siRNA沉默CTCF基因对牛角膜内皮细胞p27蛋白表达的影响  

Effects of small interfering RNA approach to silence the connective tissue growth factor gene on the expression of the protein p27

作　　者：陈宏[1] 张明昌[1] 张红旭[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院眼科,武汉430022

出　　处：《中华眼科杂志》2010年第7期631-634,共4页Chinese Journal of Ophthalmology

摘　　要：目的 探讨RNA干扰技术沉默结缔组织生长因子（CTGF）基因表达对p27蛋白表达的影响.方法 实验研究.设计并合成CTGF特异性小干扰RNA（siRNA）,用脂质体转染入培养的牛角膜内皮细胞,正常对照组仅加入培养基而未进行siRNA转染.逆转录聚合酶链反应法观察基因沉默效果,免疫印迹法观察1.00μg/L转化生长因子β2作用下角膜内皮细胞内p27蛋白表达.采用单因素方差分析进行统计学分析.结果 CTGF siRNA成功转染入培养的牛角膜内皮细胞并有效沉默CTGF mRNA表达,转染24、48及72 h CTGF mRNA表达分别为正常对照组的17.3%、24.4%及41.7%,与正常对照组比较,差异有统计学意义（F=389.9,P〈0.05）.在转染36 h时,p27蛋白表达明显下降（F=299.3,P〈0.05）.结论 CTGF特异性siRNA能有效沉默细胞内CTGF mRNA表达,下调角膜内皮细胞p27蛋白的表达.Objective To investigate the effects of the small interfering RNA （ siRNA） approach to silence the expression of connective tissue growth factor （ CTGF） gene expression on the expression of the protein p27. Methods Experimental research. The specific siRNA of CTGF was designed and synthesized, then transfected into cultured bovine corneal endothelial cells by liposomes. The blank control group was only treated with the culture medium and not be transfected with siRNA. Reverse transfection-polymerase chain reaction（RT-PCR） was applied to show the effect of gene-silencing, and western blot was used to observe the protein p27 expression of bovine comeal endothelial cells, which were treated with 1. 00 μg/L transforming growth factor β2 （ TGF-β2 ）. The results were analyzed by one-factor analysis of variance. Results CTGF siRNA was successfully transfected into cultured bovine corneal endothelial cells and effectively silence the expression of CTGF mRNA. The CTGF mRNA expression decreased to 17. 3% , 24.4% and 41.7% of control values at 24, 48 and 72 h after transfection, there was significant difference between these groups （ F = 389.9 ,P 〈 0.05 ）. Significant decrease of protein p27 expression was detected at 36 h after transfection （F=299.3,P 〈0.05）. Conclusion CTGF-specific siRNA was able to inhibit the expression of CTGF mRNA effectively and down-regulate the expression of p27.

关 键 词：结缔组织生长因子 转化生长因子Β2 RNA 小分子干扰 内皮 角膜 周期素依赖激酶抑制剂p27结缔组织生长因子 转化生长因子Β2 RNA 小分子干扰 内皮 角膜 周期素依赖激酶抑制剂P27 

分 类 号：R772.2[医药卫生—眼科]