糖基化终末产物介导的牙周膜干细胞成骨分化过程中Wnt经典信号通路相关作用机制的研究  

Effects of advanced glycation end products on the osteogenic influence of WNT classic pathway in human periodontal ligament stem cells

作　　者：崔晓霞[1] 杨琨[1] 伍燕[1] 邓超[1] 刘琪[1] 金岩 

机构地区：[1]遵义医学院附属口腔医院,贵州遵义563003 [2]第四军医大学口腔医院组织工程中心,陕西西安710032

出　　处：《牙体牙髓牙周病学杂志》2013年第9期564-568,580,共6页Chinese Journal of Conservative Dentistry

基　　金：国家自然基金(30725042;81020108019);贵州省省长资金(C_397);贵州省科技厅自然资金(C_393);遵义市科技资金(E_063)

摘　　要：目的:探讨人牙周膜干细胞(human periodontal stem cells,HPDLSCs)成骨分化过程中在糖基化终末产物(AGE-HSA)介导下Wnt经典通路β-catenin的变化。方法:体外组织块法和有限稀释法克隆化培养牙周膜干细胞,流式细胞仪进行干细胞表型分子鉴定后,根据不同刺激环境随机分为OS组(单纯成骨诱导对照组)、OSA组(成骨诱导液+10μg/mL AGEs)、OSDKK-1组(成骨诱导液+100μg/mL DKK-1)、OSA+DKK-1组(成骨诱导液+10μg/mL AGEs+100 ng/mL DKK-1)4组,并进行相应的诱导培养。诱导培养7 d后,碱性磷酸酶染色检测ALP活性;RT-PCR检测Wnt经典通路基因β-catenin mRNA以及成骨相关基因Runx2、ALP mRNA的表达变化情况;Western Blot检测细胞核蛋白β-catenin和细胞总蛋白Runx2的表达变化情况。结果:纯化的人牙周膜干细胞各表型分子CD29、CD90、CD146、stro-1均阳性表达,ALP染色结果显示:OSA组颜色最浅,OSDKK-1组颜色最深,OSA+DKK-1组颜色介于两者之间;RT-PCR及Western Blot结果显示:与OS组相比,OSDKK-1组β-catenin的表达水平明显降低,Runx2、ALP的表达水平明显升高(P<0.05),表明DKK-1抑制了经典wnt通路,而使HPDLSCs的成骨能力增强;而OSA组β-catenin的表达水平明显升高,Runx2、ALP表达水平明显降低(P<0.05),表明激活了经典wnt通路,而使HPDLSCs的成骨能力下降;当联合加入AGEs和DKK-1(OSA+DKK-1组)时,成骨能力与OSDKK-1组相比明显降低,而与OSA组相比明显升高(P<0.05)。结论:在HPDLSCs成骨分化过程中,抑制经典wnt通路可以促进其骨向分化;而10 ng/mL的AGEs可通过激活wnt经典通路从而抑制其成骨分化。AIM： To investigate the effects of advanced glyeation end products （AGEs） on the osteogenic influence of WNT classic pathway in human periodontal ligament stem cells （HPDLSCs）. METHODS： HPDLSCs were isolated by limited dilution for cell cloning. Flow cytometery was employed to detect the cell surface markers. HP DLSCs were divided into 4 groups： OS group （only osteogenic induction）, OSA group （osteoinduction ＋ 10 p,g/mLAGEs）, OSDKK - 1 group （ osteoinduction ＋ 100 ng/mL DKK- 1 ） and OSA ＋ DKK- 1 group （ osteoinduction ＋ 10 ~g/ mL AGEs ＋ 100 ng/mL DKK-1 ）. The osteogenic differentiation capacity of HPDLSCs was evaluated by ALP staining, RT-PCR and Western Blot. RESULTS： hPDLSCs showed positive expression for mesenchymal stem cell markers of STRO-1, CD146 and CID0. After 7 days osteogenic induction, the expression of 13-catenin in HPDLSCs decreased and the expression of osteogenic marker （ALP, Runx-2） increased in OSDKK-1 group. However, the expression of 13 -catenin in HPDLSCs increased and the expression of osteogenic markers （ALP, Runx-2） decreased in OSA group. Moreover, the osteogenic markers in OSA ＋ DKK-1 group were higher than those in OSA group （P 〈 0.05） and lower than in OSDKK-1 group （P 〈 0.05）. CONCLUSION ： Inhibition of WNT pathway may promore the osteogenic dif ferentiation of HPDLSCs. AGEs can activate WNT pathway and inhibite the osteogenic differentiation of HPDLSCs.

关 键 词：糖基化终末产物 人牙周膜干细胞 骨向分化 Wnt经典通路 

分 类 号：R780.2[医药卫生—口腔医学]