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目的 :观察单纯疱疹病毒胸苷激酶基因 (HSV - tk基因 ,自杀基因 )与阿昔洛韦 (ACV)系统对人肝癌细胞的体外杀伤效果。方法 :体外培养已感染含 HSV- tk基因的重组逆转录病毒的 PA317- tk细胞 ,获取含 HSV- tk基因的重组逆转录病毒颗粒。用后者感染人肝癌细胞株 SMMC- 772 1,G418培养基筛选抗性克降 SMMC- 772 1- tk,并以 PCR技术检测 HSV- tk基因判断转染成功与否。用形态学方法和 MTT法检测 ACV对 SMMC- 772 1- tk细胞的杀伤作用。结果 :SMMC- 772 1- tk细胞经 PCR证实含 PCR- tk基因。形态学观察显示 ,ACV浓度在 0 .0 1～ lμg/ ml时 ,SMMC- 772 1- tk细胞小部分死亡 ,在浓度自 1μg～ 10 0 0μg/ m l时 ,细胞大部分死亡 ;HE染色可见细胞凋亡。MTT法测定显示 ,ACV浓度自 1μg/ ml开始即对 SMMC- 772 1- tk细胞产生明显抑制作用 ,抑制率自 49%到 78% ,作用强度呈 ACV浓度依赖性。各种浓度的 ACV对 SMMC- 772 1细胞没有或只有轻度抑制作用。结论 :HSV- tk/ACV系统体外对肝癌细胞有明显的杀伤作用 ,有可能成为临床基因治疗方案。
Objective: To observe the in vitro killing effect of HSV - tk gene, suicide gene and aciclovir (ACV) on human hepatocellular carcinoma cells in vitro. Methods: PA317-tk cells infected with recombinant retrovirus containing HSV-tk gene were cultured in vitro to obtain recombinant retroviral particles containing HSV-tk gene. The latter was infected with human hepatocellular carcinoma cell line SMMC-772 1 and G418 medium to screen for resistance to SMMC-772 1- tk. HSV-tk gene was detected by PCR to determine whether the transfection was successful or not. The cytotoxicity of ACV on SMMC-772 1- tk cells was detected by morphological methods and MTT assay. Results: SMMC-772 1-tk cells were confirmed by PCR to contain the PCR-tk gene. Morphological observation showed that SMMC-772 1-tk cells died at a small fraction of ACV from 0 to 1μg / ml. Most of the cells died when the concentration was from 1μg to 100μg / ml. HE staining showed that cells Apoptosis. MTT assay showed that ACV concentration from 1μg / ml on SMMC-772 1- tk cells had a significant inhibitory effect, inhibition rate from 49% to 78%, the intensity of action was ACV concentration-dependent. ACV at various concentrations showed no or only mild inhibition of SMMC-772 1 cells. Conclusion: The HSV-tk / ACV system has a significant cytotoxic effect on hepatocellular carcinoma cells in vitro and may become a clinical gene therapy regimen.