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

邢玉洁; 祝领; 项羽; 马美娟; 徐晶; 王军奎; 马宏喜

doi:10.12125/j.chj.201905032

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

摘要

摘要:
目的探讨Wnt/β-catenin信号通路在大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells, 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24 h，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Western blot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。Western blot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。

Abstract:
AIM To investigate the function of 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 5-aza group (angiotensin II, final concentration 0.1 μmol/L + 5-azacytidine, final concentration 10 μmol/L) and 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 respectively detected by inverted phase contrast microscopy, MTT, flow cytometry, immunofluorescence staining and transmission electron microscopy. The expression level of Wnt and β-catenin was detected by Western blot.
RESULTS BMMSCs showed various morphologies in primary culture. After passage, BMMSCs grew in long shuttle shape and showed uniform growth after induction. MTT assay showed that the growth rate of Ang II combined with 5-aza group was faster than that in control group. Flow cytometry showed that the induction rate of cardiomyocytes in Ang II combined with 5-aza group was (31.2 ± 1.7)% and that in control group was (1.1 ± 0.2)%. Immunofluorescence staining showed that after induction BMMSCs expressed α-actin positively in Ang II combined with 5-aza group. Myofilament and gap junction were seen by transmission electron microscopy. Western blot results showed that the expression levels of Wnt and β-catenin in Ang II combined with 5-aza group were significantly higher than those in 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.

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