小鼠干细胞成骨和成软骨分化特异性微小RNA的初步研究  

PRELIMINARY STUDY ON microRNA REGULATED OSTEOGENIC AND CHONDROGENIC DIFFERENTIATION OF MOUSE STEM CELLS

作　　者：幸嵘[1] 孔清泉[1] 项舟[1] 杨婧[1] 罗静聪[2] 邓力[2] 解慧琪[2] 

机构地区：[1]四川大学华西医院骨科,成都610041 [2]四川大学华西医院生物治疗国家重点实验室.干细胞与组织工程研究室,成都610041

出　　处：《中国修复重建外科杂志》2014年第8期1009-1016,共8页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(81171731;30700837)~~

摘　　要：目的通过对C3H10T1/2细胞进行成骨及成软骨分化,利用微小RNA(microRNA,miRNA)芯片技术筛选成骨分化和成软骨分化中差异miRNA,以期寻找调节C3H10T1/2细胞成骨及成软骨分化的特异性miRNA。方法取C3H10T1/2细胞系,分别行成骨和成软骨诱导分化,对诱导组和对照组进行鉴定。然后通过miRNA芯片技术筛选分化前后2倍变化幅度且平均标准化信号值>2的差异miRNA,并行实时荧光定量PCR(real-time fl uorescence quantitative PCR,RT-qPCR)验证。结果诱导7 d成骨诱导组较对照组有明显ALP表达(P<0.05);诱导7、14、21 d,RT-qPCR检测成骨诱导组成骨标志物Runx2、丝氨酸蛋白酶7(serine protease 7,Sp7)、Ⅰ型胶原、骨桥蛋白(osteopontin,OPN)mRNA相对表达量均显著高于诱导0 d时(P<0.05);诱导21 d,Western blot检测成骨诱导组Runx2、Sp7、Ⅰ型胶原、OPN蛋白表达量明显高于对照组(P<0.05)。成软骨诱导5、10、15 d,RT-qPCR检测成软骨诱导组软骨标志物SOX9、Ⅱ型胶原、蛋白多糖(Aggrecan)、透明质酸合成酶(Has2)mRNA相对表达量均显著高于诱导0 d时(P<0.05);诱导15 d,Western blot检测成软骨诱导组SOX9、Ⅱ型胶原、Aggrecan、Has2蛋白表达量明显高于对照组(P<0.05)。通过miRNA芯片技术分别筛选出10个成骨和3个成软骨2倍变化幅度且平均标准化信号值>2的差异miRNA。除miR-455-3p之外,其余差异miRNA验证结果与芯片结果比较有较好一致性。结论通过miRNA芯片技术对C3H10T1/2细胞系以及其诱导成骨和成软骨分化细胞进行检测对比,发现部分miRNA表达量在诱导后细胞中发生变化,为进一步挑选成骨和成软骨特异性miRNA进行功能验证和靶基因预测奠定了基础。Objective To investigate the specific microRNA （miRNA） in osteogenic and chondrogenic differentiations of C3H10T1/2 cells. Methods C3H10T1/2 cells were induced to differentiate into osteoblasts and chondrocytes. Specific miRNA more than 2 fold change and 2 average normalized probe signal between C3H10T1/2 and C3H10T1/2-derived osteoblast, and between C3H10T1/2 and C3H10T1/2-derived chondrocytes were screened out by miRNA microarray, and verified by real-time fluorescence quantitative PCR （RT-qPCR）. Results Alkaline phosphatase expression of osteogenic induced group was significantly higher than that of control group at 7 days after induced （P 〈 0.05）. RT-qPCR results showed the expressions of Runx2, serine protease （Sp7）, collagen type I, and osteopontin （OPN） genes were significantly increased at 7, 14, and 21 days after induced when compared with before induced （P 〈 0.05）. Western blot results showed the expressions of Runx2, Sp7, collagen type I, and OPN proteins of osteogenic induced group were significantly higher than those of control group at 21 days after induced （P 〈 0.05）. The expressions of SOX9, collagen type II, Aggrecan, and Has2 were significantly increased at 5, 10, and 15 days after induced when compared with before induced （P 〈 0.05）. The expressions of SOX9, collagen type 2, Aggrecan, and Has2 proteins of chondrogenic induced group were significantly higher than those of control group at 15 days after induced （P 〈 0.05）. Totally, 10 osteogenic and 3 chondrogenic miRNA more than 2 fold change and 2 average normalized probe signal were screened out by miRNA microarray. RT-qPCR results of these specific miRNAs were similar to microarray results except miR-455-3p. Conclusion Specific miRNAs are screened out by microarray and it is a good foundation for the future study on miRNA functional verification and target gene prediction.

关 键 词：C3H10T1 2细胞 成骨分化 成软骨分化 微小RNA 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]