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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 adenovirus system expressing enhanced green fluorescent protein (EGFP) regulated by human telomerase reverse transcriptase (hTERT) promoter and observe its expression in bladder cancer cells. Methods Adenovirus shuttle vector pDC315-Tp-EGFP with hTERT promoter was constructed. The recombinant adenovirus Ad-hTERT-EGFP was transfected into bladder cancer cell line 253J and human embryo lung at an MOI of 100 The cell line MRC-5 was used to detect the expression of EGFP by inverted fluorescence microscopy and RT-PCR. Ad-EGFP, a mouse cytomegalovirus (mCMV) promoter, was used as a positive control. Results The recombinant adenovirus Ad-hTERT-EGFP was successfully constructed and its titers were 3.8 × 10 ~ (10) pfu / ml. In 253J cells infected with Ad-hTERT-EG-FP, ) (%), And did not show any green fluorescence in MRC-5 cells (P <0.01). The control virus Ad-EGFP was (87.1 ± 2.2)% and (92.5 ± 3.1% of cells can see green fluorescence. The results of RT-PCR showed that EGFP gene was expressed in 293J cells transfected with Ad-hTERT-EGFP and Ad-EGFP, and Ad-EGFP transfected with MRC-5 cells. However, MRC-5 cells transfected with Ad-hTERT- No EGFP gene expression was observed. Conclusion The downstream genes regulated by hTERT promoter in this adenovirus system can be selectively expressed in bladder tumor cells and not expressed in normal cells.