单纯疱疹病毒胸苷激酶/丙氧鸟苷系统对人骨肉瘤细胞杀伤作用的研究

The killing effect of herpes simplex virus thymidine kinase/ganciclovior system on osteosarcoma cells

目的 :观察单纯疱疹病毒胸苷激酶 /丙氧鸟苷 (HSV -TK/GCV)基因治疗系统对骨肉瘤细胞株OS732的杀伤作用并研究其作用机制。方法 :构建含HyTK基因的逆转录病毒载体DORHyTK ,用DOTAP将其转入骨肉瘤细胞株OS732 ;提取阳性转染细胞 (OS732TK)的DNA和总RNA ,分别用PCR和斑点杂交的方法检测转染的效果 ;用MTT比色法测定OS732TK对GCV的敏感性及旁观者效应 ;Hoeschst332 2 5 8染色 ,电镜和流式细胞仪分析HSV -TK/GCV系统对OS732TK细胞杀伤作用的机制。结果 :成功构建含HyTK基因的重组逆转录病毒载体DORHyTK。OS732TK细胞整合了TK基因并有稳定的转录产物。 5mg/LGCV作用 5d即可杀伤所有OS732TK细胞。旁观者效应在高、低两种细胞密度接种条件下均存在 ,且前者强于后者。Hoeschst 332 2 5 8染色及电镜均显示典型的细胞凋亡特征 ,但也有少量坏死细胞的存在。流式细胞仪分析的结果说明在GCV作用下 ,OS732TK细胞阻滞于细胞周期的G0 -G1期。结论 :HSV -TK/GCV基因治疗系统对骨肉瘤细胞株OS732有很强的杀伤作用 ,且存在明显旁观者效应。此系统导致了OS732细胞周期的紊乱 ,引起细胞的凋亡和坏死。HSV

AIM: To investigate killing effect of herpes simplex virus thymidine kinase/ganciclovior system(HSV-TK/GCV) on osteosarcoma cell and its mechanisms.METHODS: Recombinant retroviral vector (DORHyTK) containing hygromycin phosphotransferase-thymidine kinase fusion gene(HyTK) was constructed and introduced into human osteosarcoma cell line OS732 with DOTAP. DNA and total RNA extracted from HyTK expressing cells (OS732TK) were tested by PCR and RNA dot blot analysis. A chemosensitivity of OS732TK cells to GCV and 'bystander effect' were measured by means of MTT colorimetric assay. Hoeschst 332258 staining and flow cytometric analysis were performed for mechanism of HSV-TK/GCV system gene therapy. RESULTS: DORHyTK was constructed successfully; the HyTK gene existed and expressed in OS732TK cells; All OS732TK cells were killed after 5 days of exposure to 5 mg/L GCV. The 'bystander effect' was observed in both a high population and a low population, but the former was stronger than the latter. Hoeschst 332258 staining revealed the characteristic hallmarks of apoptosis, however necrosis also existed. Flow cytometric analysis of DNA content showed a G 0-G 1 phase blockade. CONCLUSION: HSV-TK/GCV gene therapy system showed its strong killing effects on osteosarcoma cells OS732; The phenomenon of 'bystander effect' was also very apparent. GCV exposure induced both necrotic and apoptotic death in HSV-TK expressing cells, and perturbed the cell cycle. HSV-TK/GCV gene therapy system may provide a new therapeutic approach for treatment of osteosarcoma.