无翅型MMTV整合位点家族蛋白通路在调控脂肪干细胞成骨及成软骨分化中的分子机制  

Molecular mechanism of Wnt pathway in regulating osteogenic and chondrogenic differentiation of adipose stem cells

作　　者：代万武 张波 刘俊佳 范世昌 梁春华 孙桂丽 揭旭日 杜勇军 李兴艳 Dai Wanwu;Zhang Bo;Liu Junjia;Fan Shichang;Liang Chunhua;Sun Guili;Jie Xuri;Du Yongjun;Li Xingyan(Department of Joint Surgery,the Third Affiliated Hospital of Guangxi Medical University,Nanning 530031 China;Traditional Chinese Medicine orthopedics,Ruikang Clinical Medical College,Guangxi University of Traditional Chinese Medicine,Nanning 530031,China)

机构地区：[1]广西医科大学第三附属医院关节外科,南宁530031 [2]广西中医药大学瑞康临床医学院中医骨科,南宁530031

出　　处：《中华实验外科杂志》2019年第9期1634-1637,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目(81760389);南宁市科学技术局项目(20173018-2).

摘　　要：目的探讨无翅型MMTV整合位点家族蛋白(Wnt)通路和SRY相关高迁移率族盒基因9(Sox9)、骨形态发生蛋白2(BMP-2)相互反馈调节在调控脂肪干细胞(ADSCs)成软骨和成骨分化中的作用机制.方法通过氯化锂(LiCl)、Dickkopf相关蛋白1(DKK-1)刺激ADSCs成软骨及成骨分化,检测早中晚后期成软骨和成骨分化的各项指标如Ⅱ型胶原蛋白(Collagen2a)、聚集蛋白聚糖(Aggrecan)、核心结合因子α1(Runx2)、无翅型MMTV整合位点家族蛋白成员5A(Wnt5a)及Wnt通路关键蛋白Sox9、BMP-2、β-连环蛋白(β-catenin)、糖原合酶激酶-3β(GSK-3β)表达情况.随后用慢病毒作为载体,将全长基因序列Sox9和BMP-2基因分别转染进ADSCs使其稳定表达后再次检测相关各项指标的表达水平.应用SPSS 20.0统计软件进行分析,并探讨分子机制.结果ADSCs成软骨诱导分化过程中,Wnt通路早中晚期促进其快速增殖并诱导其成软骨分化.Collagen 2a、Aggrecan的表达早期增高不明显,中晚期明显增高(t=1.484、0.792、8.598、8.788、3.333、8.285,P<0.05);后期则抑制成熟软骨表型并促进软骨肥大和早期软骨骨化;过表达则反馈抑制Wnt通路活性.Sox9表达早中期上调晚期下调(0.14±0.03比0.45±0.09、0.33±0.15比0.84±0.05、0.27±0.02比0.58±0.06,t=5.897,7.726,12.175,P<0.01).成骨诱导分化过程中,Wnt通路前期促进其快速增殖并诱导其成骨分化,Runx2、Wnt5a的表达与对照组比较早期无明显差异(t=0.543、1.755,P>0.05),中晚后期Runx2表达均明显增强(t=13.452、12.148、16.535,P<0.05),而Wnt5a表达均下降(t=-4.017、-19.901、-7.181,P<0.05);过表达则反馈激活Wnt通路,上调β-catenin(0.26±0.08比0.64±0.05,t=8.789,P<0.05),下调Wnt5a(1.35±0.23比0.51±0.26,t=-5.233,P<0.05).BMP-2表达在两组各时期均上调(0.26±0.03比0.64±0.05、0.47±0.03比1.15±0.15、0.63±0.11比1.31±0.09,t=16.340、8.750、29.721,P<0.01).结论Wnt通路分别通过调控Sox9和BMP-2来调节ADSCs成软骨及成骨分�Objective To explore the mechanism of Wingless-type MMTV integration site family(Wnt)pathway and SRY-related high mobility group-box gene 9(Sox9)and Bone morphogenetic protein 2(BMP-2)feedback regulation in regulating chondrogenic and osteogenic differentiation of adipose-derived stem cells(ADSCs).Methods The cartilage and osteogenic differentiation of ADSCs were stimulated by Lithium Chloride(LiCl)and Dickkopf related protein 1(DKK-1).The markers of cartilage and osteogenic differentiation Collagen2a,Aggrecan,Core binding factor alphal 1(Runx2),wingless-type MMTV integration site family,member 5A(Wnt5a)and Wnt pathway key proteins Sox9,BMP-2,β-catenin,Glycogen synthase kinase-3β(GSK-3β)were detected in early,middle and late stages.The full-length gene sequences of Sox9 and BMP-2 were transfected into ADSCs by lentivirus,respectively,then the expression levels of various indexes were detected again while stably expressed.Apply SPSS 20.0 statistical software for analysis and explore molecular mechanisms.Results During the cartilage differentiation of ADSCs,the Wnt pathway promotes its rapid proliferation and induces chondrogenic differentiation in the early and middle stages.The expression of Collagen 2a and Aggrecan was not significantly increased in the early,while significantly increased in the middle and late stages(t=1.484,0.792,8.598,8.788,3.333,8.285,P<0.05).At the later stage,the mature cartilage phenotype was inhibited and promote its hypertrophy and early cartilage ossification.Overexpression feedback inhibition of Wnt pathway activity.Sox9 expression was up-regulated in the early and middle stages,down-regulated in the late stage(0.14±0.03 vs.0.45±0.09;0.33±0.15 vs.0.84±0.05;0.27±0.02 vs.0.58±0.06,t=5.897,7.726,12.175,P<0.01).During osteogenic differentiation of ADSCs,Wnt pathway promoted its rapid proliferation and induced osteogenic differentiation in the early stage.The expression of Runx2 and Wnt5a was not significantly different from that of the control group(t=0.543,1.755,P>0.05).The expr

关 键 词：脂肪干组织 成软骨分化 成骨分化 无翅型MMTV整合位点家族蛋白/β-连环蛋白信号通路 

分 类 号：R684.3[医药卫生—骨科学]