摘要
目的:构建带有肿瘤特异性DF3启动子、针对hTERT基因的RNA干扰表达载体pGenesil-10-miR30-DF3-hTERT,探讨该表达载体的肿瘤靶向性基因抑制作用。方法:分别用针对hTERT基因的RNA干扰寡核苷酸序列及DF3启动子取代质粒pGenesil-10-Micro30中原有的miR30序列及CMV启动子,构建pGenesil-10-miR30-DF3-hTERT载体并进行酶切及测序鉴定。将该载体分别转染人乳腺癌MCF-7细胞、肝癌HepG2细胞及造血干细胞,ELISA法检测hTERT蛋白的表达情况。结果:重组质粒经酶切及测序鉴定证实符合设计要求,构建成功。ELISA法检测结果显示,实验组MCF-7细胞及HepG2细胞的hTERT蛋白下降显著(P<0.05),其中以MCF-7细胞下降更为明显;造血干细胞实验组则无明显变化(P>0.05)。结论:DF3启动子调控的hTERT基因的RNA干扰表达载体能有效抑制DF3阳性肿瘤细胞中hTERT的表达,以乳腺癌细胞效果最为显著,而对端粒酶阳性的正常细胞不产生影响。
Objective： To construct hTERT gene-targeted RNAi expression vector driven by tumor specific DF3 promoter and investigate its tumor-targeted gene suppression effects.Methods： The miR30 sequence and CMV promoter in the plasmid pGenesil-10-Micro30 were replaced respectively by hTERT gene-targeted RNAi oligonucleotides sequence and DF3 promoter.The new recombinant vector pGenesil-10-miR30-DF3-hTERT was confirmed by enzyme digestion and sequencing,and then transfected into three types of cells： human breast cancer cells（MCF-7）,human hepatocarcinoma cells（HepG2） and haematopoietic stem cells（HSC）.Finally,ELISA was performed to evaluate the expression of hTERT protein.Results： Enzyme digestion and sequencing confirmed that the recombinant plasmid was constructed successfully;ELISA showed that the expression level of hTERT protein in the experimental groups of MCF-7 and HepG2 cells decreased significantly（P〈0.05） and the former was more obvious,but there was no apparent change in the experimental group of HSC（P〉0.05）.Conclusion： The hTERT gene-targeted RNAi expression vector mediated by DF3 promoter can inhibit expression of hTERT protein in DF3 positive tumor cells effectively,and the effect is the most obvious on breast cancer cells.However,no effect on telomerase-positive normal cells is observed.
出处
《泸州医学院学报》
2011年第5期521-525,共5页
Journal of Luzhou Medical College
基金
四川省科技厅资助项目(04JY029-020-1)
