hMAM-EP调控HSV-TK腺病毒载体的构建及其对乳腺癌细胞的靶向杀伤

Construction of recombinant adenovirus carrying HSV-TK controlled by hMAM enhancer and promoter and its targeted killing effect on human breast cancer cells

目的:分别构建人乳腺珠蛋白(human mammaglobin,hMAM)基因增强子和启动子(enhancer and promoter ofhMAM,hMAM-EP)调控的增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)报告基因和单纯疱疹病毒胸苷激酶(herpes simple virus thymidine kinase,HSV-TK)自杀基因两种重组腺病毒载体,探讨hMAM-EP调控的HSV-TK在乳腺癌细胞特异性表达及其对乳腺癌的靶向治疗作用。方法:构建hMAM-EP-EGFP和hMAM-EP-TK重组质粒载体,将重组质粒目的基因转移到腺病毒骨架黏粒载体pAxcwit2,并转染HEK 293细胞获得重组腺病毒载体Ad-EP-EGFP和Ad-EP-TK。将Ad-EP-EGFP感染乳腺癌细胞T-47D、ZR-75-30和鼻咽癌细胞5-8F,荧光显微镜观察EGFP的表达。将Ad-EP-TK感染T-47D细胞,给予1、10、20、50μg/ml前药更昔洛韦(ganciclovir,GCV),观察TK基因对乳腺癌细胞的特异杀伤作用。结果:成功构建hMAM-EP调控的重组腺病毒载体Ad-EP-EGFP和Ad-EP-TK。Ad-EP-EGFP感染后,乳腺癌T-47D细胞可见EGFP表达,但ZR-75-30细胞和5-8F细胞无表达。与未感染组或感染Ad-EP-EGFP组相比,Ad-EP-TK重组腺病毒联合GCV(50μg/ml)组T-47D细胞存活率显著降低[(35.69±0.07)%vs(91.74±0.02)%,(87.69±0.11)%;P<0.05],且随GCV质量浓度的增加,T-47D细胞存活率逐渐下降,在MOI=100、GCV质量浓度分别为1、10、20、50μg/ml条件下,细胞存活率分别为(94.34±0.04)%、(86.26±0.02)%、(66.51±0.09)%、(35.69±0.07)%。结论:hMAM-EP调控的HSV-TK自杀基因在乳腺癌T-47D细胞中特异性表达,Ad-EP-TK联合GCV可靶向杀伤乳腺癌T-47D细胞。

Objective ： To construct two recombinant adenovirus vectors carrying reporter gene enhanced green fluorescent protein （EGFP） or suicide gene herpes simple virus thymidine kinase （HSV-TK） at the downstream of enhancer and promoter of human mammaglobin （hMAM-EP）. To explore breast-cancer-cell-specific regulation effect of hMAM-EP and new methods of targeted therapy for breast cancer. Methods： Two recombinant plasmid vectors, hMAM-EP-EGFP and hMAM-EP-TK, were constructed, which respectively carried reporter gene EGFP and suicide gene HSV-TK at the downstream of hMAM-EP. Recombinant adenovirus vectors Ad-EP-EGFP and Ad-EP-TK were obtained after the target genes from the recombinant plasmids were transferred into adenovirus skeleton cosmid pAxcwit2; recombinant adenovirus vectors Ad-EP-EGFP and Ad-EP-TK were then transfected into breast cancer T-47D cells, ZR-75-30 cells and nasopharyngeal cancer 5-8F cells. The expression of EGFP was observed under a fluorescence microscope. Recombinant adenovirus Ad-EP-TK-infected T-47D cells were cultured with 1, 10, 20 and 50 μg/ml prodrug GCV to observe specific cell-killing effect on breast cancer cells. Results： The recombinant plasmid vectors Ad-EP-EGFP and Ad-EP-TK controlled by hMAM-EP were successfully constructed. EGFP could be observed in human breast cancer T-47D cells infected with Ad-EP-EGFP recombinant adenovirus, and could not be detected in ZR-75-30 and 5-8F cells. Compared with un-infected and Ad-EP-EGFP-infected groups, the survival rate of T-47D cells in Ad-EP-EGFP-infection combined with GCV （50 μg/ml） group was significantly decreased （／[35.69±0.07／]% vs ／[91.74±0.02／]%, ／[87.69±011／]%, P〈0.05）. With an increase in mass concentration of GCV, the survival rate decreased. Cell survival rates were （94.34±0.04）%, （86.26±0.02）%, （66.51±0.09）% and （35.69±0.07）% when T-47D cells were infected with hMAM-EP-TK in a MOI of 100 and cultured with 1, 10, 20, and 50 μg/ml GCV. Conclusion： HSV-TK suicide gene controlled by hMAM-EP is specifically expressed in breast cancer T-47D cells, and T-47D cells can be killed by Ad-EP-TK combined with GCV.