骨髓间充质干细胞外泌体微小RNA-200a通过Wnt/β-连环蛋白通路抑制肾小管上皮细胞上皮-间充质转化  

Exosomes microRNA-200a secreted by bone marrow mesenchymal stem cells suppress epithelial-mesenchymal transition of renal tubular epithelial cells via Wnt/β-catenin signaling pathway

作　　者：孙雄林[1] 林婷婷[1] 陈少豪 董汝男 朱君明 陈航 陈佳茵 林云知 蔡海[1] 黄金杯[1] 郑清水[1] 许宁[1] 陈烨辉 Sun Xionglin;Lin Tingting;Chen Shaohao;Dong Runan;Zhu Junming;Chen Hang;Chen Jiayin;Lin Yunzhi;Cai Hai;Huang Jinbei;Zheng Qingshui;Xu Ning;Chen Yehui(Departments of Urology,Urology Research Institute,the First Affiliated Hospital of Fujian Medical University,Fuzhou 350005,China)

机构地区：[1]福建医科大学附属第一医院泌尿外科福建医科大学泌尿外科教研室,福州350005

出　　处：《中华实验外科杂志》2020年第12期2207-2210,共4页Chinese Journal of Experimental Surgery

基　　金：福建省自然科学基金(2018J01172);福建医科大学启航基金(2017XQ1063)。

摘　　要：目的探讨骨髓间充质干细胞(BMMSCs)来源外泌体miR-200a对肾小管间质纤维化的影响及其机制。方法2019年3月至2020年7月,收集从4周龄SD大鼠(福建医科大学动物实验中心)提取的BMMSCs来源外泌体,与从新西兰兔提取的肾小管上皮细胞共培养,提取BMMSCs来源外泌体,与肾小管上皮细胞共培养,蛋白免疫印迹(Western blot)检测上皮-间充质转化(EMT)标志物E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)和波形蛋白(Vimentin)蛋白的相对表达水平,明确外泌体对EMT的影响。构建过表达和低表达miR-200a的BMMSCs,分别与肾小管上皮细胞共培养,Western blot检测E-cadherin、N-cadherin和Vimentin蛋白及Wnt/β-连环蛋白(β-catenin)信号通路各标志蛋白的表达水平。双荧光素酶报告实验验证肾小管上皮细胞中miR-200a与β-catenin基因CTNNB1是否直接结合。使用方差分析和独立样本t检验分析组间差异。结果高剂量外泌体组E-cadherin表达水平较低剂量组更低(0.51±0.09比0.78±0.12,t=3.115,P<0.05),而N-cadherin(2.05±0.22比1.40±0.13,t=6.478,P<0.001)和Vimentin(1.01±0.13比0.75±0.12,t=2.987,P<0.05)表达水平高于后者。高表达miR-200a外泌体处理组E-cadherin表达水平较低表达miR-200a外泌体处理组降低(0.71±0.09比1.48±0.14,t=4.751,P<0.05),而N-cadherin(1.55±0.12比0.75±0.10,t=11.603,P<0.01)和Vimentin(0.88±0.07比0.37±0.04,t=6.039,P<0.05)表达水平前者较后者更高。Wnt/β-catenin信号通路标志蛋白糖原合成酶激酶3β(GSK3β)表达量在组间差异无统计学意义(0.89±0.21比0.92±0.15,t=0.130,P>0.05),而低表达miR-200a外泌体处理组p-GSK3β、β-catenin和T细胞因子-1(TCF-1)表达水平均高于高表达miR-200a外泌体处理组(1.89±0.17比1.15±0.12,t=4.125,P<0.05;1.32±0.09比0.74±0.15,t=4.962,P<0.05;0.96±0.08比0.38±0.05,t=6.740,P<0.05)。双荧光素酶报告实验显示,野生型β-CTNNB1肾小管上皮细胞中,miR-200a组细胞荧光活性显著低于miR-NC组(0.51±0.Objective To investigate the effects and mechanism of exosomes microRNA(miRNA,miR)-200a secreted by bone marrow mesenchymal stem cells(BM-MSCs)on epithelial-mesenchymal transition(EMT)of renal tubular epithelial cells.Methods This study was conducted from March 2019 to July 2020.Exosomes from BMMSCs,which were seperated from SD rats,were extracted and were co-cultured with renal tubular epithelial cells,which were seperated from New Zealand rabbits.Exosomes from BM-MSCs were extracted and were co-cultured with renal tubular epithelial cells.The expression levels of E-cadherin,N-cadherin and Vimentin,which were biomarkers of EMT were detected by Western blotting to determine the effect of exosomes on EMT.BM-MSCs with high and low expression level of miR-200a were constructed and were co-cultured with renal tubular epithelial cells,respectively.The expression levels of E-cadherin,N-cadherin,Vimentin,and Wnt/β-catenin signaling pathway markers were detected using Western blotting.Dual-luciferase reporter assay was performed to verify whether miR-200a combined with CTNNB1 directly.ANOVA and Student t test were used for statistical analysis.Results The expression level of E-cadherin in high-dose exosomes group was lower than that in low-dose exosomes group(0.51±0.09 vs.0.78±0.12,t=3.115,P<0.05);while the expression levels of N-cadherin(2.05±0.22 vs.1.40±0.13,t=6.478,P<0.01)and Vimentin(1.01±0.13 vs.0.75±0.12,t=2.987,P<0.05)were higher than those in the latter.The expression level of E-cadherin in the group treated with exosomes over-expressing miR-200a was lower than that in the group treated with exosomes,in which miR-200a was silenced(0.71±0.09 vs.1.48±0.14,t=4.751,P<0.05);while the expression levels of N-cadherin(1.55±0.12 vs.0.75±0.10,t=11.603,P<0.01)and Vimentin(0.88±0.07 vs.0.37±0.04,t=6.039,P<0.05)in the former one were lower than those in the latter one.The expression level of glycogen synthase kinase 3β(GSK3β)had no significant difference between two groups(0.89±0.21 vs.0.92±0.15,t=0.130,P>0.

关 键 词：肾间质纤维化 上皮-间充质转化 骨髓间充质干细胞 微小RNA-200a Wnt/β-连环蛋白通路 

分 类 号：R692.6[医药卫生—泌尿科学]