南方医科大学学报

南方医科大学学报 ›› 2020, Vol. 40 ›› Issue (07): 988-994.doi: 10.12122/j.issn.1673-4254.2020.07.11

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脐带间充质干细胞来源的外泌体通过抑制上皮间质转化缓解肺纤维化

杨 静，胡华钟，张书勤，姜琳瑞，程远雄，谢浩俊，王小燕，姜交华，王 红，张 群   

  • 出版日期:2020-07-20 发布日期:2020-07-20
  • 基金资助:

Human umbilical cord mesenchymal stem cell-derived exosomes alleviate pulmonary fibrosis in mice by inhibiting epithelial-mesenchymal transition

  

  • Online:2020-07-20 Published:2020-07-20

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摘要： 目的 研究人脐带间充质干细胞外泌体（hUCMSC-EXOs）的抗纤维化作用及其潜在机制。方法 采用随机数字表法将24只C57 BL/6小鼠平均分为4组，6只/组。空白组：气管内注射生理盐水（3 mg/kg）；肺纤维化组：气管内注射1.5 mg/mL博来霉素溶液（生理盐水配成，3 mg/kg）；EXOs1组：气管内注射1.5 mg/mL博来霉素溶液（3 mg/kg）和hUCMSC-EXOs（造模第2天尾静脉注射，100 μg/250 μL）；EXOs2组：气管内注射1.5 mg/mL博来霉素溶液（3 mg/kg）和hUCMSC-EXOs（造模第11天尾静脉注射，100 μg/250 μL）。造模后21 d处死小鼠，取小鼠双侧肺组织，计算肺系数，分别通过HE染色和Masson染色进行肺组织病理 学和胶原蛋白沉积检查，ELISA实验检测血清中TGF-β1的表达水平，免疫组化法检测vimentin、E-cadherin和p-Smad2/3的表达水平。在体外A549细胞模型上，CCK8实验观察hUCMSC-EXOs对细胞增殖的影响，Western blotting观察hUCMSC-EXOs 对p-Smad2/3、vimentin和E-cadherin的表达水平的影响。结果 hUCMSC-EXOs显著降低肺纤维化小鼠的肺系数（P<0.05）、改善肺组织切片胶原沉积和肺组织病理变化，减少小鼠TGF-β1的表达（P<0.05），抑制p-Smad2/3、vimentin的表达而增加E-cadherin的表达，且外泌体一组的治疗效果优于外泌体二组；hUCMSC-EXOs对细胞增殖无明显影响（P>0.05），且可抑制p-Smad2/3、 vimentin的表达而促进E-cadherin的表达。结论 hUCMSC-EXOs可以通过抑制TGF-β1/Smad2/3信号通路激活的上皮-间质转化而缓解小鼠肺纤维化程度，且在第2天给予缓解效果更加明显。

关键词: 人脐带间充质干细胞外泌体, 肺纤维化, 上皮间质转化, TGF-β1/Smad2/3

Abstract: Objective To study the anti- fibrotic effect of human umbilical cord mesenchymal stem cell-derived exosomes (hUCMSC-EXOs) and explore the mechanism. Methods Twenty-four C57 BL/6 mice were divided into 4 groups (n=6), including the control group treated with intratracheal injection of saline (3 mg/kg); lung fibrosis model group with intratracheal injection of 1.5 mg/mL bleomycin solution (prepared with saline, 3 mg/kg); EXOs1 group with intratracheal injection of 1.5 mg/mL bleomycin solution (3 mg/kg) and hUCMSC-EXOs (100 μg/250 μL, given by tail vein injection on the next day after modeling); and EXOs2 group with intratracheal injection of 1.5 mg/mL bleomycin solution (3 mg/kg) and hUCMSC-EXOs (100 μg/250 μL, given by tail vein injection on the 10th day after modeling). At 21 days after modeling, pulmonary index, lung tissue pathology and collagen deposition in the mice were assessed using HE staining and Masson staining. The expression level of TGF-β1 was detected using ELISA, and vimentin, E-cadherin and phosphorylated Smad2/3 (p-Smad2/3) were detected using immunohistochemical staining. CCK8 assay was used to evaluate the effect of hUCMSCEXOs on the viability of A549 cells, and Western blotting was used to detect the expression levels of p-Smad2/3, vimentin, and E-cadherin in the cells. Results Compared with those in the model group, the mice treated with hUCMSC-EXOs showed significantly reduced the pulmonary index (P<0.05), collagen deposition, lung tissue pathologies, lowered expressions of TGF-β1 (P<0.05), vimentin, and p-Smad2/3 and increased expression of E-cadherin. hUCMSC-EXOs given on the second day produced more pronounced effect than that given on the 11th day (P<0.05). CCK8 assay results showed that hUCMSC-EXOs had no toxic effects on A549 cells (P>0.05). Western blotting results showed that hUCMSC-EXOs treatment significantly increased the expression of E-cadherin and decreased the expressions of p-Smad2/3 and vimentin in the cells. Conclusion hUCMSC-EXOs can alleviate pulmonary fibrosis in mice by inhibiting epithelialmesenchymal transition activated by the TGF-β1/ Smad2/3 signaling pathway, and the inhibitory effect is more obvious when it is administered on the second day after modeling.

Key words: human umbilical cord mesenchymal stem cell derived exosomes, idiopathic pulmonary fibrosis; epithelial-mesenchymal transition, TGF-β1/Smad2/3