低表达LATS1通过抑制Hippo信号通路促进间充质干细胞的分化增殖和迁移  

Under-expression of LATS1 promotes the differentiation, proliferation and migration of mesenchymal stem cells by inhibition the Hippo signaling pathwayin vitro

作　　者：李朗[1] 董亮[1] 惠姣洁[1] 高飞[1] 王秋卉[1] 杨岚[1] 张江茜 严洁[1] 

机构地区：[1]南京医科大学附属无锡人民医院重症医学科,江苏无锡214023

出　　处：《中华危重病急救医学》2017年第8期731-737,共7页Chinese Critical Care Medicine

基　　金：国家自然科学基金（81400054）;江苏省自然科学基金（BK20140122）;江苏省青年医学重点人才项目（QNRC2017179）

摘　　要：目的 探讨低表达大肿瘤抑制因子1（LATS1）基因抑制Hippo信号通路对小鼠骨髓来源间充质干细胞（mMSCs）分化、增殖和迁移能力的影响.方法 将C57BL/6小鼠mMSCs分为正常对照组（MSC组）、空载体对照组（MSC-GFP组）、过表达LATS1实验组（MSC-LATS1组）、空干扰病毒转染组（MSC-shControl组）、转染干扰LATS1病毒组（MSC-shLATS1组）,分别构建过表达或低表达LATS1的慢病毒载体及其空载体对照并转染mMSCs.用荧光显微镜和流式细胞仪评估慢病毒转染效率;用实时荧光定量聚合酶链反应（qRT-PCR）检测LATS1 mRNA表达,用蛋白质免疫印迹试验（Western Blot）检测LATS1、磷酸化YAP（p-YAP）、14-3-3蛋白表达,以明确LATS1对Hippo信号通路的调控作用;用茜素红、油红O染色及qRT-PCR检测成骨转录因子Runx2、OSX及成脂转录因子C/EBPα、PPAR-γ的mRNA表达,以明确Hippo信号通路对mMSCs成骨及成脂分化的影响;用四甲基偶氮唑盐（MTT）比色法检测mMSCs的数量,以评估Hippo信号通路对mMSCs增殖能力的影响;用划痕实验及Transwell小室迁移实验评估Hippo信号通路对mMSCs水平及垂直迁移能力的影响.结果 慢病毒载体转染mMSCs效率可达94.74%-96.10%.与MSC-GFP组比较,MSC-LATS1可激活Hippo信号通路〔LATS1 mRNA（2-ΔΔCT）：4.37±0.21比1.20±0.04,LATS1蛋白（灰度值）：2.21±0.06比1.09±0.10,p-YAP/YAP蛋白比值（灰度值）：1.51±0.13比0.98±0.05,14-3-3蛋白（灰度值）：1.92±0.18比1.10±0.09,均P〈0.05〕,抑制mMSCs成骨及成脂分化〔钙质沉积（A值）：0.13±0.02比0.40±0.03,Runx2 mRNA（2-ΔΔCT）：0.51±0.02比0.98±0.09,OSX mRNA（2-ΔΔCT）：0.41±0.04比1.04±0.09,脂质沉积（A值）：0.10±0.02比0.25±0.03,C/EBPαmRNA（2-ΔΔCT）：0.33±0.03比1.11±0.09,PPAR-γmRNA（2-ΔΔCT）：0.29±0.02比1.04±0.10,均P〈0.05〕,抑制4-7 d的MSC增殖及mMSCs水平和垂直迁移能力〔划痕弥合程度：（18.65±3.53）%比（40.29±1.87�Objective To explore the effects of under-expression of large tumor suppressor 1 （LATS1） on activation of Hippo signaling pathway and differentiation, proliferation, migration of bone marrow mesenchymal stem cells （mMSCs） of micein vitro.Methods mMSCs of C57BL/6 mice were divided into normal control （MSC） group, empty vector control （MSC-GFP） group, LATS1-over-expressing （MSC-LATS1） group, empty vector without LATS1 shRNA control （MSC-shControl） group and LATS1-under-expressing （MSC-shLATS1） group. Lentiviral vectors with activated,inactivated LATS1 （the key molecule of Hippo signaling pathway） modifications and empty vectors were constructed and were used to infect mMSCsin vitro. The transduction efficiencies mediated by the lentiviral vectors were evaluated by fluorescence microscopy and flow cytometry. The mRNA expression of LATS1 was quantified by quantitative real-time polymerase chain reaction （qRT-PCR）, and the protein expressions of LATS1, YAP （p-YAP）, 14-3-3 were quantified by Western Blot to evaluate the activation of Hippo signaling pathway. Osteogenic and adipogenic differentiation of mMSCs were evaluated through measurement of Runx2, OSX and C/EBPα, PPAR-γ mRNA by qRT-PCR, as well as Alizarin Red S and Oil red O staining. Proliferation of mMSCs was evaluated using methy thiazdyl tetrazolium （MTT） assay. The scratch test and Transwell chamber test were used to analyze the horizontal and vertical migration ability of mMSCs.Results The transduction efficiencies mediated by the lentiviral vectors were 94.74%-96.10%. Compared with MSC-GFP group, the activation of Hippo signaling pathway was promoted in MSC-LATS1 group [LATS1 mRNA （2-ΔΔCT）： 4.37±0.21 vs. 1.20±0.04, LATS1 protein （gray value）： 2.21±0.06 vs. 1.09±0.10, p-YAP/YAP protein （gray value）： 1.51±0.13 vs. 0.98±0.05, 14-3-3 protein （gray value）： 1.92±0.18 vs. 1.10±0.09, allP 〈 0.05], osteogenic and adipogenic differentiation of mMSCs were decreased in MSC-LATS1 group [m

关 键 词：LATS1基因 Hippo信号通路 间充质干细胞 分化 增殖 迁移 

分 类 号：R563.8[医药卫生—呼吸系统]