EphA1基因可控性双稳转内皮祖细胞系EPCsTet-On-EphA1SiRNA的建立

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。

To construct an EphA1 gene regulatable endothelia progenitor cells (EPCs) by Tet-On system

Objective To develop the EPCsTet-On-EphA1SiRNA cell line which can regulate the EphA1 gene expression by doxycycline.Methods EPCs were transfected with pWHE146 vector by liposome transfection reagent.The transfected cells were screened in medium containing G418 and G418-resistant clones were isolated.All individual G418-resistant clones were selected by transient transfection with plasmid pTRE-hyg-luc.And the low background and high induction of luciferase in response to doxycycline clones were selected.The isolated clones were named EPCsTetOn.Followed,EPCsTet-On cells were transfected with pTRE-EphA1SiRNA vector by liposome transfection reagent.The transfected cells were selected in medium containing hygromycin(hyg) and the double-stable cell lines(G418-and hygresistant) EPCsTet-On-EphA1SiRNA were isolated.Induced by doxycycline,RT-PCR and Western blotting were used to test the expression of EphA1.Results Low background and high induction EPCsTet-On-EphA1SiRNA were established successfully.EphA1 mRNA could be induced to down-expressed in EPCsTet-On-EphA1SiRNA by doxycycline.Compared with no doxycycline group which has statistical significance(P<0.05).In addition the expression rate of EphA1 was decreased significantly by doxycycline in a concentration-dependent manner.Conclusion The double-stable cell line EPCsTet-On-EphA1SiRNA was successfully established,which could be induced EphA1 down-expressed by doxycycline and provided an ideal experimental platform for further study of EPCs in angiogenesis of liver cancer.