慢病毒介导的GFP-Hsp90αE47A基因在HepG2细胞表达及对细胞增殖性影响的研究

目的：建立真核细胞表达的GFP-Hsp90αE47A基因重组慢病毒载体三质粒包装细胞系统,并检测其对细胞增殖性的影响,为进一步研究HSP90分子伴侣功能奠定基础。方法：制备完整的重组慢病毒载体三质粒系统：转移质粒（Hsp90αE47A/psin-GFP）,包装质粒（ΔNRF）及包膜蛋白质粒（VSV-G）。磷酸钙法将三质粒共转染293T包装细胞,48h后收集病毒上清。将制备好的慢病毒颗粒感染HepG2细胞,在荧光显微镜下观察报告基因GFP的表达情况,Westernblot检测HepG2细胞GFP-Hsp90α表达。MTT法检测细胞增殖情况。结果：转染后的293T和感染后的HepG2细胞能观察到较强的绿色荧光,培养液上清病毒滴度约为3.0×103ifu/μl,HepG2细胞中有GFP-Hsp90α蛋白的表达。内源性Hsp90α表达无明显上升（为对照组的1.05±0.15倍,P〈0.05,t检验）,有明显外源性GFP-Hsp90αE47A蛋白的表达,为对照组内源性Hsp90α的0.68±0.12倍。外源性GFP-Hsp90αE47A蛋白的表达HepG2细胞增殖活性于第4d有明显抑制。（1.051±0.03vs1.349±0.05,P〈0.05,t检验）。结论：成功建立重组慢病毒载体的三质粒包装细胞系统,并将GFP-Hsp90αE47A基因在HepG2细胞中稳定表达,且并未引起细胞明显的热休克反应而导致的内源性Hsp90α增高;且能明显抑制细胞增殖,为后期Hsp90α分子伴侣功能进行研究奠定基础。

Expression of Recombinant GFP-Hsp90P E47A Gene by Lentiviral Vector Transfection and Its Effect on Cell Proliferation in HepG2 Cells

Objective： To construct the three-plasmid packaging cell line of the recombinant lentiviral vector encoding Hsp90αE47A/psin-GFP gene and investigate the molecular chaperon function of hsp90α. Methods： The three-plasmid recombinant lentiviral vector, which was made up of the vector plasmid （Hsp90αE47A/psin-GFP）, the packaging plasmid （ΔNRF） and the envelop plasmid encoding the vesicular stomatitis virus-glycoprotein （VSV-G）, was isolated and purified. Human embryonic kidney 293T cells were co-transfected with the three plasmids by calcium phosphate method. 48 hours after the transfection, the viral supernatant was col- lected to infect HepG2cells. The expression of reporter gene GFP was detected by fluorescence microscope, and the GFP-Hsp90αE47A protein expressed in Hepg2 cells was detected by Western blot. Cell proliferation was tested by CCK-8 method. Results： There was strong expression of GFP s in 293T and Hepg2 cells after transfection. The viral titer was 3.0 ×103 ifu /μl. The expression of GFP-Hsp90α in Hepg2 cells was confirmed by Western blot. Cell proliferation of GFP-Hsp90αE47A protein expressing Hepg2 cells was obviously inhibited at 4d. （1.051±0.03 vs 1.349±0.05, P0.05,t test）. Conclusion： The three-plasmid packaging cell line system of recombinant lentiviral vector was successfully established, cell proliferation was obviously inbited by GFP-Hsp90αE47A gene transfec- tion, which will provide a basis for exploring the molecular chaperon function of Hsp90α.