辛伐他汀诱导的骨髓间充质干细胞成骨分化过程中Hedgehog信号通路的作用  

The role of Hedgehog signaling pathway in the simvastatin-induced osteogenic differentiation of bone marrow mesenchymal stem cells

作　　者：郭志斌 迟博婧 张钰英 吴春芳 刘子洪[3] 邢磊 张国彬 GUO Zhi-bin;CHI Bo-jing;ZHANG Yu-ying;WU Chun-fang;LIU Zi-hong;XING Lei;ZHANG Guo-bin(Linxi Hospital of Kailuan General Hospital,Tangshan 063000,China;North China University of Science and Technology,Tangshan 063200,China;Kailuan General Hospital,Tangshan 063000,China)

机构地区：[1]开滦总医院林西医院,唐山063000 [2]华北理工大学,唐山063200 [3]开滦总医院,唐山063000

出　　处：《国际药学研究杂志》2019年第11期832-839,共8页Journal of International Pharmaceutical Research

基　　金：河北省医学科学研究重点课题资助项目(20190152,20160722);河北省自然科学基金资助项目(H2013209255)

摘　　要：目的在研究辛伐他汀(SIM)诱导骨髓间充质干细胞(BMSC)成骨分化作用的基础上,结合应用Hedgehog信号通路阻断剂环巴胺cyclopamine(Cpn),探讨Hedgehog通路是否参与辛伐他汀刺激BMSC成骨分化的过程。方法采用全骨髓贴壁培养法提取大鼠BMSC,在诱导培养基(CM)、含SIM或SIM+Cpn的诱导培养基中培养。采用碱性磷酸酶(alkaline phosphatase,ALP)染色法,检测细胞中ALP的表达;通过免疫荧光染色观察细胞中Gli1及骨钙素(osteocalcin,OCN)的表达;采用实时荧光定量PCR(RT-PCR)法,检测细胞中Gli1、ALP、Ⅰ型胶原(collagen typeⅠ,COLΙ)及OCN mRNA的表达;采用蛋白质印迹技术,分析细胞中Gli1、Runx2、COLΙ及OCN的表达,通过茜素红染色检测细胞钙结节形成及基质矿化能力。结果SIM组ALP表达水平显著高于CM组(P<0.05),而SIM+Cpn组ALP表达水平则显著低于SIM组(P<0.05),但仍高于CM组。免疫荧光检测显示,SIM在有(无)Cpn存在的情况下均可促进Gli1及OCN的表达;给药第10和18天SIM组Gli1、ALP、OCN及COLΙmRNA表达水平均显著高于CM组(P<0.05),且这种改变不能被Cpn完全阻断;给药第10和18天SIM组Gli1、Runx2、COLΙ及OCN蛋白表达显著高于CM组(P<0.05),SIM+Cpn组显著低于SIM组(P<0.05)。茜素红染色结果提示,SIM组基质矿化能力显著高于CM组、SIM+Cpn组(P<0.05)。结论SIM可通过上调Gli1及ALP、Runx2、COLΙ及OCN等成骨标志物表达,诱导BMSC成骨分化,且Cpn无法完全阻断该作用。相关机制有待进一步研究阐明。Objective To investigate the osteogenic differentiation of bone marrow mesenchymal stem cells(BMSC)induced by simvastatin(SIM)in vitro and then further investigate whether the Hedgehog signaling pathway is involved in the SIM-induced osteogenic differentiation of BMSC using the Hedgehog signaling pathway blocking agent cyclopamine(Cpn).Methods The rat BMSC was extracted by the whole-bone marrow adherent culture method and cul⁃tured in the induction medium(control medium,CM)and the induction medium containing SIM or SIM+Cpn.The expression of alkaline phosphatase(ALP)in induced cells was detected by the ALP staining.Immunofluorescence stain⁃ing was performed to detect the espressions of Gli1 and osteocalcin(OCN)in the induced cells.The Gli1,ALP,colla⁃gen typeⅠ(COLⅠ)and OCN mRNA expression was detected by real-time quantitative PCR(RT-PCR).Western blot(WB)was used for assessing the expression level of Gli1,Runx2,COLⅠand OCN proteins.The cell calcium nodule for⁃mation and matrix mineralization ability were detected by alizarin red staining.Results The ALP expression was signifi⁃cantly higher in SIM group than in CM group(P<0.05),while the ALP expression was lower in SIM+Cpn group than in the SIM group(P<0.05)but higher than in the CM group.Immunofluorescence assay showed that SIM could promote the expression of Gli1 and OCN in the conditions with or without Cpn.On the 10th and 18th day of intervention,the mRNA expression level of Gli1,ALP,OCN and COLⅠwas significantly higher in the SIM group than in the CM group(P<0.05),and the higher mRNA expression in the SIM group could be completely blocked by Cpn.Further,on the 10th and 18th day of intervention,the expressed Gli1,Runx2,COLⅠand OCN protein level was significantly higher in the SIM group in the CM group(P<0.05),while the level of these expressed proteins in the SIM+Cpn group was significantly lower than in the SIM group(P<0.05).The alizarin red staining showed that SIM group had stronger matrix mineralization ability than the other groups(P<0.0

关 键 词：HEDGEHOG信号通路 GLI1 骨髓间充质干细胞 辛伐他汀 成骨分化 

分 类 号：R681.4[医药卫生—骨科学] R979.9[医药卫生—外科学]