糖基化终末产物对人牙周膜干细胞骨向分化过程中的Wnt经典信号通路研究

doi:10.7518/hxkq.2015.06.016

华西口腔医学杂志 ›› 2015, Vol. 33 ›› Issue (6): 627-632.doi: 10.7518/hxkq.2015.06.016

糖基化终末产物对人牙周膜干细胞骨向分化过程中的Wnt经典信号通路研究

伍燕^1,2,邓超¹,杨琨¹,崔晓霞¹,刘琪¹,金岩³

1.遵义医学院附属口腔医院，遵义 563003；2.贵阳市口腔医院牙周科，贵阳 550002；3.第四军医大学组织工程中心，西安 710032

收稿日期:2015-05-11 修回日期:2015-09-08 出版日期:2015-12-01 发布日期:2015-12-01
通讯作者: 刘琪，教授，博士，E-mail：liuqi1964@hotmail.com
作者简介:伍燕，主治医师，硕士，E-mail：330796865@qq.com
基金资助:
国家自然科学基金资助项目（81360168）

Canonical Wnt signaling pathway of the osteogenic differentiation of human periodontal ligament stem cells induced by advanced glycation end products

Wu Yan^{1, 2}, Deng Chao¹, Yang Kun¹, Cui Xiaoxia¹, Liu Qi¹, Jin Yan³

1. Stomatological Hospital Affiliated to Zunyi Medical College, Zunyi 563003, China; 2. Dept. of Periodontology, Guiyang Stomatological Hospital, Guiyang 550002, China; 3. Tissue Engineering Center, The Fourth Military Medical University, Xi’an 710032, China)

Received:2015-05-11 Revised:2015-09-08 Online:2015-12-01 Published:2015-12-01

摘要/Abstract

摘要： 目的探讨Wnt经典通路相关因子Dickkopf蛋白1（DKK-1）、β-链蛋白（β-catenin）在糖基化终末产物（AGEs）介导的人牙周膜干细胞（hPDLSCs）骨分化过程中的变化。方法通过体外组织块法和有限稀释法克隆化培养获取hPDLSCs，实验分两组：正常hPDLSCs组（N-hPDLSCs组）和AGEs刺激的正常hPDLSCs组（A-hPDLSCs组）。对2组细胞成骨矿化诱导后行碱性磷酸酶（ALP）和茜素红染色；实时聚合酶链反应（real time PCR）检测成骨基因，以及Wnt经典通路中相关因子DKK-1、β-catenin；Western blot检测相关成骨蛋白、细胞核蛋白β-catenin。结果成骨诱导后，A-hPDLSCs组ALP染色明显浅于N-hPDLSCs组；茜素红染色A-hPDLSCs组钙化结节少于N-hPDLSCs组；real time PCR与Western blot的结果显示A-hPDLSCs组BSP、ALP、Runx-2的表达都较低，差异有统计学意义（P<0.05）。Wnt经典信号通路中相关因子：A-hPDLSCs组β-catenin表达高于N-hPDLSCs组，A-hPDLSCs组DKK-1表达明显低于N-hPDLSCs组，并且Weston blot显示A-hPDLSCs组核蛋白β-catenin的表达高于N-hPDLSCs组。结论AGEs可以使hPDLSCs骨向分化过程中Wnt经典通路的抑制因子DKK-1下调，细胞核中的β-catenin表达升高，激活了Wnt经典通路，并且抑制了骨分化。

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

Abstract: Objective The effect of advanced glycation end products (AGEs) on the osteogenic differentiation of human periodontal ligament stem cells(hPDLSCs) was discussed. Changes in the Wnt signaling pathway during glycation were also determined. Methods In vitro tissue explanting method was primarily applied. Limiting diluted clone was cultured to obtain hPDLSCs in vitro. The subjects were divided into two groups: the healthy group (N-hPDLSCs) and the AGEs-stimulating group (A-hPDLSCs). Osteoblast mineralization was induced in the experimental groups. The following processes were performed: alizarin red staining; alkaline phosphatase (ALP) staining; real time polymerase chain reaction (real time PCR) for detecting osteogenic genes and Wnt classical pathway-related factors, DKK-1 and β-catenin; Western blot analysis. Bone protein and β-catenin were correlated in the nuclear expression. Results The cells were osteogenically induced. ALP staining showed that the N-hPDLSCs displayed the deepest color. Alizarin red staining indicated that the A-hPDLSCs group had less calcified nodules than the N-hPDLSCs group. The real time PCR results suggested that the expression of relative osteogenic genes in A-hPDLSCs was quite low. Statistically significant differences in differentiation were found between groups (P<0.05). The Western blot result was similar to that of real time PCR. Classical Wnt signaling pathway-related factor β-catenin was higher in A-hPDLSCs than in N-hPDLSCs. By contrast, DKK-1, which is an inhibitor in the Wnt pathway, had a significantly lower expression rate in A-hPDLSCs than in N-hPDLSCs. The Western blot result also showed that β-catenin expression in the nucleoprotein in A-hPDLSCs was notably higher than in N-hPDLSCs. Conclusion AGEs can inhibit hPDLSCs osteogenic differentiation. AGEs induce changes in the normal periodontal ligament stem cells classical Wnt pathway. Canonical Wnt pathway is reactivated because of AGEs stimulation.

Key words: advanced glycation end products, human periodontal ligament stem cells, osteogenic differentiation, classical Wnt pathway

中图分类号:

Q 257

伍燕,邓超,杨琨,崔晓霞,刘琪,金岩. 糖基化终末产物对人牙周膜干细胞骨向分化过程中的Wnt经典信号通路研究[J]. 华西口腔医学杂志, 2015, 33(6): 627-632.

Wu Yan, Deng Chao, Yang Kun, Cui Xiaoxia, Liu Qi, Jin Yan. Canonical Wnt signaling pathway of the osteogenic differentiation of human periodontal ligament stem cells induced by advanced glycation end products[J]. West China Journal of Stomatology, 2015, 33(6): 627-632.

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糖基化终末产物对人牙周膜干细胞骨向分化过程中的Wnt经典信号通路研究

Canonical Wnt signaling pathway of the osteogenic differentiation of human periodontal ligament stem cells induced by advanced glycation end products

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