雌激素对大鼠骨髓间充质干细胞成骨分化及微小RNA表达的影响  

Effect of estrogen on osteogenic differentiation and microRNAs expression in rat bone marrow mesenchymal stem cells

作　　者：刘冠琪[1] 麦志辉[1] 黄锦华[1] 陈琳[1] 陈奇[1] 陈正[1] 艾虹[1] 

机构地区：[1]中山大学附属第三医院口腔医学部正畸科,广州510630

出　　处：《中华口腔医学研究杂志（电子版）》2017年第1期17-24,共8页Chinese Journal of Stomatological Research(Electronic Edition)

基　　金：国家自然科学基金(81470766)

摘　　要：目的研究雌激素对大鼠骨髓间充质干细胞(BMSC)成骨分化以及对微小RNA(miR-20a、miR-29a)表达水平的影响。方法分离培养大鼠BMSC,将细胞分3组分别进行处理,使用完全培养基培养组作为空白对照记为CM,成骨诱导液培养组作为对照记为OM,成骨诱导液+17β雌二醇(E2)培养组作为实验组记为E2。细胞处理第1、5、9天进行碱性磷酸酶(ALP)活性检测;第5、9天,蛋白免疫印迹法(Western blot)检测成骨相关蛋白如Runt相关转录因子2(RUNX2)、骨形态发生蛋白2(BMP2)、骨桥蛋白(OPN)的表达情况,定量聚合酶链反应(PCR)检测miR-20a及mi R29a的表达情况;第19天,茜素红染色检测矿化结节的形成情况。两组数据的结果进行两组独立样本的t检验,两组以上的数据进行单因素方差分析,检验水准为P<0.05。结果处理1 d后,各组ALP活性较低,组间差异无统计学意义(CM1 d:3.49,OM1 d:2.82,E21 d:2.92;F=2.002,P=0.216);5 d后,E2组及OM组ALP活性较1 d时明显提高(E21 d:2.92,E25 d:15.83;t=5.065,P=0.007;OM1 d:2.82,OM5 d:8.38;t=13.39,P=0.0002),5 d时E2的值为OM组的2倍、CM组的4倍,差异有统计学意义(F=13.95,P=0.0055);9 d后,各组ALP活性水平有明显增高(CM9 d:14.66,OM9 d:22.17,E29 d:45.99),约为5 d时的3倍(CM:t=11.830,P=0.0003;OM:t=8.068,P=0.0013;E2:t=9.332,P=0.0007),组间差异变化不明显,OM组约为CM组1.5倍,E2组约为OM组2倍,差异有统计学意义(F=119.1,P<0.0001)。第5、9天,E2组成骨相关蛋白表达水平在均比OM、CM组高。第19天,E2组比OM组形成更多矿化结节。E2组miR-20a-5p表达水平在第5天为OM组的2倍(Mann-Whitney U=20.000,P=0.013),在第9天为OM组的1.5倍(Mann-Whitney U=32.000,P=0.079);而miR-29a-3p的表达水平相比OM组无明显变化(5 d:Mann-Whitney U=0.000,P>0.999,Mann-Whitney U=10.000,9 d:P=0.680)。结论 E2能促进大鼠BMSC的成骨分化,同时提高mi R-20a-5p的表达水平。Objective To investigate the effect of estrogen on osteogenic differentiation and the expression levels of miR-20a, miR-29a in rat bone marrow mesenchymal stem cells （BMSCs）. Methods BMSCs were isolated and cultured, and were divided into 3 groups; Blank control group was cultured in complete medium and was named CM group; Control group was cultured in osteogenic differentiation medium and was named OM group; Treatment group was cultured in osteogenic differentiation medium containing 1713- estradiol （E2） and was named E2 group. Cells＇ alkaline phosphatase activities were detected after being incubated for 1, 5, 9 days and the expression levels of osteogenic related proteins such as Runt-related transcription factor 2 （RUNX2）, bone morphogenetic protein 2 （BMP2）, Osteopontin （OPN） were detected in day 5 and 9 by western blot. Expression levels of miR-20a-5p, miR-29a-3p were detected in day 5 and 9 by quantitative polymerase chain reaction （qPCR）. The production of mineralized nodule were stained by Alizarin Red in day 19. The t test was used to compare differences between 2 groups and the One- Way ANOVA was used to compare differences among more than 2 groups. P 〈 0.05 was considered statistically significant. Results Alp activities ： In day 1, the figures were low in every group and there were no significant differences between each group （CM1 d： 3.49, OM1 d： 2.82, E21 d： 2.92; F = 2.002, P = 0.216） ; In day 5, the alp activities in OM group and E2 group raised markedly （E21 d： 2.92, E25 d ： 15.83 ; t = 5.065, P = 0.007 ; OMt a ： 2.82, OM5 d ： 8.38 ; t = 13.39, P = 0.0002） ; The figure in E2 group was significantly twice as in OM group and was four times as in CM group （F= 13.95 ,P= 0.0055） ; In day 9, alp activities increased significantly by 3 folds in all the groups compared with the figures in day 5 （CM9a： 14.66, OM9 a＂ 22.17, E29 d ： 45.99 ; CM ： t = 11.830, P = 0.0003 ; OM ： t = 8.068, P = 0.0013 ; E2 ： t = 9.332, P = 0.0007） ; The fig

关 键 词：雌二醇 大鼠 间质干细胞 成骨分化 微小RNA 

分 类 号：R783.5[医药卫生—口腔医学]