模拟微重力影响人牙髓干细胞的矿化能力与RhoA-Rho激酶信号通路相关性研究  

Effects of simulated microgravity on mineralization of hDPSCs via RhoA-Rho signaling pathway

作　　者：牛玉梅[1] 张巍巍[1] 曹涛[1] 李艳萍[1] 刘会梅[1] 贾丛辉[1] 

机构地区：[1]哈尔滨医科大学口腔医学院牙体牙髓科,黑龙江哈尔滨150001

出　　处：《口腔医学》2016年第5期399-402,共4页Stomatology

基　　金：黑龙江省教育厅科学技术研究项目(12531234)

摘　　要：目的初步研究Rho A-Rho激酶信号通路在模拟微重力(Simulated Microgravity,SMG)影响人牙髓干细胞(human dental pulp stem cells,h DPSCs)矿化过程中的作用。方法将经酶消化法分离、纯化后的人牙髓干细胞体外扩增培养,以1×106细胞接种于PLGA支架上,分别于模拟微重力下及普通重力下矿化诱导液中培养,72 h后收集细胞,采用Western Blot检测Rho A蛋白表达;此外,在重力条件下采用Rho激酶抑制剂Y-27632,以矿化诱导液培养组作为对照组,检测矿化诱导3、5、7、10d两组碱性磷酸酶(Alkaline phosphatase,AKP)活性变化,采用Western Blot检测矿化诱导10 d,成牙本质相关蛋白牙本质基质蛋白(dentin matrix protein-1,DMP-1)、牙本质涎蛋白(dentin sialoprotein,DSP)、牙本质涎磷蛋白(dentin sialophosphoprotein,DSPP)的表达,采用茜素红染色法检测矿化诱导21 d,矿化结节形成水平。结果模拟微重力下,人牙髓干细胞中Rho A蛋白的表达下调;在普通重力矿化诱导条件下,加入抑制剂Y-27632后,各时间点细胞AKP表达水平均较对照组有所降低;矿化诱导10 d,成牙本质相关蛋白DSP、DSPP、DMP-1表达明显下调,矿化诱导21 d,矿化结节形成同样有所减少。结论模拟微重力下,Rho A蛋白表达水平下调;抑制Rho激酶的表达,可降低h DPSCs矿化能力,提示Rho A-Rho激酶信号通路可能参与模拟微重力对h DPSCs矿化能力的影响。Objective To investigate the effect of Rho A-Rho kinase signaling pathway on differentiation of human dental pulp stem cells（ h DPSCs） in simulated microgravity（ SMG）. Methods HDPSCs were seeded in the PLGA scaffolds,and divided into normal gravity group and microgravity group. After 72 hours＇ culture,cells were seeded in plate and divided randomly into mineralization induced with or without Y-27632 groups. AKPase activity test was established after 3,5,7,10 days. DMP-1（ dentin matrix protein-1）,DSP（ dentin sialoprotetin） and DSPP（ dentin sialophosphoprotein） were detected by Western Blot for 10 d,and mineralized nodules were detected by Alizarin red staining for 21 d. Results The expression level of Rho A was down-regulated under microgravity. Treated with Y-27632 in normal gravity after 3,5,7 and 10 days,the AKPase activity was significantly lower than control. After 21 days＇ culture,the Alizarin red staining showed that the positive granules were hardly detected. The expression levels of DSP,DSPP and DMP-1 were lower than those in the control group after 10 days of culture. Conclusion Simulated microgravity inhibited the expression of Rho A. Inactivation of Rho kinase by addition of inhibitor Y-27632 caused lower mineralization ability of h DPSCs. It is possible that Rho A-Rho kinase signaling pathway is involved in the effect of simulated microgravity on mineralization process of h DPSCs.

关 键 词：人牙髓干细胞 模拟微重力 RhoA-Rho激酶信号通路 矿化 

分 类 号：R780.2[医药卫生—口腔医学]