摘要
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 ,并研究其在前列腺癌细胞株PC 3中的表达。方法 构建带有hTERT启动子的腺病毒穿梭载体pDC3 15 Tp EGFP ,细胞重组技术获得腺病毒AdhTERT EGFP ,体外转染PC 3及人胚肺成纤维细胞株MRC 5 ,通过RT PCR和倒置荧光显微镜分别在mRNA和蛋白质水平检测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 成功构建出腺病毒AdhTERT EGFP ,滴度为 4.2× 10 9空斑形成单位 (pfu) /ml,分别转染PC 3和MRC 5 ,前者表达EGFP的阳性细胞数为 (66.4% 3 .3 ) % ,而后者不表达EGFP(P <0 .0 1) ;对照病毒Ad EGFP在PC 3和MRC 5中分别有 (88.1± 2 .2 ) %及 (89.2± 3 .1) %的细胞表达EGFP ,两者表达量差异无统计学意义。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前列腺肿瘤细胞中表达 ,在正常细胞中不表达 ,具有明显的靶向性。
Objective To construct an EGFP labeled recombinant defective adenovirus vector driven by human telomerase reserve transcriptase (hTERT) promoter and study its expression in prostate carcinoma cell line PC 3.Methods The shuttle plasmid pDC315 Tp EGFP which contained the hTERT promoter was generated.By using AdEasy system,the replication defective adenovirus AdhTERT EGFP was assembled.The recombinant adenovirus was transfected into the prostate carcinoma cell line PC 3 and normal human lung fibroblast cell line MRC 5.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 AdhTERT EGFP was generated successfully and its titer was 4.2× 10 9 pfu/ml.The EGFP protein was highly expressed in (66.4± 3.3)% PC 3 cells which was transfected with AdhTERT EGFP,but not in MRC 5 ( P < 0.01). While transfected with the Ad EGFP,they had expressed the EGFP protein in (88.1± 2.2)% PC 3 and (89.2± 3.1)% MRC 5 cells ( P < 0.01). Conclusion The downstream gene controlled by hTERT promoter in adenovirus system can be expressed selectively in prostatic carcinoma cell line PC 3,which showed the obvious targeting character.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2005年第1期23-24,i001,共3页
Chinese Journal of Experimental Surgery
