人端粒酶催化亚单位启动子调控自杀基因HSV-TK靶向性表达治疗肺癌的体内外实验研究

An Experimental Study on Targeting Suicide Gene Therapy for Lung Cancer with HSV-TK Driven by hTERT Promoter

目的研究人类单纯疱疹病毒的胸苷激酶(HSV-TK)/更昔洛韦(GCV)治疗系统在人端粒酶催化亚单位(hTERT)启动子的调控下对肺癌细胞A549的体外靶向性增殖抑制作用和对裸鼠体内A549移植瘤的治疗效果。方法1用已构建的hTERT启动子和SV40启动子调控的TK基因表达质粒pGL3-hTp-TK和pGL3-SV40-TK转染端粒酶阳性的人肺腺癌细胞A549及端粒酶阴性的人胚肺细胞MRC-5,RT-PCR方法检测转染细胞中TK基因的表达情况;2MTT法检测GCV对上述转染细胞体外增殖的抑制作用;并应用流式细胞仪检测GCV对上述转染细胞凋亡的影响;3将A549细胞接种于裸鼠皮下,研究pGL3-hTp-TK/GCV和pGL3-SV40-TK/GCV对移植瘤的体内生长抑制作用。结果1转染pGL3-SV40-TK的A549和MRC-5细胞均有TK mRNA表达,转染pGL3-hTp-TK的A549细胞也有TK mRNA表达,但MRC-5细胞无TK mRNA表达;2GCV对转染pGL3-SV40-TK的A549、MRC-5细胞和转染pGL3-hTp-TK的A549细胞的体外增殖均有明显抑制作用,对转染pGL3-hTp-TK的MRC-5细胞无明显抑制作用;转染pGL3-SV40-TK和pGL3-hTp-TK的A549细胞经GCV处理后细胞凋亡指数(分别为21.58%和23.19%)均显著高于转染pGL3-hTp的A549细胞及空白对照,转染pGL3-SV40-TK的MRC-5细胞凋亡指数(9.35%)显著高于对照组,而转染pGL3-hTp-TK的MRC-5细胞凋亡指数(0.88%)无明显升高;3pGL3-SV40-TK/GCV和pGL3-hTp-TK/GCV治疗系统对裸鼠A549移植瘤的生长具有明显抑制作用,抑瘤率分别为46.7%和40.5%,均显著高于各阴性对照组(分别为9.7%,14.3%,7.0%和8.0%)。结论hTERT启动子可以调控HSV-TK基因在肺癌细胞中靶向性表达,HSV-TK/GCV治疗系统在hTERT启动子调控下对肺癌细胞A549的体外增殖和裸鼠体内的移植瘤生长均有明显的抑制作用。

Objective To study the approach of targeting expression of suicide gene HSV-TK driven by human telomerase catalytic subunit （hTERT） promoter in lung cancer cells, and to investigate inhibitory effect of HSV-TK/GCV driven by hTERT promoter on proliferation of lung cancer cell line A549 in vitro and in vivo. Methods ①Reeombinant expression vectors of HSV-TK driven by hTERT promoter and SV40 promoter （pGL3- hTp-TK and pGL3-SV40-TK） were transfeeted into telomerase-positive human lung adenoeareinoma cell A549 and telomerase-negative human embryonic lung fibroblast cell MRC-5. The mRNA expression of TK gene was detected with RT-PCR method; ② With the treatment of GCV, the proliferation of above transfected cells was investigated by MTT assay; Influence of GCV on apoptosis and cell cycle of these ceils was evaluated with flow eytometry; ③ After the subcutaneously transplantation of A549 cells into nude mice, intra-tumor injection of plasmid-liposome as well as intra-peritoneal injection of GCV were performed to study antl-tumor effects of pGL3- hTp-TK/GCV and pGL3-SV40-TK/GCV in vivo. Results ① Enzyme digestion and PCR suggested that recombinant plasmids of pGL3-hTp-TK and pGL3-SV40-TK were sueeessfully constructed; TK mRNA expression was detected in both A549 and MRC-5 cells transfected with pGL3-SV40-TK, also in A549 transfeeted with pGL3-hTp-TK, but not in MRC-5 transfected with pGL3-hTp-TK; ② GCV showed significant inhibition effect on proliferation of A549 and MRC-5 transfected with pGL3-SV40-TK in vitro, also on that of A549 transfected with pGL3-hTp-TK, but not of MRC-5 transfected with pGL3-hTp-TK; With the treatment of GCV, apoptosis index （AI） of A549 cells transfeeted with pGL3-SV40-TK and pGL3-hTp-TK （21.58%and 23. 19% respectively） increased significantly, compared with that of A549 transfeeted with pGL3-hTp and blank control;GCV enhanced the effects on AI in MRC-5 transfected with pGL3-SV40-TK（9.35%）, but not with pGL3-hTp- TK（O. 88%）; ③ Inhibition ratio of pGL3-SV40-TK/GCV and pGL3-hTp-TK/GCV to transplanted tumor of A549 in nude mice （46.7% and 40. 5% respectively） were significantly higher than that of negative control groups （9.7% ,14. 3%,7.0% and 8. 0% respectively）. Conclusion TK gene driven by hTERT promoter could express selectively in lung cancer cell. Lung cancer cell could be specifically inhibited by HSV-TK/GCV driven by hTERT promoter in vitro and in vivo.