IFN-γ诱导的自噬抑制人胎盘胎儿侧来源间充质干细胞增殖

The autophagy induced by IFN-γ inhibits proliferation of human placental mesenchymal stem cells of fetal side

目的探讨IFN-γ诱导无血清培养人胎盘胎儿侧来源MSCs自噬的发生,并分析自噬对细胞增殖的影响。方法用酶消化法和无血清培养体系分离培养人胎盘胎儿侧来源MSCs,利用流式细胞仪和分化培养体系鉴定细胞属性;用质量浓度50μg/L的IFN-γ处理人胎盘胎儿侧来源MSCs,以未处理细胞作为对照组,3-Ma处理为自噬抑制组;分别提取总蛋白,Western blot检测自噬标志基因LC3Ⅰ/Ⅱ的表达;mRFP-GFP-LC3腺病毒感染细胞,观察细胞内点状聚集的情况;MTT法检测IFN-γ对细胞增殖的影响。结果所分离细胞呈CD73、CD90和CD105阳性细胞,不表达CD14、CD34和CD45,具有向脂肪和成骨细胞分化的能力;IFN-γ可提高LC3Ⅱ的表达量(P<0.05),荧光共聚焦显微境观察到IFN-γ处理细胞中的点状聚集显著增加;3-Ma可解除IFN-γ对MSCs增殖能力的抑制(P<0.05)。结论 IFN-γ诱导的自噬负调控人胎盘胎儿侧来源MSCs的增殖能力。

Objective To investigate the activation of autophagy induced by IFN-γin fPMSCs cultured in ser- um-free medium, so as to determine the role of autophagy in the capacity of proliferation in MSCs. Methods Enzyme dissociation was used in the isolation of MSCs of fetal side from human placenta. The characteristic of MSCs was identified by flow cytometry analysis and differentiation culture system. After fPMSCs being treated with 50 μg/L IFN-γ, the expression of autophagy marker gene LC3 Ⅰ /Ⅱ was measured by western blot as- say; fPMSCs was infected with mRFP-GFP-LC3 adenovirus, the puncta light was observed by confocal fluores- cence microscopy ; MTT assay was employed to identify the effect of autophagy induced by IFN-γin fPMSCs for the capacity of proliferation. The normal cells and the cells treated with 3-Ma was as control. Results The cells isolated from human placenta of fetal side expressed MSCs surface marker CD73, CD90 and CD105, but not express CD14, CD34 and CD45, and had the ability of differentiation into adipogenic and osteogeniccells. IFN-γ increased the transfer ratio of LC3 Ⅰ to LC3 Ⅱ （P 〈0. 05） , and induce more puncta light exis- ted in cytoplasm by Confocal fluorescence microscope. 3-Ma alleviated the inhibition of proliferation carried by IFN-γin fPMSCs （P 〈0. 05 ）. Conclusions The autophagy induced by IFN-γ negatively regulated the abili- ty of proliferation in fPMSCs.