miR-145-5p在正常大鼠与2型糖尿病大鼠骨髓间充质干细胞成骨分化中的表达比较  

Comparison of expression of miR-145-5p in osteogenic differentiation of bone marrow mesenchymal stem cells derived from normal versus type 2 diabetic rats

作　　者：戴雅文 鄂玲玲[2] 郑颖[1,2] 马小草 张戎 时权[2] 刘洪臣[2] DAI Yawen;E Lingling;ZHENG Ying;MA Xiaocao;ZHANG Rong;SHI Quan;LIU Hongchen(Graduate School,Chinese PLA General Hospital,Beijing 100853,China;Institute of Stomatology&Oral Maxilla Facial Key Laboratory,the First Medical Center,Chinese PLA General Hospital,Beijing 100853,China)

机构地区：[1]解放军总医院研究生院,北京100853 [2]解放军总医院第一医学中心口腔医学研究所、口腔颌面战创伤军队重点实验室,北京100853

出　　处：《解放军医学院学报》2023年第5期549-557,F0003,共10页Academic Journal of Chinese PLA Medical School

基　　金：国家自然科学基金项目(82170985,81930026)。

摘　　要：背景糖尿病会导致骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMMSCs)的成骨分化能力降低,miRNA在此过程中发挥重要作用,其中miR-145-5p对细胞成骨成软骨分化有重要的调节作用。然而miR-145-5p对糖尿病源BMMSCs成骨分化的影响尚不清楚。目的比较正常大鼠与2型糖尿病大鼠BMMSCs成骨分化能力,初步探讨在成骨分化中miR-145-5p及其靶基因SEMA3A和Wnt通路关键蛋白β-catenin表达的差异。方法12只GK大鼠采用高糖高脂饲料喂养构建2型糖尿病大鼠模型,12只Wistar大鼠常规饲养作为对照组。无菌条件下分离大鼠股骨,全骨髓培养法培养正常大鼠和2型糖尿病大鼠BMMSCs(分别对应WT-BMMSCs和GK-BMMSCs);CCK-8检测细胞增殖能力;结晶紫染色检测细胞集落形成能力;碱性磷酸酶染色及半定量分析检测碱性磷酸酶活性;茜素红染色检测矿化基质形成;qRT-PCR和Western blot检测成骨相关标志物、miRNA-145-5p、SEMA3A和β-catenin的表达。结果干细胞培养4~9 d时,GKBMMSCs的增殖水平低于WT-BMMSCs,差异有统计学意义(P<0.01);10 d时,其集落形成率显著低于WT-BMMSCs(P<0.01);成骨诱导7 d时,GK-BMMSCs碱性磷酸酶活性(P<0.001)、成骨相关标志物碱性磷酸酶基因(P<0.01)和蛋白(P<0.001)表达、骨钙素基因(P<0.001)和蛋白(P<0.001)表达均显著低于WT-BMMSCs,1型胶原蛋白(P<0.01)基因的表达显著低于WT-BMMSCs,而Runt相关转录因子2(P<0.001)蛋白表达显著高于WT-BMMSCs。在WT-BMMSCs成骨分化中,miR-145-5p表达下调,SEMA3A表达上调,而在GK-BMMSCs成骨分化中,miR-145-5p和SEMA3A表达均上调,β-catenin(P<0.001)在GK-BMMSCs中的表达显著降低。成骨诱导21 d时,WT-BMMSCs矿化基质染色较深。结论2型糖尿病大鼠BMMSCs细胞增殖、集落形成及成骨分化能力降低,推测在正常大鼠BMMSCs成骨分化中miR-145-5p起抑制作用,在2型糖尿病大鼠BMMSCs成骨分化中miR-145-5p起促进作用。Background Diabetes reduces the osteogenic differentiation ability of bone marrow mesenchymal stem cells(BMMSCs),and miRNAs are essential in this process,during which miR-145-5p plays a vital role in osteogenic and chondrogenic differentiation of cells.However,the effect of miR-145-5p on the osteogenic differentiation of diabetic BMMSCs remains unclear.Objective To compare the osteogenic differentiation ability of BMMSCs derived from normal and type 2 diabetic rats respectively,and preliminarily explore the differences in the expression of miR-145-5p,its potential target gene SEMA3A and the key proteinβ-catenin of Wnt pathway in osteogenic differentiation.Methods Twelve GK rats were fed on high-fat and high-sugar diet to establish type 2 diabetes rat model as the experimental group.Twelve Wistar rats were fed on standard diet and served as the control group.Rat femur was isolated under aseptic condition.BMMSCs derived from normal and type 2 diabetic rats(WT-BMMSCs and GK-BMMSCs)were cultured by whole bone marrow culture method.CCK-8 was used to detect the cell proliferation.Colony formation ability was detected by the crystal violet stain assay. Alkaline phosphatase activity was detected by alkaline phosphatase staining and semi-quantitative analysis. Alizarin red staining was used to detect the formation of the mineralized matrix. The expression levels of bone-related markers, miRNA-145-5p, SEMA3A and β-catenin were detected by qRT-PCR and Western blot. Results When the stem cells were cultured for 4-9 days, the proliferation of GK-BMMSCs was significantly lower than that of WT-BMMSCs (P<0.01). On day 10, colony formation rate was significantly lower than that of WT-BMMSCs (P<0.01). On day 7 of osteogenic induction, alkaline phosphatase activity of GK-BMMSCs (P<0.001), osteogenesis-related markers alkaline phosphatase (ALP) gene (P<0.01) and protein (P<0.001) expressions, osteocalcin (OCN) gene (P<0.001) and protein (P<0.001) expressions were significantly lower than those of WT-BMMSCs, type 1 collagen (COL1)

关 键 词：2型糖尿病 microRNA-145-5p 骨髓间充质干细胞 成骨分化 大鼠 

分 类 号：R587.1[医药卫生—内分泌]