摘要
目的:构建可同时表达野生型p53(wt-p53)及siRNA-mdm2的重组真核表达载体用于激素非依赖性前列腺癌的联合基因治疗。方法:利用亚克隆、T-A克隆和PCR技术合成并构建pcDNA3.1-p53、siRNA-mdm2、p53/siRNA-mdm2和siRNA-scramble重组质粒。脂质体法将上述重组质粒转染至PC-3细胞,半定量RT-PCR和Western blotting检测共表达质粒对mdm2和p53表达的影响,MTT法检测共表达质粒转染后PC-3细胞增殖活性。结果:克隆出wt-p53全长及带有U6启动子的siRNA-mdm2序列,经DNA测序证实序列正确,通过连接成功构建上述真核重组表达载体;转染细胞48h后可见siRNA-mdm2组GFP明显表达;RT-PCR和Western blotting检测显示转染共表达质粒后PC-3细胞中wt-p53表达增强,mdm2表达下调;MTT检测显示共表达质粒转染后PC-3细胞生长抑制率为40.4%。结论:成功构建p53与siRNA-mdm2共表达质粒,共表达质粒可抑制前列腺癌细胞PC-3的增殖。
Objective To construct the recombinant vectors that coexpress wild type p53 （wt-p53） and siRNA- mdm2 for gene therapy in androgen independent prostate cancer . Methods The suhcioning, T-Acloning, and PCR technique were used to construct the recombinant vectors, named pcDNA3.1-p53, siRNA-mdm2, p53/siRNA- mdm2, and siRNA-scramble. The recombinant expression vectors mentioned above were transfected into PC-3 cells, the expression levels of the mdm2 siRNA and p53 after transfection were detected by the semi-quantltative RT-PCR analysis and Western blotting analysis. MTT assay was used to detect the proliferation inhibition of PC-a cells after tl：ansfeetion. Results The recombinant plasmids containing both wt-p53 and siRNA-mdm2 were successfully constructed, and confirmed by DNA sequence analysis. After transfected for 48 h, the expression of GFP was observed. The expression level of wt-p53 was increased, and the expression of mdm2 was decreased detected by RT-PCR and Western blotting. MTT assay showed that the inhibitory rate of proliferation of PC-3 cells was 40.4 after transfection. Conclusion The recombinant plasmids containing both wt-p53 and siRNA-mdm2 are successfully constructed, they can remarkablely inhibit the proliferation of PC-3 cells.
出处
《吉林大学学报（医学版）》
CAS
CSCD
北大核心
2010年第1期30-34,I0001,共6页
Journal of Jilin University：Medicine Edition
基金
教育部博士学科专项基金资助课题(20070183012)
吉林省科技厅科技发展计划项目资助课题(200705360)
