siRNA沉默小鼠原代成骨细胞LIMK2基因的实验研究  

An experimental study on silencing the expression of LIMK2 gene by small interfering RNAs in mouse primary osteoblasts

作　　者：李友瑞[1] 覃峰[1] 张艺平[1] 邵敏锋[1] 陈睿 沈韵 付强[1] 

机构地区：[1]中山大学光华口腔医学院.附属口腔医院.口腔医学研究所,广州510055 [2]广州宜康医疗投资管理有限公司人力资源部 [3]深圳瑞尔齿科诊所

出　　处：《中华口腔医学研究杂志（电子版）》2010年第2期17-20,共4页Chinese Journal of Stomatological Research(Electronic Edition)

基　　金：广东省卫生厅医学科研基金(A2008227);广东省科技计划项目(2009B050700027;2008B030301121)

摘　　要：目的探讨采用RNA干扰抑制原代成骨细胞中LIMK2的表达及其抑制效率,为进一步研究LIMK2基因的作用奠定基础。方法针对小鼠LIMK2基因,化学合成3条siRNA,分别编号为R1、R2、R3,用脂质体分别介导转染到小鼠原代成骨细胞中,转染后24、48h提取细胞的总RNA和总蛋白,分别进行RT-PCR和Western blot检测,研究3条siRNA在不同时间点对LIMK2基因的抑制效率。结果RT-PCR结果显示编号为R3的siRNA对LIMK2mRNA表达的抑制效率最高,两组应用R3siRNA后的LIMK2 mRNA表达量分别为对照组的19.30%(转染后24h)、18.63%(转染后48h);Westernblot检测显示编码为R3的siRNA对LIMK2蛋白表达的抑制效果最明显,两组应用R3siRNA的LIMK2蛋白表达量分别仅为空白对照组的1.32%(转染后24h)、1.19%(转染后48h)。结论编号为R3的siRNA可以很好地抑制小鼠原代成骨细胞的LIMK2表达。Objective To silence the expression of LIMK2 gene in primary mouse osteoblasts by small interfering RNAs （siRNAs） and evaluate their inhibition efficiency. Methods Three siRNAs which target mouse LIMK2 gene were chemically synthesized and named R1, R2 and R3, respectively. The siRNAs were transfected into primary osteoblasts by liposome. Reverse transcriptase polymerase chain reaction （RT-PCR） and Western blot analysis were performed to test the LIMK2 gene expression and then the most efficient siRNA sequence was chosen. Results The mRNA and protein levels of LIMK2 in the groups using R3 siRNA were much lower than those of other groups. Compared with the negative control group, the relative expression of LIMK2 mRNA in the the groups using R3 siRNA were 19.30% （24 h after transfection） and 18.63% （48h after transfection）, respectively. Compared with the blank control group, the relative expression of LIMK2 protein in the Group 7 and Group 8 using R3 siRNA were only 1.32%（24 h after transfection）and 1.19% （48 h after transfection）, respectively. Conclusion The expression of LIMK2 gene in mouse primary osteoblasts can be successfully silenced by the R3 siRNA.

关 键 词：LIMK2基因 RNA干扰 成骨细胞 

分 类 号：R78[医药卫生—口腔医学]