摘要
目的观察体外化学合成的小干扰RNA(small interfering RNA,siRNA)对原代培养的人视网膜色素上皮(retinal pigment epithelium,RPE)细胞缝隙连接蛋白43(connexin43,cx43)基因和蛋白表达的影响。方法设计合成针对人cx43基因的小干扰片段(siR-NA1、siRNA2、siRNA3)和1条阴性对照siRNA0,在脂质体的介导下转染培养的人RPE细胞,采用RT-PCR确定最有效的抑制片段;针对siRNA的有效片段,分为不同浓度(50～30nmol.L-1)转染培养细胞,培养72h后进行MTT增生试验,观察其对人RPE细胞增生能力的影响,确定最有效的浓度;siRNA转染细胞后,分别培养12h、24h、48h、72h后进行Westonblot试验,观察不同时间点其对cx43蛋白表达的影响。结果体外合成的3条siRNA均在一定程度上抑制cx43基因的合成,其中siRNA1是最有效的抑制片段,根据条带灰度比其抑制率达到72%;siRNA1可以显著促进人RPE细胞的增生,增生率达到146%;明显抑制其cx43蛋白的表达(P<0.01),抑制率达到61%;从浓度组间结果可以看出,siRNA的最佳作用浓度是250nmol.L-1,在此浓度之前效率与浓度呈正比,超过此浓度后各浓度组间差异无统计学意义;比较不同时间点,siRNA转染人RPE细胞24h后开始抑制cx43表达,48h后对cx43蛋白合成抑制率最明显。结论化学合成的siRNA可以有效抑制培养的人RPE细胞cx43基因的表达,促进细胞的增生,这种基因沉默作用可能对于视网膜色素上皮组织的再生有一定作用。
Objective To investigate the effect of chemical synthetic small interfering RNA（siRNA） targeting against connexin 43（ cx43 ） gene on its mRNA synthetic and protein expression in cultured human retinal pigment epithelium （RPE） cells cultured in vitro. Methods Three different siRNA （ siRNA1, siRNA2,siRNA3 ） targeting against human cx43 gene and 1 negative control siRNA were designed and transfected into cultured human RPE cells via liposome reagent. The most effective siRNA could be determined by semi.quantitative reverse transcription PCR. To the most effective siRNA,72 hours after transfected into human RPE cells with different concentration（50 - 300 nmol · L^-1 ） ,the cellular proliferate activities were assayed by MTr colorimetry and the optimization concentration was determined;At 12 hours,24 hours,48 hours,72 hours after transfected （ 50 - 300 nmol · L^-1 ）, the expression of cx43 were studied by Weston blot analysis. Results Three pairs of siRNA were reduced the synthesis of cx43 mRNA at different levels,and the most effective siRNA was siRNA1. The inhibited rate was up to 72% according to the gray scale of strap.After siRNA1 transfected into human RPE cells, the cellular proliferate activities enhanced significantly,but the protein expression of cx43 was decreased obviously （P〈0.01）.The maximal growth rate was 146% and the largest inhibited rate was 61% ;Compared with different concentration groups, the optimization concentration was 250 nmol · L^-1 . The efficiency of siRNA1 showed a concentrationdependent manner within 50 -250 nmol · L^-1 ;Higher than 250 nmol · L^-1 , there was no significant difference. Compared with different timing, the protein expression of cx43 was depressed at 24 hours after transfection and the depression rate was the most significant at the 48 hours. Conclusion Chemically synthetic siRNA could significantly inhibit cx43 gene expression in human RPE cells and enhance the proliferation of RPE cells, which may has potential effect on regeneration o
出处
《眼科新进展》
CAS
2008年第6期412-415,共4页
Recent Advances in Ophthalmology
基金
北京市自然科学基金资助(编号:7032046)~~
