摘要
目的:探讨转化生长因子-β(TGF-β)信号通路抑制剂SB431542在体外对人牙龈间充质干细胞(hGMSCs)增殖及成骨分化能力的影响。方法:组织块法原代培养hGMSCs,采用流式细胞术检测表面标志物,然后给予不同浓度(0、0.1、1、10μmol/L)SB435142处理,CCK-8及流式细胞仪分别检测增殖活性及凋亡比例;成骨培养基分组诱导培养21 d后,进行茜素红染色,检测成骨分化;hGMSCs给予1μmol/L SB431542处理,采用Western blot检测成骨诱导过程中的成骨相关蛋白的表达及TGF-β信号通路信号分子的变化。结果:原代培养的hGMSCs高表达CD105、CD90和CD73,而阴性表达CD14、CD34、CD19、CD45和人类白细胞DR抗原(HLA-DR)。与对照组相比,各处理浓度SB431542并不引起hGMSCs凋亡率的明显改变(P>0.05)。而10μmol/L SB431542作用于hGMSCs第7天时,细胞增殖能力较对照组降低(P<0.05)。1μmol/L SB431542能显著增加hGMSCs矿化结节的形成(P<0.05),且在成骨诱导11 d后,Runt相关转录因子2、碱性磷酸酶及Ⅰ型胶原蛋白表达明显高于对照组及空白组(P<0.05)。成骨诱导30、60 min时,SB431542处理组的TGF-β信号通路下游Smad3信号分子磷酸化水平明显被抑制(P<0.05)。结论:SB431542可能通过抑制成骨分化过程中Smad3的磷酸化水平来诱导hGMSCs体外成骨。
Objective:To explore the effects of SB431542,a TGF-βsignaling inhibitor,on the growing status and osteogenic differentiation of human gingival mesenchymal stem cells(hGMSCs)in vitro.Methods:The hGMSCs were isolated and cultured from clinically discarded gingival tissues and the surface markers were analyzed by flow cytometry.Then the hGMSCs were treated with various concentrations of SB435142(0,0.1,1,10μmol/L).hGMSCs proliferation and apoptosis were analyzed using CCK-8 assay and Apoptosis Detection Kit,respectively.Osteogenic differentiation of hGMSCs was investigated using Alizarin Red staining.In addition,osteoblastic differentiation-related protein and downstream molecules of TGF-βsignaling pathway in hGMSCs were measured by Western blot.Results:The hGMSCs were positive for CD105,CD90 and CD73,but negative for CD14,CD34,CD19,CD45 and HLA-DR.Apoptosis rates of SB431542 treated hGMSCs were not statistically different from that of control group(P>0.05).But the proliferation was inhibited in 10μmol/L SB431542 group,compared with control group at day 7(P<0.05).Comparing with control group,1μmol/L SB431542 treatment dramatically accelerated osteogenesis,increased COL-1,ALP and Runx2 protein expressions of hGMSCs(P<0.05).In addition,SB431542 treatment markedly inhibited the Smad3 phosphorylation in hGMSCs(P<0.05).Conclusions:1μmol/L SB431542 treatment could induce a robust osteogenic differentiation in hGMSCs in vitro,which may be regulated by inhibiting the phosphorylation level of Smad3 during osteogenic differentiation.
作者
石安源
鲍东昱
童昕
秦海燕
王斌
SHI Anyuan;BAO Dongyu;TONG Xin;WANG Bin;QIN Haiyan(Department of Dental Implantology,Nanjing Stomatological Hospital,Medical School of Nanjing University,Nanjing 210008,China;Clinical Stem Cell Center,The Affiliated Drum Tower Hospital of Nanjing University Medical School,Nanjing 210008,China)
出处
《口腔生物医学》
2019年第1期6-11,共6页
Oral Biomedicine
基金
国家自然科学基金(81200770
81571213)
