Exendin-4通过PI3K/Akt信号通路促进脂肪干细胞旁分泌细胞因子

Exendin-4 promotes paracrine action of adipose- derived stem cells through PI3K/Akt signaling pathways

目的探讨Exendin-4促进脂肪来源干细胞(ADSCs)旁分泌细胞因子的机制。方法混合酶消化法提取和培养SD大鼠腹股沟处的脂肪干细胞,使用流式细胞学检测有或无Exendin-4处理后的第4代ADSCs表面标记物,MTT法绘制0、1、5、10、20 nm/L不同浓度Exendin-4下的ADSCs生长曲线;qPCR法检测不同浓度Exendin-4处理12 h后bFGF、VEGF、HGF、IGF-1的表达变化。Western blotting和qPCR检测Exendin-4对于Akt通路的作用。同时添加通路阻断剂LY-294002,使用ELISA检测Akt通路阻断后Exendin-4对于旁分泌蛋白的影响。结果分离培养的ADSCs高表达CD29、CD90、CD105,低表达CD34、CD45,并且能够多向分化为脂肪细胞、骨细胞,符合间充质干细胞的表型特点。Exendin-4处理后,不仅不会改变ADSCs的表型,还能以浓度依赖方式促进ADSCs在体外增殖(P<0.05)。不同浓度Exendin-4处理ADSCs 12 h后,旁分泌蛋白bFGF、VEGF、HGF、IGF-1的表达量逐渐提高,其中10 nm/L Ex-4处理12 h可能为最佳处理浓度(P<0.05)。Western blotting和qPCR提示Exendin-4能够显著提高胞内Akt的磷酸化水平,而给予LY-294002后,磷酸化Akt表达减弱,同时ADSCs培养液上清中bFGF、VEGF、HGF、IGF-1的含量也明显降低(P<0.05)。结论短暂Exendin-4处理不会改变ADSCs表型,但能加强细胞的增殖能力,且以剂量依赖方式促进干细胞旁分泌细胞因子bFGF、VEGF、HGF、IGF-1。10 nm/L可能为Exendin-4最适促旁分泌浓度,其扩大干细胞旁分泌的机制部分是通过激活PI3K/Akt信号通路来介导。

Objective To investigate the mechanism by which exendin-4 promotes paracrine secretion of cytokines by adipose-derived stem cells （ADSCs）. Methods In vitro cultured SD rat ADSCs （fourth passage） with or without exendin-4 treatment underwent flow cytometry to characterize the surface markers. MTT assay was performed to assess the proliferation of the cells exposed to different concentrations （0-20 nm/L） of exendin-4, and the paracrine secretion of cytokines （bFGF, VEGF, HGF, and IGF-1） by the ADSCs was evaluated by qPCR. The changes in the expressions of p-Akt in the cells were analyzed by Western blotting and qPCR in response to exendin-4 （10 nm/L） with or without exposure to PI3K/Akt inhibitor LY-294002 （50 nm/L）; bFGF, VEGF, HGF, and IGF-1 production in the cells were detected using ELISA kits. Results Treatment with exendin-4 for 12 h did not affect the surface marker profile of the ADSCs but promoted the cell proliferation （P〈0.05）. Exendin-4 significantly increased the mRNA expressions of VEGF, bFGF, HGF, and IGF-1 in a concentration-dependent manner, and 10 nm/L was the optimum concentration （P〈0.05）. Exendin-4 treatment resulted in significantly increased p-Akt expressions in the ADSCs, and PI3K/Akt inhibitor not only reversed such effects of exendin-4 on p-Akt but also diminished the exendin-4- mediated up-regulation of the paracrine cytokines. Conclusion Exendin-4 can concentration-dependently promote the proliferative and paracrine capacities of ADSCs partially through the PI3K/Akt signaling pathway without affecting the surface marker profile of the cells.