罗格列酮与全反式维甲酸诱导小鼠胚胎成纤维细胞定向骨分化研究  

Effect of rosiglitazone and all-trans retinoic acid on osteogenic differentiation in mouse embryonic fibroblasts

作　　者：王涵[1,2] 邵英[1,2] 朱茄慧 廖云鹏[1,2] 马妍 任文艳[1,2] 周林云[1,2] 吴柯[1,2] 何百成[1,2] WANG han;SHAO Ying;ZHU Jia-hui;LIAO Yun-peng;MA Yan;REN Wen-yan;ZHOU Lin-yun;WU Ke;HE Bai-cheng(Chongqing Key Laboratory of Biochemistry and Molecular Pharmcology,Chongqing 400016,China;Dept of Pharmcology,Pharmacy School of Chongqing Medical University,Chongqing 400016,China)

机构地区：[1]重庆市生物化学与分子药理学重点实验室 [2]重庆医科大学药理学教研室,重庆400016

出　　处：《中国药理学通报》2018年第10期1384-1391,共8页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No 81572226)

摘　　要：目的探讨罗格列酮(RSG)与全反式维甲酸(ATRA)对干细胞定向骨分化的影响,并初步解析这一作用的可能分子机制。方法通过定量PCR、组织化学染色及Western blot等方法,检测相关成骨与成脂分化标志物的变化情况;利用荧光素酶报告质粒,检测Runx2的转录水平以及BMP/Smad信号的激活情况。结果在C2C12、C3H10T1/2和MEFs中,PPARγ以及RAR和RXR各亚型受体均有表达。单独使用RSG或ATRA均能增加MEFs细胞中碱性磷酸酶(ALP)的活性,且RSG能明显增加ATRA所诱导的ALP活性。RSG或ATRA单独应用不能诱导钙盐沉积,两者合用不但增加OPN和OCN的表达,而且还诱导钙盐沉积。RSG可诱导脂滴生成,ATRA不能诱导脂滴生成,且抑制RSG促进脂滴生成的作用。RSG在MEFs中促进C/EBPα表达,但ATRA能抑制RSG的这种作用。ATRA与RSG合用可促进MEFs中Runx2的蛋白表达。同时,ATRA与RSG合用增加了MEFs中BMP/Smad的转录和Smad1/5/8的磷酸化水平,同时也增加了Smad6的mRNA表达,但对Samd7的mRNA表达有抑制作用。结论RSG与ATRA合用能诱导MEFs定向骨分化,该作用可能与ATRA抑制RSG诱导的成脂分化,并增加BMP/Smad信号转导活性有关。To investigate whether the combination of rosiglitazone(RSG)and all-trans retinoic acid(ATRA)can induce osteogenic differentiation in mouse embryonic fibroblasts(MEFs),as well as the possible mechanism underlying this process.Methods We introduced real-time PCR,histochemical staining and Western blot to determine the endogenous expression of PARγ,RAR,RXR and the markers for adipogenic or osteogenic differentiation.We used luciferase reporter assay,Western blot and PCR assay to detect the level of Runx2,the activation of BMP/Samd signal and the mRNA expression of Smad6 and Smad7 in MEFs.Results PPARγ,RAR and RXR were all detectable in C2C12,C3H10T1/2 and MEFs.RSG and ATRA could increase the alkaline phosphatase(ALP)activity in MEFs,repectively.But,the combination of RSG and ATRA greatly increased the ALP activity,much stronger than that of RSG or ATRA.No apparent mineralization nodal was found in the groups treated with RSG or ATRA.However,the combination of RSG and ATRA increased not only the level of OPN and OCN,but also the matrix mineralization in MEFs.RSG induced adipogenesis apparently in MEFs and ATRA showed no effect on it,but the combination of RSG and ATRA almost diminished the RSG induced adipogenesis in MEFs.Same results were found in the detection of C/EBPα.For mechanism,the combination of RSG and ATRA increased the transcriptional activity of Runx2 reporter and the level of Runx2.Besides,the combination of RSG and ATRA also increased the transcriptional activity of BMP/Smad reporter,the level of of p-Smad1/5/8 and the mRNA level of Samd7,but decreased the mRNA level of Smad7.Conclusions The combination of RSG and ATRA can induce osteogenic differention in MEFs,and this process may be related with the inhibitory effect of ATRA on adipogenic differentiation and promotion effect on the activation of BMP/Smad signal.

关 键 词：罗格列酮 全反式维甲酸 小鼠胚胎成纤维细胞 成脂分化 成骨分化 BMP/Smad信号 

分 类 号：R-332[医药卫生] R322.71