Rho蛋白激酶抑制剂对体外ECM介导的牙周膜细胞功能的作用  

Effect of Rho protein kinase inhibitor on the function of periodontal ligament cells mediated by ECM in vitro

作　　者：兰婷 贾如 LAN Ting;JIA Ru(Department of Stomatology,North-West Women and Children’s Hospital,Xi’an 710061,China;Department of Prosthodontics,Hospital of Stomatology,Xi’an Jiaotong University)

机构地区：[1]西北妇女儿童医院口腔科,西安710061 [2]西安交通大学附属口腔医院修复科

出　　处：《山西医科大学学报》2021年第10期1332-1340,共9页Journal of Shanxi Medical University

基　　金：西安交通大学基本科研业务费自由探索与创新-教师类项目(xzy012019106)。

摘　　要：目的探究Rho蛋白激酶抑制剂在细胞外基质(ECM)介导的牙周膜细胞(PDL细胞)增殖、迁移、愈合和成骨分化过程中的作用。方法收集西北妇女儿童医院口腔科的5名牙周组织健康提供者的牙周膜组织样本,通过消化获取PDL细胞,并分为4组:无ECM包被的PDL细胞对照组(PDL组),ECM包被的PDL细胞组(PDL+ECM组),ECM包被的PDL细胞含盐酸土根碱(EmD)组(PDL+ECM+EmD组),无ECM包被的PDL细胞含EmD组(PDL+EmD组)。通过细胞增殖检测(MTS)分析PDL细胞活力及增殖能力;通过ALP试剂盒测定PDL细胞中碱性磷酸酶(ALP)的活性;通过实时RT-PCR测定ALP的mRNA表达水平;免疫荧光和蛋白质印迹实验分析PDL细胞的Ⅰ型胶原和纤连蛋白的免疫原性及表达水平;Transwell实验和伤口愈合实验分析PDL细胞迁移和愈合能力。结果MTS检测结果表明,10μmol/L EmD对PDL细胞的活力无显著影响。与PDL组相比,PDL+EmD组PDL细胞中ALP的mRNA表达水平和活性均显著降低(P<0.05)。免疫荧光分析结果表明,与PDL组相比,PDL+EmD组细胞出现更细的肌动蛋白丝,Procollagen-Ⅰ和Fibronectin蛋白的免疫反应性和表达水平均显著降低(P<0.05)。与PDL组相比,PDL+ECM组ALP活性、细胞增殖、迁移及愈合能力显著升高(P<0.01)。与PDL+ECM组相比,PDL+ECM+EmD组ALP阳性菌落及活性、细胞增殖、迁移及愈合能力显著降低(P<0.05)。结论牙周ECM可能通过依赖于Rho蛋白激酶(ROCK)并影响ECM相关基因表达的机制来介导PDL细胞的成骨分化,并保证细胞正常的增殖及愈合。Objective To explore the role of Rho protein kinase(ROCK)inhibitor in the process of extracellular matrix(ECM)-mediated osteogenic differentiation of periodontal ligament(PDL)cells.Methods The samples of periodontal ligament from 5 volunteers with healthy periodontal tissues in the Department of Stomatology of North-west Women and Children’s Hospital were taken to obtain PDL cells through digestion,and divided into four groups:PDL cells without ECM coating group(PDL group),ECM-coated PDL group(PDL+ECM group),ECM-coated PDL cells containing Emetine dihydrochloride(EmD)group(PDL+ECM+EmD group),PDL cells without ECM coating EmD group(PDL+EmD group).The viability and the proliferation of PDL cells were determined by MTS analysis.The activity of alkaline phosphatase(ALP)in PDL cells was determined by ALP kit,and the mRNA expression level of ALP was determined by real-time RT-PCR.The immunogenicity and the expression levels of typeⅠcollagen and fibronectin in PDL cells were determined by immunofluorescence and Western blotting.Transwell analysis and wound healing experiments were used to analyze the migration and healing abilities of PDL cells.Results The results of MTS test showed that 10μmol/L EmD had little effect on the viability of PDL cells.Compared with PDL group,the mRNA expression level and the activity of ALP were significantly reduced in PDL+EmD group(P<0.05).The results of immunofluorescence analysis showed that PDL cells grew in multiple layers at high density in PDL group.Compared with PDL group,there were finer actin filaments in PDL+EmD group,and the immunoreactivity and the expression levels of typeⅠcollagen precursor and fibronectin were significantly reduced in PDL+EmD group(P<0.05).Compared with PDL group,ALP activity,the cell proliferation,migration and healing abilities were significantly increased in PDL+ECM group(P<0.01).Compared with PDL+ECM group,the ALP-positive colony activity,and the cell proliferation,migration and healing abilities were significantly reduced in PDL+ECM+EmD group(P<

关 键 词：Rho蛋白激酶 细胞外基质 牙周膜细胞 成骨分化 盐酸土根碱 

分 类 号：R781.42[医药卫生—口腔医学]