miR-30a抑制BMP9诱导间充质干细胞C3H10T1/2的成骨分化  

BMP9-induced Osteogenic Differentiation is Partially Inhibited by miR-30a in Mesenchymal Stem Cell Line C3H10 T1/2

作　　者：李宝林[1] 白慧丽[1] 张汝益[1] 严树涓[1] 何方[1] 杨丹丹[1] 刘晨[1] 施琼[1] 

机构地区：[1]重庆医科大学检验医学院临床检验诊断学教育部重点实验室,重庆400016

出　　处：《中国生物工程杂志》2013年第11期14-20,共7页China Biotechnology

基　　金：国家自然科学基金(NSFC 31200971);重庆市科委自然科学基金计划(CSTC 2011BB5131);重庆市教育委员会科学技术研究(KJ120327)资助项目

摘　　要：目的:确认miR-30a在BMP9诱导间充质干细胞C3H10T1/2成骨分化中的作用。方法:首先用Ad-BMP9处理间充质干细胞C3H10T1/2,通过碱性磷酸酶(ALP)检测试剂盒检测早期成骨指标ALP、Western blot和PCR检测晚期成骨指标OPN,茜素红S染色检测钙盐沉积了解BMP9对C3H10T1/2细胞的成骨分化作用,同时用Real-time PCR检测miR-30a在该成骨分化中的变化。然后用BMP9条件培养基分别作用Ad-mmu-miR-30a和Ad-RFP预处理10h的C3H10T1/2细胞,检测早期成骨指标ALP、晚期成骨指标钙盐沉积和骨钙素OPN的表达。最后探讨miR-30a在BMP9诱导C3H10T1/2细胞成骨分化中的可能作用机制。结果:Ad-BMP9处理C3H10T1/2细胞5d,7d后,ALP的活性的表达增高;第9d后,OPN的表达增加;第14d后,钙盐的沉积明显增多。而miR-30a过表达后,抑制了这一现象。同时发现过表达miR-30a后,Runx2蛋白水平较对照组下降而mRNA水平基本没有变化。结论:miR-30a可以抑制BMP9诱导C3H10T1/2细胞的成骨分化,其作用机理可能和Runx2相关。Objective：To study the role of miR-30a in bone morphogenetic protein 9 （BMP9） induced osteogenic differentiation of mesenchymal stem cell line C3H10T1/2. Method：C3H10T1/2 cells were treated with Adenovirus GFP and BMP9, the early osteogenic marker Alkaline phosphatase （ALP） activity was detected by ALP quantitative assay and staining assay, the later osteogenic marker calcium precipitation was detected by Alizarin Red S staining, OPN was another later marker, which was detected by PCR and Western blot. The expression of miR-30a in C3H10T1/2 ceils quantified by Real-time PCR. Effects of miR-30a on BMP9-induced osteogenic differentiation of C3H10T1/2 cells was detected by ALP, OPN and calcium precipitation. Runx2 was detected in order to study its mechanism. Results ： BMP9 was not only increased the activity of ALP on days 5,7, the calcium precipitation on 14 day was also increased compared with Ad-GFP group, miR-30a reduced the expression of ALP on BMP9-induced osteogenic differentiation. The expression of OPN and calcium precipitation were also inhibited by miR-30a. Conclusion： BMP9-induced osteogenic differentiation is partially inhibited by miR-30a in mesenchymal stem cell line C3H10 T1/2.

关 键 词：miR-30a 骨形成蛋白9 成骨分化 

分 类 号：R273[医药卫生—中西医结合]