Wnt/β-catenin信号通路在低氧促进hBMSCs体外增殖中的作用

Roles of Wnt/β-catenin Signaling in the Proliferation of Human Bone Marrow Mesenchymal Stem Cells Improved by Hypoxia in Vitro

目的探讨持续低氧环境对人骨髓间充质干细胞(human bone marrow mesenchymal stem cells,h BMSCs)体外增殖的影响,并初步探讨Wnt/β-catenin信号通路在其中的作用。方法将购自江阴齐氏生物科技有限公司第2代h BMSCs进行传代、纯化,取第4代h BMSCs进行流式细胞仪检测鉴定;根据培养条件不同分为常氧组(20%氧浓度)和低氧组(3%氧浓度);CCK-8法测定各组生长曲线;采用RT-PCR检测Wnt/β-catenin信号通路关键组件β-catenin、cyclin D1 m RNA表达水平;Western blot检测β-catenin、P-β-catenin蛋白表达水平。结果第4代h BMSCs流式鉴定结果显示:骨髓基质标志CD90表达为98.7%,而造血细胞标志CD19表达为0.3%;与常氧组比较,低氧组增殖速率更快,β-catenin、cyclin D1 m RNA表达升高(P<0.05),β-catenin蛋白表达升高(P<0.05),P-β-catenin蛋白表达下降(P<0.05)。结论持续低氧环境可能通过激活Wnt/β-catenin信号通路而促进h BMSCs体外增殖。

Objective To investgate the effect of hypoxia on the proliferation of human bone marrow mesenchymal stem cells （hBMSCs）, and then explore the role of Wnt/β-catenin signaling in this process. Methods hMSCs （passage 2） were purchased from Cyagen Biosciences Inc and used to identify by flow cytometry at passage 4. hBMSCs at passage 4 were divided into normal oxygen group and hypoxia group. The growth curves of hBMSCs at passage 4 in different groups were assayed by cell counting kit 8 （CCK-8）. RT-PCR assay was used to detect the expression of several key components of Wnt/β-catenin signaling including β-catenin, cyclinD 1 at mRNA levels. Western blot assay was used to detect the expression of β-catenin and P-β-catenin. Results The identification results of flow cytometry revealed that the expression of CD90 as the markers of bone marrow stromal, was 98,7%. While the expression of CD19, as the marker of hematopoietic cell, was 0.3%. Compared to the normal oxygen group, the mRNA level of β-catenin and cyclinD1 was increased （P〈0.05）, the protein level of β-catenin was increased （P〈0.05）, and P-β-catenin was decreased （P〈0.05）. Conclusion Wnt/β-catenin signaling plays a postive role in the proliferation of hBMSCs improved by hypoxia in vitro.