2型糖尿病大鼠骨髓间充质干细胞中Dickkopf-1蛋白的表达水平及其与成骨活性的关系  

Expression level of Dickkopf-1 protein in bone mesenchymal stem cells in Type 2 diabetic rats and its relationship with osteogenic activity

作　　者：李嫣 刘志涛[1] 仝昭宸 黄河清 刘振东[1] LI Yan, LIU Zhitao, TONG Zhaochen, HUANG Heqing, LIU Zhendong(Department of Orthopedics, Third Xiangya Hospital, Central South University, Changsha 410013, Chin)

机构地区：[1]中南大学湘雅三医院骨科,长沙410013

出　　处：《中南大学学报（医学版）》2018年第9期971-981,共11页Journal of Central South University ：Medical Science

摘　　要：目的:检测2型糖尿病(Type 2 diabetesmellitus,T2DM)大鼠骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs)生长活性及成骨分化能力,以及Dickkopf-1(DKK-1)蛋白在BMSCs诱导成骨过程中的表达水平,探讨T2DM对BMSCs成骨活性的影响及其与DKK-1表达的关系。方法:提取并分离T2DM造模大鼠BMSCs,进行体外培养与成骨诱导,并将其分为T2DM组和正常对照组;采用细胞计数试剂盒检测BMSCs增殖活性;采用膜联蛋白V(annexin V)-异硫氰酸荧光素(fl uorescein isothiocyanate,FITC)/碘化吡啶(propidium iodide,PI)双染法检测细胞凋亡率;采用碱性磷酸酶(alkaline phosphatase,ALP)染色及ALP活性检测试剂盒检测BMSCs中ALP表达水平;采用茜素红染色与矿化结节定量法分析BMSCs成骨分化程度;采用实时荧光定量PCR(quantitative real time PCR,qRT-PCR)法检测大鼠BMSCs中核心结合因子a1(core binding factor alphal 1,Runx2)和DKK-1的表达。结果:T2DM组大鼠BMSCs增殖活性比正常对照组弱,细胞凋亡增加(均P<0.01);BMSCs成骨诱导过程中,T2DM组大鼠ALP表达量比正常对照组显著降低,且钙结节形成减少,Runx2表达下调(均P<0.01);T2DM组BMSCs中DKK-1蛋白和mRNA表达上调,高于正常对照组(均P<0.01);DKK-1蛋白和mRNA表达与Runx2的表达升高有关(均P<0.01)。结论:T2DM造模大鼠BMSCs的生长活性及成骨分化潜能受损,这可能与BMSCs中DKK-1表达升高有关。Objective： To examine the growth activity and osteogenic diff erentiation of bone mesenchymal stem cells （BMSCs） in rats with Type 2 diabetes mellitus （T2DM） as well as the expression level of Dickkopf-1 （DKK-1） in bone marrow, and to explore the relationship between the osteogenic activity of BMSCs and the expression of DKK-1. Methods： The BMSCs were isolated from T2DM rats and were cultured in vitro. The BMSCs were divided into a T2DM group and a control group. The proliferation of BMSCs was detected by cell counting kit-8 （CCK8）. Apoptosis rate was detected by annexin V- fluorescein isothiocyanate （FITC）/propidium iodide （PI） double staining. In the osteogenic induction phase, the expression level of alkaline phosphatase （ALP） in BMSCs was detected by ALP staining and ALP activity assay kit. The osteogenic differentiation of BMSCs was analyzed by alizarin red staining and mineralized nodule quantification. In addition, the expression of Runx2 and DKK-1 in BMSCs was detected by qRT-PCR. Results： Compared with the control group, the proliferation of BMSCs was decreased and the apoptosis was increased in the T2DM group （both P〈0.01）. In the osteogenic induction process of BMSCs, the expression of ALP significantly decreased, the formation of calcium nodules reduced, and the expression of osteoblast transcription factor Runx2 was down-regulated in the T2DM group compared with those in the control group （all P〈0.01）. The levels of DKK-1 protein and mRNA were up-regulated in the T2DM group, which were higher than those in the control group （both P〈0.01）. The levels of DKK-1 protein and mRNA were related to the increase of Runx2 （both P〈0.01）. Conclusion： The growth activity of BMSCs and the potential of osteogenic differentiation are attenuated in the T2DM rats, which may be related to the increase of DKK-1 expression in BMSCs.

关 键 词：2型糖尿病 骨髓间充质干细胞 Dickkopf-1蛋白 经典Wnt信号通路 成骨分化 

分 类 号：R587.1[医药卫生—内分泌]