The Study On The Effect Of Hypoxia Inducible Factor 1αRegulated By Tet-on Gene Expression System On Hepatoma In Vitro And In Vivo

PartⅠ: Establishment of HepG2Tet-on cell line controlled by the Tet-On regulatory systemObjective To establish tetracycline-controlled inducible system ( Tet-On) in HepG2 cell.Methods The HepG2 cells were transfected with pWHE146 vector by using liposome transfection reagent . The transfected cells were selected in medium containing G418 and G418-resistant clones were isolated. All individual G418-resistant clones were screened by transient transfection with plasmid pTRE-hyg-luc for clones with low background and high induction of luciferase in response to Dox.Results One HepG2 cell line, which exhibited high levels of induction and high gene expression levels, was obtained.Conclusion The HepG2 cell line can be used to highly express eukaryotic gene and this Tet-On system is available for use in eukaryotic gene function studies. PartⅡ: Establishment of Tet-On system in HepG2 cells to regulate HIF-1αexpressionObjective To develop the HepG2Tet-on-HIF-1αcell line which can regulate the HIF-1αexpression by doxycycline.Methods The HepG2Tet-on cells were transfected with pTRE-HIF-1αvector by using liposome transfection reagent. The transfected cells were selected in medium containing hygromycin(hyg) and the double-stable cell lines(G418- and hyg-resistant) HepG2Tet-on-HIF-1αwere isolated. Induced by Dox(1μg/ml), RT-PCR and Western-blot to test the expression of HIF-1α.Results the HIF-1αmRNA and protein of HepG2Tet-on-HIF-1αcells could be induced up to 5.899±2.176 and 2.179±0.742 folds by doxycycline(1μg/ml).Conclusion The double-stable cell line HepG2Tet-on-HIF-1αwas successfully established, which could be induced HIF-1αexpression by doxycycline and provided an ideal experimental platform for further functional study of HIF-1α. PartⅢ: Inducible Expression of Hypoxia-Inducible factor 1αon the Proliferation and Invasion Property of HepG2 cells under Normoxia in VitroObjective To study the inducible expression of HIF-1αon the proliferation and invasion property of HepG2 cells under normoxia in vitro.Methods Under normoxia in vitro, MTT assay was used to observe the proliferative and adhesive activity of cells, and the invasive activity was determined by transwell cell culture chamber method.Results There were no difference of A490nm between the Dox(+)and Dox(-)group in experiment detecting the proliferation activity(P>0.05); But in adhesive experiment, the A490nm of Dox ( + ) group was 0.662±0.058, higher than the Dox ( - ) group 0.526±0.808(P=0.008); The invasived cell number of Dox(+)group was 37.611±8.424, but in the Dox(-)group, the number was 25.333±8.117(P<0.001).Conclusion HIF-1αhas no influence with the proliferation activity, but it could enhance the adhesive and invasive properties of HepG2 cells. PartⅣ:Construction of Subcutaneous Implanted Hepatoma Model Inducible Expression of HIF-1αin Nude MiceObjective To constructe the subcutaneous implanted hepatoma model in nude mice with HepG2Tet-on-HIF-1αcell line which could be induced the expression of HIF-1αby doxycycline.Methods Constructed the subcutaneous implanted hepatoma model in nude mice with HepG2Tet-on-HIF-1αcell line; The HIF-1αmRNA and protein level of subcutaneous implanted tumors were detected with RT-PCR and Western-blot after seven weeks taking doxycycline orally.Results The HIF-1αmRNA and protein of subcutaneous implanted tumor in nude mice could be induced up to 4.303±1.004 and 1.666±0.079 folds by taking doxycycline orally(1mg/ml).Conclusion The HIF-1αmRNA and protein of subcutaneous implanted tumor with HepG2Tet-on-HIF-1αcell line in nude mice could be induced by taking doxycycline orally. It provided an ideal experimental platform for further study of the effect of HIF-1αon hepatoma in vivo. PartⅤ:Impact of Induced Hypoxia-Inducible Factor-1αon the Subcutaneous Implanted Hepatoma in Nude Mice in VivoObjective To study the impact of induced HIF-1αon the growth and apoptosis of subcutaneous implanted hepatoma in nude mice in vivo.Methods The HIF-1αof the subcutaneous implanted hepatoma in nude mice were induced by doxycycline; Observed the impact of HIF-1αinduced by doxycycline on the growth of subcutaneous implanted hepatoma in nude mice and its impact on nude mice. Tunel method was used to analyze the apoptosis index of subcutaneous implanted hepatoma. Immunohistochemistry and western blot methods were used to analyze the vascularization in subcutaneous implanted hepatoma of two groups.Results Compared with Dox(-)group, the volume, weight and growth velocity of subcutaneous implanted hepatoma were higher in Dox(+)group, but the necrosis period of the tumor were less. The weight loss of nude mice was obvious in Dox(+)group. There was no liver or lung metastasis in either group. In Dox(-)group, the AI was higher than that of Dox(+)group. And there were much more blood vessels in subcutaneous implanted hepatoma of Dox(+) groups.Conclusion HIF-1αpromote the growth of subcutaneous implanted hepatoma in nude mice in vivo. The mechanism may be that HIF-1αdecrease the cell apoptosis and promot the vascularization of subcutaneous implanted hepatoma.