前列腺特异性抗原启动子中雄激素反应元件的克隆和表达

Cloning and Expression of Androgen Response Elements of Prostate Specific Antigen Promoter

目的:利用前列腺特异性抗原(PSA)启动子的组织特异性表达的特点,克隆出DNA片段并对其表达效率和组织特异性表达进行初步研究。方法:从前列腺组织中提取染色体组,PCR扩增出PSA启动子的两个上游片段a(含ARⅠ和ARⅡ)和b(含ARⅢ),利用报告基因egfp,分别构建3个载体pa-EGFP、pba-EGFP和p△ba-EGFP,并转染细胞HepG2、SMMC-7721、Hela和PC-3,观察表达情况。结果:pba-EGFP在前列腺癌细胞PC-3中的绿色荧光强度明显高于pa-EGFP和p△ba-EGFP,说明片段b(含ARⅢ)能显著增强PSA启动子的转录能力。pa-EGFP、p△ba-EGFP和pba-EGFP转染HepG2、SMMC-7721和Hela细胞,未见表达。结论:基于PSA启动子的组织特异性和调节序列构建的靶向载体,在治疗中加上功能蛋白将为临床实验提供一个坚实的平台。

Objective： To pruvide a possible targeted gene therapy scheme for prostate cancer, and explore the expression efficiency and tissue-specific expression of prostate specific antigen （PSA） promoter. Methods： Three plasmids with egfp, pa-EGFP（ including ARⅠ ARⅡ） , pba-EGFP（ including ARⅠ, AR Ⅱ , AR Ⅲ ） anti pA ba-EGFP（ including ARⅠ, ARⅡ and mutated ARⅢ ） were designed, and the expression status was observed by transfecting into HepG2, SMMC-7721, Hela and PC-3. Results： In prostate cancer cell PC-3, pba-E（,FP expressed more GFP than pa-EGFP and p A ba-EGFP, which showed that ARm could notably increase the transcription efficiency of PSA promoter. Further, there was no GFP expression in HepG2, SMMC-7721 and Hela transfected with pa-EGFP, p△ba-EGFP and pba-EGFP. Conclusion： An expression vector based on elements of the PSA gene regulatory sequences has been developed and shown to be tightly regulated in a panel of cells from tissues of various origins. With the tissue-specific functional protein, it should provide a solid platform for clinical studies. Nail J Androl,2005,11 （12） ：930-932