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目的建立可调控EphA1基因表达的内皮祖细胞系EPCsTet-On-EphA1SiRNA。方法将pWHE146质粒转染到内皮祖细胞系中,筛选出稳定表达的细胞克隆;扩增后瞬时转染pTRE-hyg-luc质粒,强力霉素诱导表达后,检测荧光素酶活性,挑选出高表达、低背景的受强力霉素调控的EPCsTet-On细胞株;再将重组质粒pTRE-EphA1SiRNA转染入EPCsTet-On细胞株,筛选出稳定表达细胞克隆EPCsTet-On-EphA1SiRNA;通过强力霉素诱导后,利用RT-PCR和Western blotting法检测EphA1基因mRNA与蛋白的表达。结果成功构建了受强力霉素调控的高表达低背景的EPCsTet-On-EphA1SiRNA细胞株;强力霉素可诱导EPCsTet-On-EphA1SiRNA细胞株中EphA1mRNA表达下调,较之未调控组其差异有统计学意义(P<0.05);强力霉素调控EphA1蛋白表达的能力在一定范围内呈剂量依赖性关系。结论成功建立强力霉素调控EphA1基因表达的大鼠双稳转内皮祖细胞系EPCsTet-On-EphA1SiRNA,为深入研究EphA1基因在内皮祖细胞参与肝癌血管生成过程中的作用提供了有效的实验手段。
Objective To establish EPCsTet-On-EphA1SiRNA that can regulate the expression of EphA1 gene. Methods Plasmid pWHE146 was transfected into endothelial progenitor cell line and the stable cell clones were selected. After amplification, pTRE-hyg-luc plasmid was transiently transfected. After induced by doxycycline, the luciferase activity was detected and luciferase activity was selected. Expression and low background of EPCsTet-On cell line regulated by doxycycline. The recombinant plasmid pTRE-EphA1SiRNA was transfected into EPCsTet-On cell line, and the stable expression cell clone EPCsTet-On-EphA1SiRNA was screened. Afterwards, the expression of EphA1 mRNA and protein was detected by RT-PCR and Western blotting. Results Doxycycline-rich EPCsTet-On-EphA1SiRNA cell line with high expression and low background was successfully constructed. Doxycycline induced a down-regulation of EphA1mRNA expression in EPCsTet-On-EphA1SiRNA cell lines compared with the control group (P <0.05). The ability of doxycycline to regulate EphA1 expression in a dose-dependent manner. Conclusion The successful establishment of a rat biliary stable endothelial progenitor cell line EPCsTet-On-EphA1SiRNA that controls the expression of EphA1 gene by Doxycycline provides an effective experimental means for further study on the role of EphA1 gene in the process of endothelial progenitor cells participating in angiogenesis of hepatocellular carcinoma.