miR-26b调控Wnt/β-catenin信号通路促进成肝样分化MSCs迁移研究  

MiR-26b regulates Wnt/β-catenin signal pathway to promote MSCs migration in hepatoblastic differentiation

作　　者：吉杨丹[1] 罗红阳 王丹[1] 王恒[1] 杨小燕 何志旭[4,5] 刘金河 汪显耀[4,5] 赵锦 张丽娟 张玲敏[7] 秦臻 许键炜 Ji Yangdan;Luo Hongyang;Wang Dan(Dept of Pharmacology,Qiannan Medical College For Nationalities,Duyun 558013;People's Hospital of Wudang District,Guiyang 550018)

机构地区：[1]黔南民族医学高等专科学校药理学教研室,都匀558013 [2]贵阳市乌当区人民医院,贵阳550018 [3]贵州医科大学附属医院儿科,贵阳550004 [4]贵州医科大学细胞工程生物医药技术国家地方联合工程实验室,组织工程与干细胞实验中心,贵州省再生医学重点实验室,贵阳550004 [5]中国医学科学院成体干细胞转化研究重点实验,贵阳550004 [6]贵州医科大学护理学院,贵阳550004 [7]贵州医科大学基础医学院药理学教研室,贵阳550004

出　　处：《安徽医科大学学报》2020年第12期1850-1857,共8页Acta Universitatis Medicinalis Anhui

基　　金：中国医学科学院中央级公益性科研院所基本科研业务费专项资金(编号:2018PT31048);贵州省科技计划项目(编号:黔科合支撑[2017]2873);黔南州科技计划项目(编号:黔南科合社字[2017]58号);黔南民族医学高等专科学校科研基金项目(编号:QNYZ201814)。

摘　　要：目的探讨miR-26b与Wnt/β-catenin信号通路、间充质干细胞(MSCs)不同分化状态的关系及其在MSCs向肝细胞生长因子(HGF)趋化迁移过程中的作用。方法以大鼠骨髓来源MSCs作为研究对象,用无血清诱导分化培养基分别培养0、7、14、21d,建立成肝样分化不同阶段的MSCs分化模型,qRT-PCR检测miR-26b在不同分化状态MSCs细胞中的表达;重组腺病毒(Ad-26b)感染MSCs实现miR-26b的高表达,并以空病毒感染MSCs为对照(Ad)后,检测细胞中信号分子active-β-catenin(ABC)、p-β-catenin、β-catenin的表达量以及Wnt/β-catenin经典靶基因c-Myc、RUNX2的转录水平,明确其对MSCs铺展面积和黏着斑的影响以及通过Transwell、Dunnchamber实验,比较其对不同分化状态MSCs趋化迁移的影响。结果随着诱导分化时间的延长,MSCs的miR-26b表达呈上升趋势,14d时,miR-26b的表达至高峰,与对照组比较有统计学意义(P<0.05);Ad-26b组较Ad组ABC蛋白水平升高,p-β-catenin的蛋白水平下降,而β-catenin总蛋白无变化,Wnt/β-catenin信号下游靶基因c-Myc,RUNX2的表达上调。Ad-26b组高表达miR-26b后可促使MSCs铺展面积变小并呈现极性分布,MSCs细小黏着斑数目增加并向细胞前端周边分布。Transwell及Dunncham-ber实验表明MSCs诱导分化至7d,向HGF的趋化迁移能力高于诱导分化0、14和21d。高表达miR-26b组较Ad组细胞向HGF的趋化迁移能力较对照组增强(P<0.05)。结论miR-26b通过调节Wnt/β-catenin信号通路影响MSCs的分化和趋化迁移。Objective To investigate the relationship between miR-26 b and Wnt/β-catenin signal pathway, different differentiation states of mesenchymal stem cells(MSCs) and its role in the chemotaxis of MSCs to hepatocyte growth factor(HGF).Methods MSCs derived from rat bone marrow were cultured in serum-free induced differentiation medium for 0 d, 7 d, 14 d and 21 d, respectively. The models of MSCs differentiation at different stages of liver-like differentiation were established.The expression of miR-26 b in different differentiated MSCs cells was detected by qRT-PCR. The recombinant adenovirus(Ad-26 b) was used to infect MSCs to achieve high expression of miR-26 b, and the empty virus infected MSCs was used as the control(Ad), the expression of active-β-catenin(ABC), p-β-catenin, β-catenin and the transcription level of Wnt/β-catenin classical target genes c-Myc and RUNX2 were detected. The effects of Transwell and Dunn chamber on the spreading area and adhesion spot of MSCs were determined, and their effects on the chemotaxis and migration of MSCs in different differentiation states were compared.Results With the prolongation of differentiation time, the miR-26 b expression of MSCs increased, and the expression of miR-26 b reached the peak at 14 d after differentiation, and the difference was significant compared with the control group(P<0.05). After high expression of miR-26 b, the level of ABC protein increased,the protein level of p-β-catenin decreased,but the total protein of β-catenin did not change significantly. The expression of downstream target gene c-Myc and RUNX2 of Wnt/β-catenin signal was significantly up-regulated. High expression of mi R-26 b could made the spreading area of MSCs smaller and show polar distribution,and the number of fine adhesive plaques of MSCs increased significantly and distributed to the periphery of the cell front end. Transwell and Dunn chamber experiments showed that the chemotactic migration ability of MSCs to HGF in the induced differentiation group was significantly hi

关 键 词：间充质干细胞 miR-26b WNT/Β-CATENIN信号通路 成肝样分化 趋化迁移 

分 类 号：R34[医药卫生—基础医学] R575