摘要目的 探究RhoA/ROCK信号通路介导的细胞形变是否在人间充质干细胞(hMSCs)成骨分化过程中发挥调控作用.方法 取原代hMSCs培养至P3代加入成骨诱导液诱导14d后,采用免疫荧光检测RhoA和ROCK-1蛋白的表达变化.另取P3代hMSCs细胞分为诱导组、空白加药组及加药组,诱导组采取单纯成骨诱导培养,空白加药组加入成骨诱导液及溶剂二甲基亚砜(DMSO)进行诱导培养,加药组加入成骨诱导液和溶于DMSO的Y-27632(RhoA/ROCK信号通路抑制剂)进行诱导培养.利用CCK-8检测细胞的增殖,荧光显微镜观察各组细胞骨架的变化,实时聚合酶链反应(RT-PCR)及碱性磷酸酶(ALP)染色检测各组细胞成骨基因ALP、骨钙素(OCN)、侏儒相关转录因子2(RUNX-2)的表达变化.两组间数据比较采用t检验.结果 hMSCs成骨诱导前后RhoA、ROCK-1蛋白表达呈现差异性变化,成骨诱导14 d RhoA和ROCK-1蛋白表达显著高于诱导前(22.7±2.1比14.7±0.6和24.3±1.5比20.3±0.6,t=-6.414、-4.243,均P＜0.05).与诱导组相比,加药组第1、5、9、14天干细胞增殖活性无显著变化(F=0.427、1.000、0.298、1.314,均P＞0.05).加药组细胞骨架呈明显梭形改变,细胞铺展面积变小;从第14天的3组ALP染色显示加药组的染色明显比诱导组浅.RT-PCR结果显示诱导组和空白加药组细胞随诱导时间延长其成骨表型基因表达水平呈上升趋势,加药组细胞成骨表型基因ALP、OCN、RUNX-2的表达水平随诱导时间延长逐渐下调,加药组ALP表达在第14天时显著低于其他2组(F=25.891,P=0.001);加药组OCN表达在第11天和14天时显著低于其余2组(F=5.773、25382,均P＜0.05);加药组RUNX-2表达在第11天和14天时显著低于其余两组(F=34.972、10.808,均P＜0.05).结论 RhoA/ROCK信号通路可能通过介导细胞骨架形变在hMSCs成骨分化过程中起促进作用.

abstractsObjective To investigate whether the cell deformation induced by RhoA/ROCK signaling pathway plays a regulatory role in the osteogenic differentiation in human mesenchymal stem cells (hMSCs).Methods The primary hMSCs were cultured until the P3 generation was added to the osteogenic induction solution for 14 days,and the expression levels of RhoA and ROCK-1 proteins were detected by immunofluorescence.Another P3 generation hMSCs cells were divided into induction group,blank plus drug group and medicated group,and the induction group was induced by simple osteogenic induction;the blank drug-added group was added to the osteogenic induction solution and the solvent dimethyl sulfoxide (DMSO)for induction culture;the medicinal group was added with osteogenic induction solution and Y-27632 (RhoA/ ROCK signaling pathway inhibitor) dissolved in DMSO for induction culture.The proliferation of the cells was detected by CCK-8 methods.The changes of cytoskeleton in each group were observed by fluorescence microscope.The expression changes of osteogenic genes alkaline phosphatase (ALP),osteocalcin (OCN) and runt-relatedtranscriptionfactor-2 (RUNX-2) in each group were detected by real time polymerase chain reaction (RT-PCR) and ALP staining.The t test was used for data comparison between the two groups.Results There were significant differences in the expression of RhoA and ROCK-1 protein before and after osteogenic induction of hMSCs (22.7±2.1 vs 14.7±0.6 and 24.3±1.5 vs 20.3±0.6,t=-6.414,-4.243,both P＜0.05).Mesenchymal stem cell proliferation on day 1,5,9 and day 14 in the medicated group were comparable with those in the induction group (F=0.427,1.000,0.298,1.314,all P＞0.05).The cytoskeleton of the medicated group showed obvious spindle-shaped changes,and the cell spreading area became smaller.From the three groups of ALP staining on the 14th day,it can be seen that the color of the medicated group was significantly lighter than that of the induction group.The results of RT-PCR showed that the expression of osteogenic phenotypic genes increased with the induction time in the induction group and the blank drug-treated group,the expression levels of osteogenic phenotype genes ALP,OCN and RUNX-2 in the drug-treated group were gradually decreased with the induction time.The expression of ALP in the drug-treated group was significantly lower than those in the other two groups at 14th day (F=25.891,P=0.001);the expression of OCN in the drug-treated group was significantly lower than those in the other two groups at 1 1th and 14th days (F=5.773,25.382,both P＜0.05);the expression of RUNX-2 in the drug group was significantly lower than those in the other two groups at 11th and 14th days (F=34.972,10.808,both P＜0.05).Conclusion The RhoA/ROCK signaling pathway may play a role in promoting osteogenic differentiation of hMSCs through mediating cytoskeletal deformation.