人胎盘胎儿来源间充质干细胞通过下调髓样分化因子88和转化生长因子β信号通路减轻小鼠肺纤维化

Human placental mesenchymal stem cells of fetal origin relieves mouse pulmonary fibrosis via downregulating MyD88 and TGF-β signaling pathway

目的探讨无血清培养人胎盘胎儿来源间充质干细胞(hf PMSC)对博来霉素所致小鼠肺纤维化的疗效及分子机制。方法培养并采用流式细胞术鉴定hf PMSC,取无特定病原体级雄性6周龄C57BL/6J小鼠随机分为博来霉素组、hf PMSC治疗组和阴性对照组。博来霉素组和hf PMSC治疗组分别经气管内注入50μL博莱霉素(1 g/L)制备肺纤维化模型,阴性对照组经气管内注入50μL PBS。hf PMSC治疗组,在造模3 d后,经尾静脉注射200μL含5×105个hf PMSC。第21天全部处死各实验组小鼠,取肺组织进行HE染色、Masson染色以及胶原蛋白含量测定;提取各组肺组织总蛋白,Western blot法检测髓样分化因子88(My D88)、转化生长因子β(TGF-β)的表达;ELISA检测各组血清中TGF-β的浓度。结果所培养具有典型的间充质干细胞形态特征并表达表面标志分子CD73、CD90和CD105,而不表达CD14、CD34和CD45。与博来霉素组比较,HE和Masson染色显示,hf PMSC治疗组可显著性降低肺组织纤维化程度、降低肺组织胶原蛋白含量以及My D88和TGF-β蛋白水平。结论hf PMSC可通过My D88下调TGF-β的表达减轻小鼠肺纤维化。

Objective To investigate the therapeutic effect and mechanism of human placental mesenchymal stem cel s of fetal origin（ hf PMSCs） cultured in serum-free medium on mouse pulmonary fibrosis induced by bleomycin treatment. Methods Human hf PMSCs were cultured and identified by flow cytometry. Fifteen 6-week-old male SPF C57 BL /6J mice were divided into 3 groups： bleomycin treatment group,hf PMSCs transplantation group and negative control group. Pulmonary fibrosis model was induced in the mice of bleomycin treatment group and hf PMSCs transplantation group with bleomycin（ 1 μg / L,50 μL） via intratracheal instillation. The mice in negative control group were instilled with PBS（ 50 μL） through the same manner of the other two groups. Three days post-modelling,200 μL containing 5 × 10^5 hf PMSCs were injected into hf PMSCs transplantation group via tail vein. All the mice were sacrificed at day 21 after modeling in batch. Lung tissues were collected for analyzing the pathological changes by HE staining and Masson staining as well as detecting collagen content. The total protein of lung tissues was extracted for observing the expressions of myeloid differentiation factor 88（ My D88） and transforming growth factor-β（ TGF-β）; the level of TGF-β in sera was determined by Western blotting. Results The hf PMSCs possessed the morphology of mesenchymal stem cells and expressed the surface markers CD73,CD90 and CD105,but did not express CD14,CD34 and CD45. HE and Masson staining showed that hf PMSCs transplantation significantly reduced the degree of pulmonary fibrosis compared with bleomycin treatment group. The collagen content and the expression levels of My D88 and TGF-β in bleomycin treatment group were obviously higher than those in hf PMSCs transplantation group and negative control group. Conclusion hf PMSCs possess the capability of alleviating pulmonary fibrosis by down-regulating the expressions of MyD 88 and TGF-β.