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

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

目的:探讨低氧和转化生长因子β(TGF-β)对脐带间充质干细胞(UCMSC)向Ⅱ型肺上皮细胞(ATⅡ)分化的影响以及微小RNA-145(miR-145)在此过程中的调控作用,阐明UCMSC在损伤肺组织微环境中低氧和TGF-β的双重刺激下向ATⅡ分化的机制。方法:体外分离人UCMSC,与人肺癌细胞株A549共培养,诱导UCMSC向ATⅡ分化。采用氯化钴在体外模拟低氧环境,将细胞分为常氧诱导组和低氧诱导组,应用相差显微镜观察低氧诱导组细胞的形态变化,应用流式细胞术检测诱导细胞中ATⅡ所占百分比;在常氧诱导组和低氧诱导组中,采用定量PCR(qPCR)和Western blotting法分别检测ATⅡ特异性基因、细胞纤维化相关基因和miR-145表达水平。在低氧诱导组中,将细胞分为miR-145抑制剂组、miR-145抑制剂对照组和miR-145未抑制组,应用qPCR和Western blotting法检测诱导后3组细胞中纤维化相关基因的表达水平。在293T细胞中分别转入miR-145前体(pre-145)和miR-145前体对照(pre-145scramble),应用双荧光素酶报告基因系统检测TGF-βRⅡ3′-UTR野生型及突变型的相对荧光素酶活性(RLU)。结果:低氧诱导组中UCMSC由最初的成纤维样细胞逐渐变为扁平梭形,培养8d后呈铺路石样上皮细胞形态,并且高表达ATⅡ表面分子标志SpC的诱导细胞所占百分率达到(94.50±3.37)%。与常氧诱导组比较,低氧诱导下ATⅡ特异性基因KGF、CK18、SpA、SpB和SpC以及miR-145表达水平明显升高(P<0.05)。在UCMSC-ATⅡ分化过程中加入TGF-β1刺激后,与常氧诱导组比较,低氧诱导组中细胞纤维化相关基因Col-Ⅰ、TGF-βRⅡ及TGFβRⅡ下游信号因子p-Smad2和p-Smad3表达水平明显下降(P<0.05)。在低氧诱导条件下,与miR-145抑制剂对照组和miR-145未抑制组比较,miR-145抑制剂组中Col-Ⅰ和TGF-βRⅡ表达水平明显升高(P<0.05)。与TGF-βRⅡ3′-UTR突变型比较,miR-145前体作用下TGF-βRⅡ3′-UTR野生型的RLU降低(P<0.05)。结论:低氧能够促进UCMSC向ATⅡ分化并抑制TGF-β1引起的纤维化,其机制可能是低氧通过上调miR-145直接抑制TGF-βRⅡ表达从而阻断TGF-β1/TGF-βRⅡ信号通路。

Objective:To explore the influence of hypoxia and transforming growth factorβ(TGF-β)on the differentiation of umbilical cord mesenchymal stem cells(UCMSC)into the typeⅡalverolar epithelial cells(ATⅡ)and the regulatory effect of miR-145 in this process,and to illuminate the differentiation mechanism of UCMSC into ATⅡunder the double stimulation of both hypoxia and TGF-βin the damaged lung tissue microenviron ment.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(CoCl 2)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 ATⅡin the induced cells.qPCR and Western blotting methods were applied to test the expression levels of ATⅡspecific 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 ATⅡsurface 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-ATⅡdifferentiation with TGF-β1,the expression levels of fibrosis-related genes Col-Ⅰ,TGF-βRⅡand 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-Ⅰand TGF-βRⅡin 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-βRⅡ3′-UTR wild type was decreased after treated with pre-145 compared with TGF-βRⅡmutants(P<0.05).Conclusion:Hypoxia can promote the differentiation of UCMSC into the ATⅡand inhibit the TGF-β1-induced fibrosis.Its mechanism may be related to the inhibition of TGF-β1/TGFβRⅡsignaling pathway through the down-regulation of TGF-βRⅡexpression by hypoxia-induced miR-145.