论文部分内容阅读
目的:筛选Runx2特异性siRNA,优化反应条件,抑制MC3T3-E1细胞的成骨分化,探讨异位骨化基因治疗的新思路。方法:使用Ambion公司提供的网上工具,设计5条针对小鼠Runx2的mRNA的模板,用体外转录合成法合成5条siRNA;采用RT-PCR和Western blot分别从mRNA和蛋白质两个水平,在培养的MC3T3-E1成骨前体细胞中筛选有明显抑制作用的siRNA,并优化反应条件。将筛选出的siRNA转染成骨细胞,在不同时间点检测成骨细胞相关基因I型胶原、骨桥蛋白、骨钙蛋白、骨涎蛋白mRNA的表达,采用碱性磷酸酶活性分析检测碱性磷酸酶活性的变化。结果:在合成的5条siRNA中,siRNA1657-1677对Runx2的抑制效果最明显,最佳反应条件是:浓度80nM、脂质体/siRNA为1∶1、转染时间为72小时。采用RNAi技术抑制Runx2的表达可以抑制成骨相关基因如I型胶原、碱性磷酸酶、骨桥蛋白和骨钙蛋白等的表达,阻止成骨细胞分化。结论:Runx2特异性siRNA可以抑制成骨细胞分化,可用于异位骨化基因治疗的实验研究。
OBJECTIVE: To screen Runx2-specific siRNA, optimize the reaction conditions, inhibit the osteogenic differentiation of MC3T3-E1 cells and to explore new ideas of heterotopic ossification gene therapy. METHODS: Five siRNAs targeting mouse Runx2 were designed and synthesized using online tools provided by Ambion. Five siRNAs were synthesized by in vitro transcriptional synthesis. MRNA and protein levels were analyzed by RT-PCR and Western blot respectively. Of MC3T3-E1 osteoblast precursor cells were screened for significant inhibition of siRNA, and optimize the reaction conditions. The selected siRNAs were transfected into osteoblasts, and the expression of type I collagen, osteopontin, osteocalcin and bone sialoprotein mRNA were detected at different time points. The activity of alkaline phosphatase Changes in phosphatase activity. Results: Among the 5 siRNAs synthesized, siRNA1657-1677 had the most obvious inhibitory effect on Runx2. The optimal reaction conditions were as follows: concentration 80nM, liposome / siRNA 1: 1, transfection time 72h. Suppression of Runx2 expression by RNAi technology can inhibit the expression of osteogenesis-related genes such as type I collagen, alkaline phosphatase, osteopontin and osteocalcin, and prevent osteoblast differentiation. Conclusion: Runx2 specific siRNA can inhibit osteoblast differentiation and can be used in the experimental study of heterotopic ossification gene therapy.