基于Tet-On系统的EphA1基因可控性EPCs的建立

o 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 EPCsTet-On. 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 hyg-resistant) 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 additional 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.