p300特异性siRNA载体的构建及其对心肌细胞GATA4表达的影响.doc

p300特异性siRNA载体的构建及其对心肌细胞GATA4表达的影响基金项目： 基金项目：国家自然科学基金资助项目，重庆市自然科学基金资助项目（CSTC,2007BB5311）。作者简介：孙慧超(1982-),女，汉，山东省潍坊市人，在读博士研究生，研究方向为心脏发育与先心病发生机制，Tel*通讯作者，田杰，Email： HYPERLINK mailto:jietian@ jietian@ , Tel: 023 孙慧超1 , 田杰1＊, 朱静2 , 吕铁伟1 , 陈国珍1，李娅莎2 （重庆医科大学附属儿童医院 1. 心脏中心 2. 儿科研究所，重庆 400014） 【摘要】 目的 构建并筛选小鼠p300特异性siRNA载体，并观察p300表达降低对心脏特异性转录因子的调控作用，为进一步研究p300介导的组蛋白乙酰化失衡对心脏发育的影响奠定基础。方法 针对小鼠p300基因的保守序列区域设计并构建三个重组质粒p300 RNAi1，p300 RNAi2和p300 RNAi3。利用阳离子脂质体将其分别转染入体外培养的乳鼠心肌细胞，分别利用RT-PCR和免疫荧光从mRNA和蛋白水平检测p300表达，判断抑制效率，并检测心脏特异性转录因子GATA4的表达水平。结果 转染pSOS-HUS和p300 RNAi2的细胞与正常组比较，p300表达无明显变化（P0.05），而转染p300 RNAi1和p300 RNAi3的细胞内p300比正常组表达水平降低（P0.05），心肌特异性基因GATA4的表达水平也明显降低。 结论 p300特异性siRNA 质粒载体构建成功，p300 RNAi1和p300 RNAi3具有确切阻断p300蛋白表达的作用，并下调GATA4的表达。 【关键词】 RNA干扰； p300； 心脏；GATA4 ；表观遗传学；组蛋白乙酰化 【中图分类号】R34；R394.8 【文献标识码]】A Construction and expression of specific siRNA plasmid vector of p300 in SUN Hui chao1, TIAN Jie1, ZHU Jing2, Lv Tie wei1, Chen Guo zhen1，LI Ya sha2 （1 Department of Heart Centre, 2 Pediatric Institute, Childrens Hospital of Chongqing Medical University, Chongqing 400014, China) Abstract: Objective To construct and screen effective siRNA plasmid vector which can suppress the expression of p300 in mouse, research the regulation of p300 knock down on transcription factor GATA4, and to establish the foundation for the further study of the regulation by histone acetylation mediated by p300. Methods Three recombinant plasmids vectors involving siRNA, which matched the base pair of mouse p300 perfectly, were constructed and transfected into primary mice cardiocyte cultured in vitro. The expressions of p300 and GATA4 in different groups were detected by RT-PCR and immunofluorescence. Results The expression of p300 and GATA4 in experimental group transfected with p300 RNAi1 or p300 RNAi3 was depressed obviously than normal group (P0.05), while there was no obvious changes in groups of pSOS-HUS and p300 RNAi2（P0.05）. Conclusion The recombi