人端粒酶逆转录酶启动子调控的重组腺病毒的构建及其在膀胱癌细胞中的表达

Construction of an adenovirus vector driven by human telomerase reserve transcriptase gene promoter and Its expression in bladder carcimona

目的构建人端粒酶逆转录酶(hTERT)启动子调控的表达增强型绿色荧光蛋白(EGFP)的腺病毒系统,观察其在膀胱癌细胞中的表达。方法构建带有hTERT启动子的腺病毒穿梭载体pDC315-Tp-EGFP,细胞重组技术获得腺病毒Ad-hTERT-EGFP,按感染复数(MOI)100体外转染膀胱癌细胞株253J及人胚肺成纤维细胞株MRC-5,通过倒置荧光显微镜和RT-PCR分别检测EGFP的表达。带有小鼠巨细胞病毒(mCMV)启动子的腺病毒Ad-EGFP作为阳性对照。结果成功构建出重组腺病毒Ad-hTERT-EGFP,滴度为3.8×10^(10)空斑形成单位(pfu)/ml,Ad-hTERT-EG- FP感染的253J细胞中,(85.2±3.3)%发出明亮的绿色荧光;而其感染的MRC-5细胞未产生绿色荧光(P<0.01);对照病毒Ad-EGFP在253J和MRC-5中分别有(87.1±2.2)%及(92.5±3.1)%的细胞可见到绿色荧光。RT-PCR检测显示:253J细胞转染Ad-hTERT-EGFP、Ad-EGFP后及MRC-5细胞转染Ad-EGFP后均有EGFP基因表达;而MRC-5细胞转染Ad-hTERT-EGFP后未见EGFP基因表达。结论该腺病毒系统中hTERT启动子调控下游基因可选择性地在膀胱肿瘤细胞中表达,在正常细胞中不表达,具有明显的靶向性。

Objective To construct an EGFP labeled recombinant adenovirus vector driven by human telomerase reserve transcriptase （hTERT） promoter and study its expression in bladder carcinoma cell line 253J. Methods The shuttle plasmid pDC315-Tp-EGFP which contained the hTERT promoter was generated. By using AdEasy system,the replication-defective adenovirus Ad-hTERT-EGFP was assem- bled. Bladder cancer cell line 253J and human lung fibroblast cell line MRC-5 were transfected by the recombinant adenovirus of 100 multiplicities of infection （MOI）. RT-PCR and fluorescence microscopy were used to detect the mRNA and protein expression, respectively. The recombinant adenovirus Ad-EGFP with mCMV promoter served as positive controls. Results The Ad-hTERT-EGFP was generated successfully and its titer was 3.8 × 10^10 pfu/mL. The EGFP protein was highly expressed in （ 85.2 ± 3.3） % 253J cells which were transfected with Ad-hTERT-EGFP,but not in MRC-5 （P 〈 0.01 ）. While transfected with the Ad-EGFP,they expressed the EGFP protein in （ 87.1 ± 2.2 ） % 253J and （ 92.5 -＋ 3.1 ） % MRC-5 ceils respectively （ P 〈0.01 ）. By RT-PCR ,EGFP mRNA could be detected in both cell lines transfected by Ad- EGFP and 253J cells transfected by Ad-hTERT-EGFP, but couldn＇t be detected in MRC-5 cells transfected by Ad-hTERT-EGFP. Coaclnsion The downstream gene controlled by hTERT promoter in adenovirus system can be expressed selectively in bladder carcinoma cell line 253J, which showed the obvious targeting character.