Wnt信号途径促进脂肪间充质干细胞向Ⅱ型肺泡上皮细胞分化

摘要

背景：脂肪间充质干细胞向Ⅱ型肺泡上皮细胞定向分化的能力以及调节机制尚未完全阐明。　　目的：观察脂肪间充质干细胞在体外分化为Ⅱ型肺泡上皮的能力以及W nt途径对分化的调节作用。　　方法：取大鼠脂肪组织，体外分离培养脂肪间充质干细胞并通过流式细胞术进行鉴定。实验分为对照组、小气道生长液组和Wnt3a组，对照组用普通DMEM培养基培养，小气道生长液组和Wnt3a组均使用小气道生长液培养，且Wnt3a组加入Wnt信号通路激动剂Wnt3a培养。诱导10 d后分别通过qRT-PCR和免疫荧光检测Ⅱ型肺泡上皮标志物肺表面活性蛋白B，C，D的表达，并于诱导5 d和10 d时通过Western blot检测磷酸化β-catenin和GSK-3β。　　结果与结论：大鼠脂肪组织中可成功分离出纯度较高的脂肪间充质干细胞，可表达 CD44和 CD29，不表达CD11b和CD45；经小气道生长液诱导后，脂肪间充质干细胞中肺表面活性蛋白B，C，D蛋白和mRNA表达均上调(P <0.01)，表明其可被诱导为Ⅱ型上皮细胞；加入Wnt3a后，经诱导的脂肪间充质干细胞中肺表面活性蛋白B，C，D蛋白和mRNA表达均高于小气道生长液组(P <0.01)，且在诱导过程中磷酸化β-catenin表达随时间逐渐上调而GSK-3β表达逐渐下调(P <0.01)。结果证实，Wnt信号通路在脂肪间充质干细胞诱导分化为Ⅱ肺泡上皮细胞过程中激活并促进干细胞的定向分化。%BACKGROUND:Ability of adipose mesenchymal stem cels differentiating into type II alveolar epithelial cels and the regulating mechanism have not been fuly elucidated. OBJECTIVE:To study the ability of adipose mesenchymal stem cels differentiating into type II alveolar epithelial cels in vitro and the function of Wnt pathway in the regulation of differentiation. METHODS:Adipose mesenchymal stem cels were obtained from fat tissue of rats and identified by flow cytometry. The adipose mesenchymal stem cels were divided into control group, smal airway growth medium (SAGM) group and Wnt3a group. Control group was treated with normal DMEM medium; SAGM and Wnt3a groups were both treated with smal airway growth medium, and additionaly, the Wnt3a group was treated with Wnt3a, a Wnt signaling pathway agonist. After 10 days, quantitative RT-PCR and immunofluorescence detection were used to test the expression of surfactant proteins B, C, D, type II alveolar epithelial markers. Phosphorylatedβ-catenin and GSK-3β were detected using western blot after 5 and 10 days of induction. RESULTS AND CONCLUSION: Adipose mesenchymal stem cels with high purity could be successfuly isolated from the adipose tissue of rats, and expressed CD44 and CD29, but not CD11b and CD45. After SAGM treatment, protein and mRNA expressions of surfactant proteins B, C, D were al increased in adipose mesenchymal stem cels (P < 0.01), indicating the ability of adipose mesenchymal stem cels to be induced into type II epithelial cels. Surfactant proteins B, C, D expressions at protein and mRNA levels were significantly higher in the Wnt3a group than the SAGM group (P < 0.01). During the induction progress, the expression of phosphorylated β-catenin gradualy increased, but GSK-3βexpression gradualy decreased in the Wnt3a group (P < 0.01). These findings indicate that Wnt signaling pathways are involved in differentiation of adipose mesenchymal stem cels into type II alveolar epithelial cels.