胎盘间充质干细胞外泌体通过调控NF-κB信号通路抑制矽肺肺纤维化的进展  

Exosomes of Placental MSCs Inhibit the Progression of Pulmonary Fibrosis in Silicosis by Regulating the NF-κB Signaling Pathway

作　　者：铁华 曹佳伟 丁劭瑞 王林云 李锋[3] TIE Hua;CAO Jiawei;DING Shaorui;WANG Linyun;LI Feng(First Clinical Medical College of Ningxia Medical University,Yinchuan 750004,China;Key Laboratory of Stem Cell and Regenerative Medicine of Ningxia Hui Autonomous Region,Yinchuan 750004,China;General Hospital of Ningxia Medical University,First Clinical Medical College of Ningxia MedicalUniversity,Yinchuan 750004,China)

机构地区：[1]宁夏医科大学第一临床医学院,银川750004 [2]宁夏回族自治区干细胞与再生医学重点实验室,银川750004 [3]宁夏医科大学总医院,宁夏医科大学第一临床医学院,银川750004

出　　处：《宁夏医科大学学报》2024年第6期554-562,共9页Journal of Ningxia Medical University

基　　金：国家自然科学基金项目(81860566)。

摘　　要：目的探讨人胎盘间充质干细胞外泌体(hPMSCs-Exo)对二氧化硅(SiO_(2))诱导小鼠肺泡巨噬细胞(MH-S)肺纤维化(PF)的作用机制。方法采用差速超速离心法提取外泌体,采用蛋白质免疫印迹实验(Western blot)、透射电镜、纳米粒径追踪鉴定外泌体。用MTT实验检测24 h不同浓度SiO_(2)干预MH-S增殖细胞活力影响,结合实时定量聚合酶链反应(RT-qPCR)检测在MH-S细胞中胶原蛋白Ⅰ型(COL-Ⅰ)和α-平滑肌(α-SMA)的表达,选择SiO_(2)最佳刺激质量浓度进行后续实验。将MH-S细胞分为正常对照(Control)组、SiO_(2)组、SiO_(2)+DMSO组、SiO_(2)+PDTC组,SiO_(2)+DMSO+hPMSCs组、SiO_(2)+PDTC+hPMSCs组、SiO_(2)+DMSO+hPMSCs-Exo组、SiO_(2)+PDTC+hPMSCs-Exo组,采用RT-qPCR检测细胞炎症因子IL-1β、IL-6、TNF-α相对转录水平;采用Western blot检测各组NF-κB信号通路及细胞凋亡相关蛋白表达。结果SiO_(2)刺激MH-S细胞后PF相关基因COL-Ⅰ、α-SMA表达增高,炎症因子TNF-α、IL-1β、IL-6表达增加,NF-κB通路被激活,其相关蛋白表达量上调,细胞凋亡因子表达量上调(P均<0.01),促进了纤维化的发生;在加入抑制NF-κB信号通路抑制剂PDTC后,NF-κB通路被抑制,其相关蛋白表达量降低,炎症因子TNF-α、IL-1β、IL-6表达降低,PF相关基因COL-Ⅰ、α-SMA表达减弱,细胞凋亡率降低(P均<0.05),PF的发展被抑制。抑制NF-κB通路后移植hPMSCs细胞和hPMSCs-Exo,PF相关基因表达降低,炎症因子释放减少(P均<0.05),抑制了细胞凋亡,PF的发生被有效干预。结论hPMSCs-Exo可能通过调控NF-κB信号通路来抑制矽肺PF的进展。Objective To explore the mechanism of human placental mesenchymal stem cells exosomes(hPMSCs-Exo)on silica(SiO_(2))-induced pulmonary fibrosis in mouse alveolar macrophages(MH-S).Methods Exosomes were extracted by differential ultracentrifugation Western blot,transmission electron microscopy,and nanoparticle size tracking were used to identify the exosomes.MTT assay was used to detect the effect of different concentrations of SiO_(2)on the viability of MH-S proliferating cells at 24 hours,combined with RT-qPCR to detect the expression of collagen typeⅠ(COL-Ⅰ)andα-smooth muscle(α-SMA)in MH-S cells,to select the optimal stimulating mass concentration of SiO_(2)for the subsequent experiments.MH-S cells were divided into blank control group,SiO_(2)group,SiO_(2)+DMSO group,SiO_(2)+PDTC group,SiO_(2)+DMSO+hPMSCs group,SiO_(2)+PDTC+hPMSCs group,SiO_(2)+DMSO+hPMSCs-Exo group,and SiO_(2)+PDTC+hPMSCs-Exo group.RT-qPCR was used to detect the relative transcript levels of cellular inflammatory factors IL-1β,IL-6,and TNF-α.Western blot was used to detect the NF-κB signaling pathway and apoptosis of each group.Results After SiO_(2)stimulation of MH-S cells,the expression of lung fibrosis-related genes COL-Ⅰandα-SMA increased,the expression of inflammatory factors TNF-α,IL-1βand IL-6 increased.The NF-κB pathway was activated,and the expression of its related proteins and apoptotic factors were up-regulated,which contributed to the development of fibrosis(P all<0.01).After the addition of the inhibitor PDTC,which inhibits the NF-κB signaling pathway,the NF-κB pathway was inhibited,the expression of its related proteins were reduced.The expression of inflammatory factors TNF-α,IL-1β,IL-6 were reduced,the expression of pulmonary fibrosis-related genes COL-Ⅰ,α-SMA were attenuated,the apoptosis rate was reduced,and the development of pulmonary fibrosis was inhibited(P all<0.05).After inhibition of the NF-κB pathway in transplanted hPMSCs cells and hPMSCs-Exo,the expression of pulmonary fibrosis-related genes w

关 键 词：人胎盘间充质干细胞外泌体 肺纤维化 NF-ΚB信号通路 细胞凋亡 

分 类 号：R563.9[医药卫生—呼吸系统]