人间充质干细胞外泌体通过p38丝裂素活化蛋白激酶信号通路促进小鼠胚胎成骨细胞前体增殖和成骨分化  

Human mesenchymal stem cell exosomes promote proliferation and osteogenic differentiation of mouse embryo osteoblast precursor cells via p38 mitogen-actived proteinkinase signal pathway

作　　者：王新强 吴良邦 章月红 顾增辉[1] Wang Xinqiang;Wu Liangbang;Zhang Yuehong;Gu Zenghui(Third Department of Orthopaedics,No.903 Hospital of Joint Logistics Support Force of Chinese PLA,Hangzhou 310004,Zhejiang,China)

机构地区：[1]中国人民解放军联勤保障部队第903医院骨三科,杭州310004

出　　处：《脊柱外科杂志》2021年第4期253-258,288,共7页Journal of Spinal Surgery

基　　金：浙江省医药卫生科技计划项目(2019329727)。

摘　　要：目的观察人间充质干细胞(hMSC)分泌的外泌体对小鼠胚胎成骨细胞前体细胞(MC3T3-E1细胞)增殖和成骨分化的作用,并基于p38丝裂素活化蛋白激酶(p38MAPK)信号通路探讨相关分子机制。方法采用流式细胞术检测hMSC表面标志物;以成骨诱导液干预hMSC,通过茜素红染色检测钙化结节以反映其成骨能力;采用差速离心法提取hMSC外泌体,通过电镜观察检测外泌体粒径。将MC3T3-E1细胞分为4组,分别给予外泌体干预(外泌体组)、p38MAPK抑制剂SB203580干预(抑制剂组)、SB203580干预6 h后再加入外泌体干预(抑制剂+外泌体组),以未干预组作为对照组。通过蛋白质印迹法和实时荧光定量PCR检测各组细胞中Ⅰ型胶原蛋白alpha1(COL1A1)、ruh相关转录因子2(Runx2)、碱性磷酸酶(Alp)、骨桥蛋白(Opn)的蛋白和mRNA表达量,蛋白质印迹法检测p38MAPK和磷酸化p38MAPK(p-p38MAPK)的表达量,MTT法检测细胞增殖情况,茜素红染色检测细胞成骨能力,采用Alp试剂盒检测细胞上清液中Alp活力。结果 hMSC表面CD90和CD105呈阳性表达,CD34和CD45呈阴性表达;hMSC具有成骨分化能力;分离的外泌体粒径为40 ~ 160 nm。与对照组比较,外泌体组MC3T3-E1细胞增殖能力,成骨能力,Alp活力,细胞中COL1A1、Runx2、Alp、Opn的蛋白和mRNA表达量,p-p38/p38MAPK蛋白表达量比值均增高,差异有统计学意义(P < 0.05);抑制剂组结果与外泌体组相反,与对照组比较,差异均有统计学意义(P < 0.05);与抑制剂组比较,抑制剂+外泌体组MC3T3-E1细胞增殖能力、矿化结节、Alp活力无明显变化,细胞中COL1A1、Runx2、Alp、Opn的蛋白和mRNA表达量及p-p38/p38MAPK蛋白的表达量比值均无明显变化,差异无统计学意义(P > 0.05)。结论 hMSC分泌的外泌体可通过p38MAPK信号通路促进MC3T3-E1细胞的增殖和成骨分化。Objective To observe the role of exosomes secreted by human mesenchymal stem cells(hMSCs)on proliferation and osteogenic differentiation of mouse embryo osteoblast precursor cells(MC3T3-E1 cells),and to investigate the molecular mechanism based on the p38 mitogen-actived proteinkinase(p38MAPK)signaling pathway.Methods The surface markers of hMSCs were detected by flow cytometry.hMSCs were intervened with osteogenic induction solution,and the calcified nodules were detected by Alizarin Red staining to reflect their osteogenic ability.The exosomes of hMSCs were extracted by differential centrifugation,and the size of exosomes was detected by electron microscopy.The MC3T3-E1 cells were divided into 4 groups,which were treated respectively with exosome(exosome group),p38MAPK inhibitor SB203580(inhibitor group),SB203580 for 6 h and then treated with exosome(inhibitor+exosome group),and no intervention(control group).The protein and mRNA expression of collagenⅠα1(COL1A1),Runx2,Alp and Opn was detected by Western blotting and real-time quantitative PCR.Protein expression of p38MAPK and phospho-p38MAPK(p-p38MAPK)was detected by Western blotting.Cell proliferation was detected by MTT method.Cell osteogenic ability was detected by Alizarin Red staining.Alp activity in cell supernatant was detected by Alp kit.Results Among the surface markers of hMSCs,CD90 and CD105 were positively expressed,and CD34 and CD45 were negatively expressed.hMSCs had osteogenic differentiation ability.The size of the isolated exosomes from hMSCs was 40-160 nm.Compared with the control group,the proliferation ability,osteogenic ability,ALP activity,the protein and mRNA expression levels of COL1A1,Runx2,Alp and Opn,and the ratio of p-p38/p38MAPK protein expression level in the exosomes group increased significantly,and the differences were statistically significant(P<0.05).While the results of the inhibitor group were opposite,and compared with the control group,the differences were statistically significant(P<0.05).Compared with the inhibitor g

关 键 词：间质干细胞 成骨细胞 旁分泌细胞细胞间通信 细胞增殖 细胞分化 

分 类 号：R683.2[医药卫生—骨科学]