Genistein Inhibits The Immediate Early Protein Expression Of Human Cytomegalovirus

Objective:1? Detecting human cytomegalovirus'(HCMV) infecting astrocyte in early time which causing the expression change of IE72 protein2? Detecting genistein inhibits human cytomegalovirus'(HCMV) infecting astrocyte in vitro that causing the expression change of the immediate early IE72 protein and m RNA to investigate the correlation between the Genistein and HCMV.3? Testing histone acetylation level of IE72 gene promoter regions(MIEP)to deeply explore Genistein inhibits astrocyte' s mechanism of HCMV infection in the transcription stage.Methods:1? Primary human astrocytes,then use Immunofluorescence technique to detect the astrocytes marker protein-GFAP and calculate the purity of human primary astrocytes.2? Detect the activity of the experiment and control groups cells by MTT.3? The experiment is devided into five Group : HCMV group?Gen group?HCMV(MIO=5)infection after 4 hours then plus 40 ?M Gen group ?plus 40?M Gen 4 hours then infect HCMV group and control group then uses Western blot and Real-time PCR respectively to detect expression of IE protein and m RNA.4? The experiment is devided into group HCMV ? group Gen+HCMV ? group HCMV+Gen and control group and use chromatin immune coprecipitation technology(Ch IP)to test histone acetylation level changes of IE72 gene promoter regions(MIEP).Results:1? Result of Immunofluorescence technique indicate AS cells came up to 95%.2? In 40 ?M Gen+HCMV group MTT values were higher than HCMV group at 24 h?48 h?72 h post-infection( P < 0. 05). In HCMV group, Cytopathic effects(CPE) were observed at 48 h post-infection. In 40 ?M Gen+HCMV group, CPE can not be observed obviously and most cells kept normal forms.3? Real-time PCR found the expression of IE72 m RNA in the two group were lower than HCMV group at 24h?48 h?72 h and 96 h post-infection( P < 0. 05).4? Western blot assay indicated that in HCMV infection after 4 hours then plus 40 ?M Gen group and plus40?M Gen 4 hours then infect HCMV group, the expression of IE72 were lower than HCMV group( P < 0. 05).5? According to the results of chromatin immune coprecipitation(Ch IP),group HCMV+ Gen was lower than group HCMV that in histone acetylation level of IE gene promoter regions.Conclusions:1? Human primary astrocytes were isolated and cultured successfully in vitro.2? Proper concentration of Gen can inhibit abnormal proliferation of cells caused by HCMV infection, and reduce the amount of IE72 protein expression.3? Gen inhibiting HCMV infection of cells by lowering the IE gene promoter regions H3 histone acetylation level to regulate gene expression of IE thus.