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、48 h 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LIMK2 mRNA 表达的抑制效率最高，两组应用R3 siRNA 后的LIMK2 mRNA 表达量分别为对照组的19.30%（转染后24 h）、18.63%（转染后48 h）；Western blot 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R3 siRNA 的LIMK2 蛋白表达量分别仅为空白对照组的1.32%（转染后24 h）、1.19%（转染后48 h）。

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.