2型糖尿病伴牙周炎牙周膜干细胞成牙骨质分化能力的研究

The study on osteogenic differentiation ability of periodontal ligament stem cells in type 2 diabetes mellitus with periodontitis

目的探讨2型糖尿病伴牙周炎患者的人牙周膜干细胞(Periodontal ligament stem cells,PDLSCs)成牙骨质(Cementogenic)分化能力的变化。方法组织块法与单克隆法分离培养扩增健康来源牙周膜干细胞(H-PDLSCs)和2型糖尿病伴牙周炎来源的牙周膜干细胞(DP-PDLSCs);流式细胞仪鉴定其细胞表面分子CD45、CD90、CD44、CD29、CD166;CCK-8检测两组细胞7d增殖能力;ALP染色检测两组细胞成牙骨质诱导14d后矿化形成能力;两组细胞成牙骨质诱导7d后,RT-PCR检测ALP、OPN、OCN、CEMP1、CAP mRNA基因表达;两组细胞成牙骨质诱导14d后,Western blot检测OCN、CEMP1、CAP蛋白表达。结果H-PDLSCs组细胞表面分子CD45、CD90、CD44、CD29、CD166检测结果分别为0.02%、99.94%、99.59%、100.00%、99.99%,证明实验用细胞为外胚间充质来源;CCK-8检测两组细胞发现H-PDLSCs增殖能力高于DP-PDLSCs,差异具有统计学意义(P<0.05);RT-PCR结果显示,DP-PDLSCs ALP、OPN、OCN、CEMP1、CAP mRNA的表达量均低于H-PDLSCs;Western blot检测结果趋势与RT-PCR结果相一致,成牙骨质诱导14d OCN、CEMP1、CAP蛋白表达量DP-HPDLSCs低于H-PDLSCs。结论推测在2型糖尿病伴牙周炎疾病状态下,牙周膜干细胞的成牙骨质分化能力降低,可能源于全身因素和局部炎症因素的影响。

Objective To explore the changes in the differentiation ability of human periodontal membrane stem cells in patients with type 2 diabetes mellitus and periodontitis.Methods Tissue block method and single cloning method were used to isolate and culture the periodontal stem cells(H-PDLSCs)from healthy source and the periodontal stem cells(DP-PDLSCs)from type 2 diabetes mellitus with periodontitis.The cell surface molecules,such as CD45,CD90,CD44,CD29 and CD166 were identified by flow cytometry.CCK-8 was performed to detect the 7-day proliferation of the two groups of cells.ALP staining was used to detect the mineralization ability of the two groups after 14 days of induction.After 7 days of osteoblast induction,the expressions of ALP,OPN,OCN,CEMP1 and CAP mRNA were detected by RT-PCR.The expressions of OCN,CEMP1 and CAP proteins were detected by Western blot assay after 14 days of osteoblastation induction.Results H-PDLSCs were compared with DP-PDLSCs.The results of cell surface molecules were CD45(0.02%),CD90(99.94%),CD44(99.59%),CD29(100%)and CD166(99.99%).The proliferation capacity of H-PDLSCs was higher than that of DP-PDLSCs.RT-PCR results showed that the mRNA expression levels of ALP,OPN,OCN,CEMP1 and CAP in DP-PDLSCs were lower than those in H-PDLSCs.Western blotting results were consistent with RT-PCR results.The expression levels of OCN,CEMP1,and CAP proteins were lower in DP-PDLSCs than those in H-PDLSCs after 14 days of cementoblast induction.Conclusion It is speculated that in the state of type 2 diabetes with periodontitis,the reduction of the osteoblast differentiation ability of periodontal membrane stem cells might be caused by the influence of systemic factors and local inflammatory factors.