可见细胞皱缩 ,染色质浓集贴边 ;荧光显微镜下可见染色质浓集、断裂 ;DNA凝胶电泳可见 DNA呈梯状带型 ;流式细胞术分析显示有凋亡峰出现 ,凋亡细胞占 2 3%。对照组未见有细胞凋亡现象。结论 GCV能诱导转导有 HSV- tk基因的 C6 /tk细胞凋亡 ,以上结果为胶质瘤基因治疗的研究提供依据
Objective To observe whether GCV can induce apoptosis of glioma cells transduced with HSV-tk gene during the gene therapy of HSV-tk-cyclosporin (GCV) . Methods The morphological changes of rat C6 / tk glioma cells transduced with HSV-tk gene were observed by light microscope, electron microscope and fluorescence microscope. The C6 cells without HSV-tk gene were used as controls, Cytometry, DNA gel electrophoresis analysis to further verify the occurrence of apoptosis. Results C6 / tk cells showed apoptotic state after 72 h treatment with 5μg / ml GCV. Under the light microscope and electron microscope, cell shrinkage was observed and the chromatin was concentrated. The chromatin was observed under the fluorescence microscope and the DNA was broken. The DNA was observed by DNA gel electrophoresis. The DNA was analyzed by flow cytometry Appeared, apoptotic cells accounted for 23%. No apoptosis in the control group. Conclusion GCV can induce the apoptosis of C6 / tk cells transduced with HSV-tk gene. The above results provide the basis for the study of gene therapy of glioma