肝纤维化炎症微环境对间充质干细胞Hedgehog信号通路调控的研究

Study on the Regulation Effect of the Liver Fibrosis Inflammatory Microenvironment on the Hedgehog Signaling Pathway in Mesenchymal Stem Cells

目的探索脂肪间充质干细胞(adipose-derived mesenchymal stem cells,ADMSCs)的Hedgehog信号通路在肝纤维化炎症微环境中的活化情况。方法 ADMSCs原代提取及培养,流式鉴定Sca-1、CD44、CD34、CD45。利用肿瘤坏死因子-α(TNF-α),干扰素-γ(IFN-γ)和转化生长因子-β1(TGF-β1)的协同作用方式构建模拟肝纤维化炎症微环境,将ADMSCs分为对照组(PBS组)、试验组(TNF-α+IFN-γ+TGF-β1协同刺激组)。将12只雄性C57BL/6小鼠随机分为两组,对照组(橄榄油,n=6)和试验组(四氯化碳,CCl4,n=6),ADMSCs转染增强型绿色荧光蛋白(EGFP)慢病毒,用于细胞的体内示踪,稳转细胞通过小鼠尾静脉注射,3d后安乐死,取脾脏及肝脏,制作组织冰冻切片。通过qRT-PCR、Western blot、免疫荧光方法检测Hedgehog通路中Smo和Gli1的表达情况。结果实验组的Smo和Gli1mRNA和蛋白表达量高于对照组(P<0.05);肝纤维化小鼠肝脏中ADMSCs数量多于对照小鼠(P<0.05),而两组小鼠的脾脏中ADMSCs数量差异无统计学意义(P>0.05)。组织免疫荧光实验发现,肝纤维化小鼠肝脏中的ADMSCs的Smo和Gli1蛋白表达高于对照小鼠。结论肝纤维化炎症微环境能够促进ADMSCs的Gli1、Smo分子表达,激活Hedgehog信号通路。

Objective To explore the activation of the hedgehog signaling pathway in adipose-derived mesenchymal stem cells (ADMSCs) in the inflammatory microenvironment of liver fibrosis. Methods ADMSCs were cultured in primary culture, and Sca-1, CD44, CD34 and CD45 were identified by flow cytometry. The inflammatory microenvironment of liver fibrosis was constructed by the synergistic action of TNF-α, IFN-γ and TGF-β1. The ADMSCs were divided into the control group (PBS group) and the experiment group (TNF-α+IFN-γ+TGF-β1 synergistic stimulation group). Twelve male C57BL/6 mice were randomly divided into two groups including the control group with olive oil (n=6) and experiment group with carbon tetrachloride (CCl 4)( n =6). ADMSCs were transfected with enhanced green fluorescent protein (EGFP) lentivirus for the in vivo tracing of cells. The stably transfected cells were injected into the mice via the tail vein. Euthanasia was performed after 3 days. The spleens and livers were taken and made into tissue frozen slices. The expression of Smo and Gli1 in the hedgehog signaling pathway was detected by qRT-PCR, western blot and immunofluorescence. Results The mRNA and protein expression levels of Smo and Gli1 in the experiment group were significantly higher than those in the control group ( P <0.05). The number of ADMSCs in the liver of liver fibrosis mice was significantly larger than that in the liver of the control mice ( P <0.05). There was no significant differences in the number of ADMSCs in the spleen between the two groups ( P >0.05). The results of immunofluorescence showed that the protein expression levels of Smo and Gli1 in ADMSCs of liver fibrosis mice were significantly higher than those in ADMSCs of the control mice ( P <0.05). Conclusion The inflammatory microenvironment of liver fibrosis can promote the expression of Gli1 and Smo molecules in ADMSCs and activate the hedgehog signaling pathway.