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

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 figure in OM group was 1.5 times as in CM group and the figure in E2 group was 2 times as in OM group （F = 119.1, P 〈 0.0001 ）. In day 5 and 9, the expression levels of osteogenic related protein in E2 group were higher than OM or CM group. In day 19, E2 group produced more mineral nodules than OM group. The expression levels of miR-20a-5p in E2 group were high than OM group, increasing by 2 folds and 1.5 folds in day 5 and day 9 respectively （day 5： Mann-Whitney U= 20.000, P = 0.013; Mann- Whitney U = 32.000, P = 0.079）. However, the expression levels of miR-29a-3p did not have significant changes between different groups in these two time points （day 5 ： Mann-Whitney U = 0.000, P 〉 0.999, Mann-Whitney U = 10.000,9 d： P = 0.680）. Conclusions E2 promoted the osteogenic differentiation in rat BMSCs, and the expression level of miR-20a-5p increased during this process.