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目的:检测胸腺嘧啶激酶基因转染C6胶质瘤细胞后用丙氧鸟苷处理时细胞凋亡的发生。方法:用脂质体介导的基因转染将单纯疱疹病毒胸腺嘧啶激酶基因转入C6胶质瘤细胞中,用G418筛选,建立稳定表达胸腺嘧啶激酶的C6细胞株;加入丙氧鸟苷作用不同时间,用原位末端标记、透射电镜和DNA凝胶电泳法检测。结果:用丙氧鸟苷作用72h发现有大量原位末端标记阳性的细胞,阳性信号呈块状或半月形位于核膜内侧;透射电镜发现肿瘤细胞核内染色质呈块状或帽状位于核膜内侧,非染色质部分呈空泡状,细胞质有的固缩,细胞器基本保持完整;有的则高度肿胀,细胞内亚单位遭到破坏;DNA凝胶电泳发现用丙氧鸟苷作用72h细胞基因组DNA呈梯状带。结论:实验结果提示胸腺嘧啶激酶/丙氧鸟苷系统作用于表达胸腺嘧啶激酶的C6细胞时该细胞主要是以凋亡的方式死亡的。
Objective: To detect the apoptosis of thymidine kinase gene transfected C6 glioma cells with ganosine treatment. Methods: The herpes simplex virus thymidine kinase gene was transfected into C6 glioma cells by liposome-mediated gene transfection, and the C6 cell line stably expressing thymidine kinase was established by G418 screening. Different time, using in situ end labeling, transmission electron microscopy and DNA gel electrophoresis test. RESULTS: A large number of cells with positive end-labeled cells were found after 72 hours of ganoside treatment. The positive signals were massive or half-moon located inside the nuclear membrane. Transmission electron microscopy revealed that the chromatin in the nucleus of tumor cells was massive or cap-like, Medial and achromatin vacuoles, some cytoplasm shrinkage, organelles remained intact; while others were highly swollen, intracellular subunits were destroyed; DNA gel electrophoresis found that ganciclovir 72h cell genome DNA was ladder-like. CONCLUSION: The experimental results suggest that thymidine kinase / gancoside system is mainly apoptotic when it is applied to C6 cells expressing thymidine kinase.