hTERT启动子调控p53基因表达逆转录病毒的抗肿瘤作用

Antitumor Effected by Retroviral Expressing wtp53 Gene Driven by hTERT Promoter

目的:观察人端粒酶逆转录酶(hTERT)基因启动子驱动野生型p53的新型逆转录病毒对肿瘤的杀伤作用。方法:先将前期构建的逆转录病毒穿梭质粒pLHCX-hTERT-EGFP和pLHCX-hTERT-wtp53进行包装,通过浓缩获得高滴度病毒液。然后将逆转录病毒pLHCX-hTERT-wtp53感染包含突变型p53的大肠癌细胞系SW620,通过RT-PCR和Westernblot证实p53mRNA和p53蛋白的表达。最后将逆转录病毒pLHCX-hTERT-wtp53和pLHCX-hTERT-EGFP感染hTERT阳性的人大肠癌细胞株SW620,人肺癌细胞株A549和hTERT阴性的正常人胚肺成纤维细胞MRC-5,观察其特异性杀伤作用;另外通过裸鼠皮下注射肿瘤细胞SW620和A549,建立肿瘤模型,应用逆转录病毒pLHCX-hTERT-wtp53治疗后测量肿瘤体积计算肿瘤生长抑制率。结果:获得病毒液的滴度分别为2.3×107CFU.mL-1和3.5×107CFU.mL-1。逆转录病毒pLHCX-hTERT-wtp53经RT-PCR证实p53mRNA的表达,经Westernblot检测到p53蛋白表达。重组逆转录病毒导入外源的野生型p53基因后,通过流式细胞检测到hTERT阳性人大肠癌细胞株SW620,人肺癌细胞株A549凋亡率远高于hTERT阴性的正常人胚肺成纤维细胞MRC-5(P<0.05)。体内实验证实各组通过逆转录病毒pLHCX-hTERT-wtp53治疗后的肿瘤体积明显小于对照组(P<0.05)。结论:获得的新型逆转录病毒对肿瘤有特异性的杀伤作用。

Objective：To observe the antitumor effective of the retroviral expressing wild type p53（wtp53） gene driven by human telomerase reserve transcriptase（hTERT） promoter.Methods：The shuttle plasmids of pLHCX-hTERT-wtp53 and pLHCX-hTERT-EGFP that had been constructed were transfected into the packaging cell line PA317 under mediation of Lipofectamine.The high-titer retrovirus was obtained as a result by thickening.The human colon cancer cell line SW620,which the p53 gene is mutation,were infected with the retroviral pLHCX-hTERT-wtp53,the p53 mRNA and p53 protein were detected by RT-PCR and Western blot,respectively.The normal human embr lung fibroblast cell line MRC-5（hTERT negative）,the human colon cancer cell line SW620 and the human lung cancer cell line A549（hTERT positive） were infected with the retroviral,Flow cytometer（FCM） was used to observe the apoptosis of different cells.By subcutaneously injecting tumor cells SW620 and A549 into the nude mice,the animal tumor models were established,respectively.The volume of tumors in nude mice which were treated with the retroviral pLHCX-hTERT-wtp53 were measured.Results：The virus titer of retroviral pLHCX-hTERT-EGFP and pLHCX-hTERT-wtp53 were 2.3×10^7CFU·ml^-1 and 3.5×10^7CFU·ml^-1,respectively.The expression of wtp53 mRNA and protein of the retroviral pLHCX-hTERT-wtp53 were demonstrated by RT-PCR and Western blot,respectively.Through FCM,It was declared that the apoptosis of the MCR-5 cell was more lower than human cancer cell lines（P〈0.05）.The tumor size which treated with the retroviral pLHCX-hTERT-wtp53 were significantly smaller than those in the control groups（P〈0.05）.Conclusion：The neotype recombinant retroviral pLHCX-hTERT-wtp53 can kill tumor specifically.