经siRNA抑制RhoA/Rock信号通路对肺支气管上皮细胞间质转化的作用  

Effect of siRNA inhibition of RhoA/Rock signaling pathway on interstitial transformation of pulmonary bronchial epithelial cells

作　　者：刘贵钱 李颖 段燕英 张晓华 LIU Gui-qian;LI Ying;DUAN Yan-ying;ZHANG Xiao-hua(Second Department of Occupational Diseases,Hunan Prevention and Treatment Institute for Occupational Diseases,Changsha Hunan,410007,China;Xiangya School of Public Health,Central South University,Changsha Hunan,410005,China)

机构地区：[1]湖南省职业病防治院职业病二科,湖南长沙410007 [2]中南大学湘雅公共卫生学院,湖南长沙410005

出　　处：《职业与健康》2021年第12期1622-1626,共5页Occupation and Health

基　　金：湖南省自然科学基金(2017JJ3184)。

摘　　要：目的在细胞分子水平上研究RhoA/Rock信号通路在肺支气管上皮细胞发生上皮-间质转化(EMT)向肌成纤维细胞(MFB)转化发挥的作用。方法使用人外周血单核细胞系(THP-1)构建矽肺纤维化体外模型中人巨噬细胞,再使用100 mg/mL二氧化硅(SiO_(2))悬液染尘巨噬细胞12 h后,收集上清液。实验设置分为4组:对照组:人肺支气管上皮细胞(HBE)加入上清液;染尘组:HBE加入上清液后,再加入SiO_(2)悬液染尘,染尘剂量分别为50、100、200 mg/mL;RhoA沉默组:利用小干扰RNA(siRNA)在RNA干扰技术下沉默HBE内RhoA,加入上清液;RhoA沉默染尘组:采用RNA干扰技术沉默HBE内RhoA,加入上清液后,再加入SiO_(2)悬液染尘,染尘剂量分别为50、100、200 mg/mL。4组均培养24 h后,使用Western Blot法测定细胞内α-平滑肌肌动蛋白(α-SMA)、上皮性钙粘附蛋白(E-cad)、Ⅰ型胶原蛋白(collagen I)、RhoA及Rock1、肌球蛋白磷酸酶靶亚基(MBS)蛋白磷酸化表达水平。结果染尘组中染尘剂量为50、100、200 mg/mL时E-cad表达水平分别为1.34±0.60、1.21±0.17和1.01±0.30,而对照组E-cad表达水平为1.54±0.14;RhoA沉默染尘组中染尘剂量为50、100、200 mg/mL时Ecad表达水平分别为1.79±0.12、1.50±0.11和1.19±0.05,而RhoA沉默组E-cad表达水平为2.21±0.38;染尘组与对照组、RhoA沉默染尘组与RhoA沉默组比较,均显示染尘组E-cad表达水平降低,而α-SMA、collagen I、RhoA、Rock1以及MBS蛋白磷酸化表达水平增加;且随着染尘浓度增加,差异越明显。经siRNA沉默后,RhoA沉默染尘组较染尘组E-cad的表达增加,而α-SMA、collagen I、RhoA、Rock1的表达降低。结论 SiO_(2)粉尘会导致支气管上皮细胞发生EMT,RhoA/Rock信号通路介导EMT参与了矽肺纤维化,抑制该信号通路可以抑制EMT,从而对矽肺纤维化具有保护作用。Objective To investigate the role of RhoA/Rock signaling pathway in the epithelial mesenchymal transition(EMT)of pulmonary bronchial epithelial cells to myofibroblasts(MFB) at the cellular and molecular level. Methods Human derived macrophages of in vitro model of silicosis fibrosis was constructed using human peripheral blood monocyte-cell line(THP-1).Then100 mg/mL SiO_(2) suspension was used to stain the macrophages for 12 h,and the supernatant was collected.The experiment was divided into 4 groups: Control group: human bronchial epithelial cells(HBE) were added to supernatant;Dust group: after the HBE were added to supernatant,the SiO_(2) suspension was added for dust-exposure,and the dust doses were 50,100 and 200 mg/mL,respectively;RhoA silencing group: RhoA in HBE was silenced by siRNA with RNA interference technology, and supernatant was added;RhoA silencing dust group: RhoA in HBE was silenced with RNA interference technology,the SiO_(2) suspension was added for dust-exposure after the HBE were added to supernatant,and the dust doses were 50,100,and 200 mg/mL,respectively. All four groups were cultured for 24 hours. Western Blot was used to determine the protein phosphorylation levels of α-smooth muscle actin (α-SMA),epithelial cadherin(E-cad),collagen I,RhoA,Rock1 and myosin binding subunit(MBS). Results The E-cad expression level in dust group at the dust dose of 50,100 and 200 mg/mL was 1.34±0.60,1.21±0.17 and 1.01±0.30 respectively,while the E-cad expression level in control group was 1.54±0.14. The E-cad expression level in RhoA silencing dust group at the dust dose of 50,100 and 200 mg/mL was 1.79±0.12,1.50±0.11 and 1.19 ±0.05 respectively,while the E-cad expression level in RhoA silencing group was 2.21±0.38. Compared with control group,RhoA silencing dust group and RhoA silencing group,E-cad expression level in dust group was decreased,but protein phosphorylation levels of α-SMA,collagen I,RhoA,Rock1 and MBS were increased. With the increase of dust concentration,the difference was more

关 键 词：矽肺 肌成纤维细胞 上皮-间质转化 RhoA/Rock信号通路 

分 类 号：R135.2[医药卫生—劳动卫生]