低氧及其下游因子miRNA-145在脐带间充质干细胞向Ⅱ型肺泡上皮细胞分化中的作用

doi:10.13481/j.1671-587x.20180509

Abstract

Abstract: Objective:To explore the influence of hypoxia and transforming growth factor β (TGF-β) on the differentiation of umbilical cord mesenchymal stem cells (UCMSC) into the type Ⅱ alverolar epithelial cells(ATⅡ) and the regulatory effect of miR-145 in this process, and to illuminate the differentiation mechanism of UCMSC into ATⅡ under the double stimulation of both hypoxia and TGF-β in the damaged lung tissue microenvironment. Methods:The UCMSC were isolated in vitro and co-cultured with human lung cancer cell line A549 to induce the differentiation of UCMSC into ATⅡ. Cobaltous chloride (CoCl₂) was used to mimic the hypoxia condition, and the induced cells were divided into normoxia group and hypoxia group. In hypoxia group, phase contrast microscope was used to observe the changes in cell morphology and flow cytometry was used to detect the percentage of ATⅡ in the induced cells. qPCR and Western blotting methods were applied to test the expression levels of ATⅡ specific genes, fibrosis-related genes and miR-145 in normoxia group and hypoxia group. In hypoxia group, the cells were divided into inh-145 group, inh-145 scramble group and control group, the expression levels of fibrosis-related genes in the cells in three groups were tested by qPCR and Western blotting methods. The pre-145 and pre-145 scramble were transfected into the 293T cells, and then the dual luciferase reporter gene system was used to check the relative luciferase unit (RLU) of TGF-βRⅡ3'-UTR wild type and mutants. Results:In hypoxia group, the fibroblast-like UCMSC became flat and spindle, and finally showed a morphology of cobblestone-like epithelial cells 8 d later, and the percentage of the induced cells with high expression of ATⅡ surface marker SpC was up to (94.50±3.37)%. The expression levels of specific genes of ATⅡ (KGF, CK18, SpA, SpB and SpC) and miR-145 were obviously up-regulated in hypoxia group compared with normoxia group (P<0.05). After stimulating the UCMSC-ATⅡ differentiation with TGF-β1, the expression levels of fibrosis-related genes Col-Ⅰ, TGF-βRⅡ and its downstream signaling factors p-Smad2 and p-Smad3 in hypoxia group were significantly down-regulated compared with normoxia group (P<0.05). The expression levels of Col-Ⅰ and TGF-βRⅡ in inh-145 group were significantly raised compared with inh-145 scramble group and control group under hypoxia induction (P<0.05). The RLU of TGF-βRⅡ 3'-UTR wild type was decreased after treated with pre-145 compared with TGF-βRⅡ mutants (P<0.05). Conclusion:Hypoxia can promote the differentiation of UCMSC into the ATⅡ and inhibit the TGF-β1-induced fibrosis. Its mechanism may be related to the inhibition of TGF-β1/TGFβRⅡ signaling pathway through the down-regulation of TGF-βRⅡ expression by hypoxia-induced miR-145.

Key words: acute lung injury, lung fibrosis, mesenchymal stem cells, microRNA

CLC Number:

SHI Xu, YU Xin, QU Xinglong, CAO Yang, ZHANG Guangjuan, LI Changyuan. Effects of hypoxia and its downstream factor mirRNA-145 on differentiation of umbilical cord mesenchymal stem cells into type Ⅱ alveolar epithelial cells[J].Journal of Jilin University(Medicine Edition), 2018, 44(05): 935-942.

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Effects of hypoxia and its downstream factor mirRNA-145 on differentiation of umbilical cord mesenchymal stem cells into type Ⅱ alveolar epithelial cells

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