hTERT核心启动子调控HSV-TK自杀基因系统诱导肿瘤细胞的凋亡

Induction of Tumor Cell Apoptosis by HSV-TK Suicide Gene System under Control of hTERT Core Promoter

目的观察人端粒酶催化亚单位(hTERT)启动子调控逆转录病毒介导的单纯疱疹病毒-胸苷激酶基因/丙氧鸟苷(HSV-TK/GCV)自杀基因系统诱导肿瘤细胞凋亡的效果。方法采用PCR方法扩增胸苷激酶(TK)基因,并构建由hTERT核心启动子调控的胸苷激酶重组逆转录病毒载体pLNC-hTERTp-TK,转染包装细胞PT67,G418筛选阳性细胞克隆,检测病毒滴度,PCR法检测转染细胞中的TK基因。将获得的重组逆转录病毒感染人肺癌细胞株A549、肝癌细胞株SMMC-7721、宫颈癌细胞株HeLa和正常人成纤维细胞系WI-38,G418筛选阳性克隆,MTT法检测重组逆转录病毒对肿瘤细胞的生长抑制作用,流式细胞仪检测肿瘤细胞的凋亡。结果重组逆转录病毒载体pLNC-hTERTp-TK经双酶切,可见1402bp的目的基因片段,表明质粒构建正确。从转染的PT67细胞中扩增出1131bp的基因片段,与TK基因大小一致。在成功转染重组逆转录病毒的PT67细胞中,病毒滴度最高者可达7.8×105CFU/ml。重组逆转录病毒感染的3种肿瘤细胞的增殖均受到抑制,且生长抑制率随着GCV浓度的增加而升高,经较低浓度GCV(10μg/ml)处理的3种肿瘤细胞,凋亡率分别可达37.06%、34.88%和33.59%。结论由hTERT启动子调控的逆转录病毒介导的HSV-TK/GCV自杀基因系统可诱导肿瘤细胞凋亡,且具有较强的特异性和高效性。

Objective To observe the induction of tumor cell apoptosis by retrovirus-mediated herpes simplex virus thymidine kinase/ganciclovir （HSV-TK/GCV） suicide gene system under control of human telomerase reverse transcriptase （hTERT） core promoter. Methods Amplify thymidine kinase （TK） gene by PCR to construct recombinant retrovirus vector pLNC-hTERTp-TK. Packaging cells PT67 were transfected with pLNC-hTERTp-TK, and positive clones were screened with G418 for virus titration and detection of TK gene by PCR. Infect human lung cancer cell strain A549, liver cancer cell strain SMMC-7721, cervical cancer cell strain HeLa and normal fibroblast WI-38 with pLNC-hTERTp-TK and screen positive clones with G418. Determine the inhibitory effect of pLNC-hTERTp-TK on growth of tumor cells by MTT method, and the apoptosis of tumor cells by flow cytometry. Results After pLNC-hTERTp-TK was digested with BamH I and Cla I , the target gene fragment at a length of 1 402 bp was observed on agarose gel electrophoretic profile, which indicated that the recombinant retrovirus vector was constructed correctly. A gene fragment at length of 1 131 bp was amplified from the PT67 cells transfected with pLNC-hTERTp-TK, which was consistent with that of TK gene. The virus titer of transfected PT67 cells reached 7.8 × 10^5 CFU / ml. All the proliferation of 3 kinds of tumor ceils infected with pLNC-hTERTp-TK were inhibited significantly, and the inhibition rate increased with the increasing concentration of GCV. The apoptosis rates of infected HeLa, SMMC-7721 and A549 cells, after treatment with 10 μg/ml of GCV, were 37. 06%, 34. 88% and 33.59% respectively. Conclusion The retrovirus-mediated HSV-TK / GCV suicide gene system under control of hTERT core promoter may induce tumor cell apoptosis effectively and specifically.