摘要
目的构建可表达具有RNA激活功能的小激活RNA(saRNA)质粒表达载体,并对其激活前列腺癌细胞PC-3中p21WAF1/CIP1(p21)基因表达的功能进行相关研究。方法人工合成与p21基因启动子特定序列相应的DNA片段及同样长度的随机DNA序列,利用DNA重组技术将这些片段构建到真核小分子双链RNA(dsRNA)表达质粒pGenesil-1中,构建成能表达dsRNA片段的表达质粒载体(dsRNAp21-pGenesil-1)和随机对照dsRNA片段的表达质粒载体(dsRNACon-pGenesil-1)。采用脂质体介导方法,分别将dsRNAp21-pGenesil-1(实验组)、dsRNACon-pGenesil-1(随机对照组)及空白质粒pGenesil-1(空白对照组)转染到体外培养的PC-3细胞,通过荧光染色检测转染效率,采用RT-PCR和免疫组化技术检测各组细胞p21蛋白表达的差异。结果成功构建目的表达载体dsRNAp21-pGenesil-1及随机对照载体dsRNACon-pGenesil-1,将各组质粒转染到PC-3细胞后,实验组p21基因表达明显增强,而随机对照组和空白对照组无明显变化。结论本研究构建的dsRNA表达质粒载体能成功表达saRNA分子,并激活p21基因及蛋白的表达,为研究的激活效应提供有效工具。
Objective To construct the small-molecule double-stranded RNA (dsRNA) expression vector plasmid,and to investigate its activation ability of the expression of cell cycle repressor protein p21WAF1/CIP1(p21) in human prostate cancer cell line PC-3 in vitro.Methods Synthetic the sequence-specific corresponding DNA fragments of the promoter with the p21 gene,as well as the same length of random DNA fragments,by means of the recombinant DNA technology to construct these fragments into the eukaryotic expression plasmids pGenesil-1,constructed with the purpose of the expression of dsRNA fragments plasmid vectors (dsRNAp21-pGenesil-1) and the random fragments of the control dsRNA expression vectors (dsRNACon-pGenesil-1).The dsRNAp21-pGenesil-1 (experimental group),dsRNACon-pGenesil-1 (negative control group) and a blank plasmid pGenesil-1 (control group) were transfected into human prostate cancer cell line PC-3 in vitro respectively with lipofectamine reagent,and transfection efficency was detected by laser scanning confocal microscope.Immunohistochemistry and reverse transcription-PCR were performed to examine the difference expression level of p21 in each group.Results These dsRNAp21-pGenesil-1 and the dsRNACon-pGenesil-1 eukaryotic cell expression vectors were successfully constructed.For each of the groups of plasmids transfected into PC-3 cells,p21 gene expression in the experimental group significantly increased,while there was no significant difference between the negative control group and the blank control group.Conclusions The dsRNAp21-pGenesil-1 express dsRNA molecule successfully,and active the level of expression of p21 mRNA and protein,and provide an effective tool for studying the activation effect of dsRNA.
出处
《现代泌尿生殖肿瘤杂志》
2011年第5期283-286,共4页
Journal of Contemporary Urologic and Reproductive Oncology
基金
国家自然科学基金项目(30873018)