陈佳琪, 林毓嫱, 杨志前, 王致, 陈育全. 高表达RLN2的人脐带间充质干细胞来源外泌体抑制TGF-β1/Smad2通路影响肺纤维化标志物表达J. 职业卫生与应急救援, 2025, 43(6): 799-805, 819. DOI: 10.16369/j.oher.issn.1007-1326.2025.250303
引用本文: 陈佳琪, 林毓嫱, 杨志前, 王致, 陈育全. 高表达RLN2的人脐带间充质干细胞来源外泌体抑制TGF-β1/Smad2通路影响肺纤维化标志物表达J. 职业卫生与应急救援, 2025, 43(6): 799-805, 819. DOI: 10.16369/j.oher.issn.1007-1326.2025.250303
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CHEN Jiaqi, LIN Yuqiang, YANG Zhiqian, WANG Zhi, CHEN Yuquan. Exosomes derived from RLN2-overexpressing hUC-MSCs attenuated pulmonary fibrosis markers in lung cells by inhibiting the TGF-β1/Smad2 pathwayJ. Occupational Health and Emergency Rescue, 2025, 43(6): 799-805, 819. DOI: 10.16369/j.oher.issn.1007-1326.2025.250303
Citation: CHEN Jiaqi, LIN Yuqiang, YANG Zhiqian, WANG Zhi, CHEN Yuquan. Exosomes derived from RLN2-overexpressing hUC-MSCs attenuated pulmonary fibrosis markers in lung cells by inhibiting the TGF-β1/Smad2 pathwayJ. Occupational Health and Emergency Rescue, 2025, 43(6): 799-805, 819. DOI: 10.16369/j.oher.issn.1007-1326.2025.250303
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高表达RLN2的人脐带间充质干细胞来源外泌体抑制TGF-β1/Smad2通路影响肺纤维化标志物表达

Exosomes derived from RLN2-overexpressing hUC-MSCs attenuated pulmonary fibrosis markers in lung cells by inhibiting the TGF-β1/Smad2 pathway

    摘要
  • 摘要:
    目的 探讨高表达松弛素2（RLN2）的人脐带间充质干细胞（hUC-MSCs）及其外泌体（EXOs）在体外模型中抑制二氧化硅（SiO2）诱导的肺细胞促纤维化的作用及机制，分析其对TGF-β1/Smad2信号轴和肌成纤维细胞分化的影响，以探索肺纤维化疾病的潜在治疗策略。
    方法 培养人脐带间充质干细胞（hUC-MSCs）提取EXOs（hUC-MSCs-ex组）；构建慢病毒转染高表达RLN2的hUC-MSCs与巨噬细胞共孵育（macro-RLN2-hUC-MSCs），提取macro-RLN2-hUC-MSCs的EXOs（macro-RLN2-hUC-MSCs-ex组）；敲除RLN2基因的hUC-MSCs与巨噬细胞共孵育，随后提取EXOs（N-macro-RLN2-hUC-MSCs组）；上述提取的3组EXOs与SiO2诱导的人胎肺上皮细胞（BEAS-2B）、人胎肺成纤维细胞（MRC-5）共培养建立细胞模型，同时设置一组空白对照组。通过蛋白质免疫印迹法（Western blot）、免疫荧光染色、实时定量聚合酶链式反应（qPCR）分别检测细胞转化生长因子-β1（TGF-β1）/Smad2信号轴、α-平滑肌肌动蛋白（α-SMA）相关mRNA及蛋白表达水平，以及BEAS-2B、MRC-5细胞免疫组织化学的情况。
    结果 Western blot结果显示，与对照组相比，BEAS-2B和MRC-5细胞中hUC-MSCs-ex组α-SMA、Smad2、TGF-β1蛋白水平均降低（P < 0.001）；与macro-RLN2-hUC-MSCs-ex组相比，N-macro-RLN2-hUC-MSCs组α-SMA、Smad2、TGF-β1蛋白表达水平均升高（P < 0.001）。在BEAS-2B细胞中，与hUC-MSCs-ex组相比，N-macro-RLN2-hUC-MSCs组α-SMA、Smad2、TGF-β1蛋白表达水平均显著升高（P < 0.001），免疫荧光染色结果与上述结果一致。qPCR结果显示，BEAS-2B和MRC-5细胞中hUC-MSCs-ex组α-SMA、Smad2、TGF-β1的mRNA水平均低于对照组（P < 0.05），与macro-RLN2-hUC-MSCs-ex组相比，N-macro-RLN2-hUC-MSCs组α-SMA、Smad2、TGF-β1的mRNA表达水平均下调（P < 0.001）。
    结论 hUC-MSCs-ex和macro-RLN2-hUC-MSCs-ex均可抑制SiO2诱导的BEAS-2B/MRC-5细胞中TGF-β1、Smad2、α-SMA的转录及蛋白表达，减缓肌成纤维细胞活化。高表达RLN2的人脐带间充质干细胞来源外泌体能抑制SiO2诱导的肺细胞纤维化，且其机制与TGF-β1/Smad2信号通路相关。

     

    Abstract:
    Objective To investigate the inhibitory effects and mechanism of high-expression relaxin 2 (RLN2)-containing human umbilical cord mesenchymal stem cells(hUC-MSCs) and their exosomes(EXOs) on silica-induced pulmonary cell fibrosis in vitro models, and to analyze their regulation of the TGF-β1/Smad2 signaling axis and myofibroblast differentiation, to explore potential therapeutic strategies for pulmonary fibrosis.
    Methods EXOs were extracted from hUC-MSCs by culture(hUC-MSCs-ex group); macro-RLN2-hUC-MSCs were transfected with a lentivirus expressing high expression of RLN2 and co-incubated with macrophages (macro-RLN2-hUC-MSCs), and EXOs from macro-RLN2-hUC-MSCs were extracted (macro-RLN2-hUC-MSCs-ex group); another group was the N-macro-RLN2-hUC-MSCs group, which was the EXOs extracted from macro-RLN2-hUC-MSCs with the RLN2 gene knocked out; the above three groups of EXOs were co-cultured with silica (SiO2)-induced human fetal lung epithelial cells (BEAS-2B) and human fetal lung fibroblasts (MRC-5) to establish a cell model, while a blank control group was set. Western blot, immunofluorescence staining, and qPCR were used to detect the expression levels of related proteins such as α-smooth muscle actin (α-SMA), Smad2, and TGF-β1 in BEAS-2B and MRC-5 cells, and the immunohistochemical conditions of fetal lung epithelial cells/fetal lung fibroblasts.
    Results Western blot results showed that compared with the control group, the protein levels of α-SMA, Smad2, and TGF-β1 in BEAS-2B and MRC-5 cells of the hUC-MSCs-ex group were all down-regulated (P < 0.001); compared with the macro-RLN2-hUC-MSCs-ex group, the protein levels of α-SMA, Smad2, and TGF-β1 in the N-macro-RLN2-hUC-MSCs group were all up-regulated (P < 0.001). In BEAS-2B cells, compared with the hUC-MSCs-ex group, the protein levels of α-SMA, Smad2, and TGF-β1 in the N-macro-RLN2-hUC-MSCs group were all significantly increased (P < 0.001). The results of immunofluorescence staining were consistent with those of Western blot. In the qPCR results, the mRNA levels of α-SMA, Smad2, and TGF-β1 in BEAS-2B and MRC-5 cells of the hUC-MSCs-ex group were all lower than those of the control group (P < 0.05), and compared with the macro-RLN2-hUC-MSCs-ex group, the mRNA levels of α-SMA, Smad2, and TGF-β1 in the N-macro-RLN2-hUC-MSCs group were all significantly down-regulated (P < 0.001).
    Conclusions The hUC-MSCs-ex and macro-RLN2-hUC-MSCs-ex significantly inhibited the transcription and expression of TGF-β1, Smad2, and α-SMA in BEAS-2B/MRC-5 cells induced by SiO2, and slowed down the activation of myofibroblasts. The hUC-MSCs-ex with high expression of RLN2 could effectively inhibit the pro-fibrotic effect of SiO2 on lung cells, and the mechanism was related to the TGF-β1/Smad2 signaling pathway.

     

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