Wnt/β-catenin通路在大鼠骨髓间充质干细胞分化为心肌细胞过程中的作用  

Function of Wnt/β-catenin signaling pathway in inducing rat bone marrow mesenchymal stem cells to cardiomyocytes

作　　者：邢玉洁 祝领[1] 项羽 马美娟[1] 徐晶[1] 王军奎[1] 马宏喜 XING Yu-jie;ZHU Ling;XIANG Yu;MA Mei-juan;XU Jing;WANG Jun-kun;MA Hong-xi(Department of Cardiology, Shaanxi Provincial People’s Hospital, Xi’an, Shaanxi 710068, China;Department of Cardiology, Hospital of Luochuan County, Yan’an 727400, Shaanxi, China)

机构地区：[1]陕西省人民医院心内一科,陕西西安710068 [2]洛川县医院心内科,陕西延安727400

出　　处：《心脏杂志》2019年第5期501-505,共5页Chinese Heart Journal

基　　金：陕西省自然科学基础研究计划项目资助(2018JQ8076);陕西省人民医院科技发展孵化基金资助(2018YXQ-01)

摘　　要：目的 探讨Wnt/β-catenin信号通路在大鼠骨髓间充质干细胞(bonemarrowmesenchymalstemcells,BMMSCs)分化为心肌细胞过程中的作用。方法 选BMMSCs为种子细胞培养到第3代,分为血管紧张素(Ang)Ⅱ+5-氮杂胞苷(5-aza)组及对照组,共诱导24h,再用完全培养液共培养4周。用倒置显微镜、MTT法检测、流式细胞、免疫荧光染色、透射电镜依次检测细胞的形态变化、生长能力、诱导分化率、α肌动蛋白(α-actin)的表达以及超微结构,用Westernblot法检测诱导后Wnt及其下游分子β-catenin的表达水平。结果 BMMSCs原代培养时呈现多种形态,经过传代体积增大,诱导后呈长梭形且均匀一致生长。MTT结果表明AngⅡ+5-aza组细胞的生长速度比对照组快。流式细胞检测示:AngⅡ+5-aza组的心肌细胞诱导率是(31.2±1.7)%,而对照组是(1.1±0.2)%。免疫荧光染色结果示AngⅡ+5-aza组诱导后的BMMSCs阳性表达α-actin。透射电镜观察可见肌丝和缝隙连接。Westernblot提示AngⅡ+5-aza组的Wnt以及β-catenin的表达明显高于对照组(P<0.05)。结论 AngⅡ+5-aza在诱导BMMSCs向心肌细胞的分化中有促进作用,其可能与Wnt/β-catenin信号通路的激活有关。AIM To investigate the function of the Wnt/β-catenin pathway in inducing rat bone marrow mesenchymal stem cells(BMMSCs) to cardiomyocytes. METHODS Third generation BMMSCs were divided into Ang Ⅱ combined with a 5-aza group(angiotensin Ⅱ, final concentration 0.1 μmol/L+5-azacytidine, final concentration 10 μmol/L) and a control group. After induction for 24 hours, the induction medium was abandoned and the cells were cultured in complete medium for 4 weeks. Morphological changes, proliferation ability, induction differentiation rate, expression of α-actin, and ultrastructure were detected by inverted phase contrast microscopy, MTT, flow cytometry, immunofluorescence staining and transmission electron microscopy. The expression levels of Wnt and β-catenin were detected by Western blot. RESULTS BMMSCs showed various morphologies in primary culture. After passage, BMMSCs grew in long shuttle shape and demonstrated uniform growth after induction. MTT assay showed that the growth rate of Ang Ⅱ combined with a 5-aza group was faster than that in the control group. Flow cytometry demonstrated that the induction rate of cardiomyocytes in Ang Ⅱ combined with 5-aza group was(31.2±1.7)% compared with to the control group(1.1±0.2)%. Immunofluorescence staining showed that after induction BMMSCs expressed α-actin positively in Ang Ⅱ combined with the 5-aza group. Myofilaments and gap junctions were seen by transmission electron microscopy. Western blot results demonstrated that the expression levels of Wnt and β-catenin in Ang Ⅱ combined with the 5-aza group were significantly higher than those in the control group(P<0.05). CONCLUSION Ang II combined with 5-aza can promote the differentiation of BMMSCs into cardiomyocytes, which may be related to the activation of Wnt/β-catenin signaling pathway.

关 键 词：WNT/Β-CATENIN信号通路 骨髓间充质干细胞 血管紧张素Ⅱ 5-氮杂胞苷 心肌细胞 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]