论文部分内容阅读
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 investigate the inhibitory effect of RNA interference on the expression of LIMK2 in primary osteoblasts and its inhibitory efficiency, so as to lay the foundation for the further study on the role of LIMK2 gene. Methods Three siRNAs were chemically synthesized targeting LIMK2 gene in mouse and were respectively numbered as R1, R2 and R3. Liposomes were transfected into mouse primary osteoblasts respectively. After 24 h and 48 h of transfection, Total RNA and total protein were detected by RT-PCR and Western blot respectively to study the inhibitory effect of three siRNAs on LIMK2 gene at different time points. Results The results of RT-PCR showed that the siRNA targeting R3 showed the highest inhibitory effect on LIMK2 mRNA expression. The mRNA expression of LIMK2 in both groups was 19.30% (24h after transfection), 18.63% (48h after transfection) ). The results of Western blot showed that siRNA targeting R3 showed the most obvious inhibitory effect on LIMK2 protein expression. LIMK2 protein expression of R3 siRNA was only 1.32% (24h after transfection) and 1.19% (transfected) After 48h). Conclusion The siRNA of R3 could inhibit the expression of LIMK2 in mouse primary osteoblasts.