Gcn5基因shRNA的载体构建及其对干细胞分化中组蛋白乙酰化修饰的干扰作用

目的构建组蛋白乙酰化酶转录辅激活酶Gcn5基因表达的干扰shRNA质粒并初步检测其性质，为研究干细胞诱导分化过程中乙酰化修饰所起的调控作用奠定基础。方法选择7条组蛋白乙酰化修饰关键因子Gcn5基因片段，用带有绿色荧光蛋白（green fluorescence protein，GFP）基因的pGenesil-1作为载体，构建相应7个shRNA质粒。脂质体共转染5-氮杂胞苷（5-azacytidine，5-aza）诱导的大鼠间充质干细胞（mesenchymal stem cells，MSCs），利用乙酰化酶特异性抗体Gcn5（H75）对转染细胞进行免疫化学检测，荧光显微镜图象分析系统，记数细胞，在同视野中记数转染阳性细胞以及干扰的阳性细胞，初步计算出其转染率和干扰效率。结果通过限制性内切酶酶切和DNA序列分析，重组质粒shRNA与设计相吻合；免疫细胞化学检测，发现转染阴性对照HK的细胞显现“饱满核”，乙酰化作用蛋白表达明显；转染shRNA质粒的细胞呈现十分明显表达减弱的“淡染核”或不表达的“空洞核”，转染率和干扰效率分别为93．7％和46．6％。结论构建的shRNA质粒能够达到干扰乙酰化作用的目的，为深入研究乙酰化作用奠定了一定的试验基础。

Constructions of Gcn5 shRNAs interfere the histone acetylation modification with stem cell differentiation

OBJECTIVE: To construct the Gcn5 shRNA plasmids and to explore the Gcn5 shRNA role in histone acetylation modification with the differentiation of stem cells. METHODS: Seven shRNA fragments were recombined into pGenesil-1 vector to form 7. Gcn5 shRNA constructions. The mesenchymal stem cells (MSCs) induced for two weeks with 5-aza were transfected by the plasmids with lipofectamine2000. Polyclonal antibodies labeled with TRITC were used to identify the acetylation in MSCs with or without Gcn5 shRNA constructions. The efficiencies of transfection and RNAi were calculated based on the ratio of GFP (green fluorescence)/DAPI (blue fluorescence) and TRITC (red fluorescence)/DAPI, respectively. RESULTS: Seven Gcn5 shRNA plasmids or constructions were identified by restriction endonucleases Pst I/Sal I and DNA sequencing. Acetylation block was observed after Gcn5 shRNA plasmids transfected into cells. Fluorescent intensity of TRITC in nucleuses were decreased remarkably, or even disappeared in MSCs. The efficiencies of transfection and RNAi were 93.7% and 46.6%, respectively. CONCLUSION: The Gcn5 shRNA plasmids constructed in the present study can decrease the histone acetylation during cell differentiation. It sets the basis for further exploring the role of acetylation in the regulation of cell differentiation.