EphA1基因可控性双稳转内皮祖细胞系EPCs^(Tet-On-EphA1)SiRNA的建立

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

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。

Obiective To develop the EPCs^Tet-ON-EphalSiRNA cell line which can regulate lhe EphAl geneexpression by doxvcvcline. Methods EPCs were transfected with pWHE146 vector by liposome transfection reagent. The transfected eel ls were screened in medium containing C418 and G41 g-resistant clones were isolated. All individual G418-resistant clones were gelected by transient transfectlon with plasmid pTRE-hyg-lnc. And the low background and high induction of luciferase in response to doxycycline clones were selected. The isolated climes were named EPCsTet- On. Followed, EPCsTet-On cells were transfected with p^Tet-ON-EphalSiRNA vector bv liposome transfection reagent. The transfected cells were selected in medium containing hygTomvcin（hw） and the double-stable cell lines（G418- and hw- resistant） EPCsT^Tet-ON-EphalSiRNA were isolated. Induced by doxvcveline, RT-PCR and Western blotting were used to lest the expression of EphAl. Results Low background and high induction EPCs^Tet-ON-EphalSiRNA were established successfully. EDhA 1 mRNA could be induced to down-expressed in EPCsT^Tet-ON-EphalSiRNA by doxvcvcline. Compared with no doxycycline group which has statistical significance（P〈 0.05）. In addition the expression rate of EphA 1 was decreased significantly by doxvcvcline in a concentration-dependent manner. Conclusion The double-stable cell line EPCsr^Tet-ON-EphalSiRNA was successfully established, which could be induced EphA1 down-expressed by doxvcvcline and provided an ideal experimental platform for further study of EPCs in angiogenesis of liver cancer.