肺泡Ⅱ型上皮细胞过表达OC-STAMP诱导肌动蛋白细胞骨架重塑促进上皮-间质转化的机制  

The mechanism of OC-STAMP overexpression induced actin cytoskeleton remodeling in promoting epithelial-mesenchymal transition in the alveolar typeⅡepithelial cell

作　　者：耿菲 蔡玉浩 赵莹 魏中秋 徐洪 杨方 Geng Fei;Cai Yuhao;Zhao Ying;Wei Zhongqiu;Xu Hong;Yang Fang(Hebei Key Laboratory for Organ Fibrosis Research,Department of Medical Experimental Technology,School of Public Health,North China University of Science and Technology,Tangshan 063000,China;Hebei Key Laboratory for Chronic Diseases,Department of Pathology,School of Basic Medical Sciences,North China University of Science and Technology,Tangshan 063000,China)

机构地区：[1]华北理工大学公共卫生学院医学实验技术系,河北省器官纤维化重点实验室,唐山063000 [2]华北理工大学基础医学院病理学系,河北省慢性疾病重点实验室,唐山063000

出　　处：《中华劳动卫生职业病杂志》2023年第11期801-807,共7页Chinese Journal of Industrial Hygiene and Occupational Diseases

基　　金：国家自然科学基金资助项目(82204006);河北省自然科学基金项目(H2022209058);河北省高等学校科学技术研究项目(QN2022009);河北省在读研究生创新能力培养资助项目(CXZZBS2022104);大学生创新训练计划项目(R2022035)。

摘　　要：目的:探讨过表达破骨细胞刺激性转膜蛋白(osteoclast stimulatory transmembrane protein,OC-STAMP)对上皮-间质转化的作用及机制。方法:于2021年4月,将小鼠肺泡Ⅱ型上皮细胞MLE-12细胞分为过表达对照组(NC组)、 Ocstamp过表达组(over- Ocstamp组)、法舒地尔(Fasudil)干预组(over- Ocstamp+Fasudil组)、沉默对照组(si-NC组)、 Ocstamp沉默组(si- Ocstamp组)。采用蛋白免疫印迹(Western blotting)法、免疫细胞化学染色法检测OC-STAMP、上皮标志蛋白E-钙黏蛋白(E-cadherin,E-cad)、间质标志蛋白α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、Ras基因家族成员A(Ras homolog gene family member A,RhoA)、Rho GDP解离抑制因子α(Rho GDP dissociation inhibitor α,Rho GDIα)、Rho相关蛋白激酶(Rho-associated protein kinase,ROCK)、磷酸化肌球蛋白磷酸酶(phosphate myosin phosphatase,p-MYPT)蛋白表达,采用鬼笔环肽法检测细胞骨架肌动蛋白(filamentous actin,f-actin)的变化。采用 t检验比较两组间各蛋白的相对表达量。 结果:Western blotting及免疫细胞化学染色法结果显示,与NC组比较,over- Ocstamp组E-cad蛋白表达下调,α-SMA、Rho GDIα、RhoA、ROCK、p-MYPT蛋白表达增加,F-actin表达增强,差异均有统计学意义( P<0.05);与si-NC组比较,si- Ocstamp组E-cad和α-SMA蛋白表达差异均无统计学意义( P>0.05);与over- Ocstamp组比较,over- Ocstamp+Fasudil组E-cad蛋白表达上调,α-SMA、Rho GDIα、RhoA、ROCK、p-MYPT蛋白表达下降,F-actin表达减弱,差异均有统计学意义( P<0.05)。 结论:肺泡Ⅱ型上皮细胞过表达OC-STAMP,可能通过激活Rho GDIα/RhoA/ROCK信号通路,诱导肌动蛋白细胞骨架重塑,进而促进上皮-间质转化。ObjectiveTo explore the mechanism of osteoclast stimulatory transmembrane protein(OC-STAMP)overexpression on epithelial-mesenchymal transition(EMT).MethodsIn April 2021,mice alveolar typeⅡepithelial cells MLE-12 were divided into five groups:overexpression control group(NC group),Ocstamp overexpression group(over-Ocstamp group),Fasudil intervention group(over-Ocstamp+Fasudil group),silence control group(si-NC group),Ocstamp silence group(si-Ocstamp group).The protein expressions of OC-STAMP,epithelial marker protein-E-cadherin(E-cad),interstitial marker protein-α-smooth muscle actin(α-SMA),Ras homolog gene family member A(RhoA),Rho GDP dissociation inhibitorα(Rho GDIα),Rho-associated protein kinase(ROCK),phosphate myosin phosphatase(p-MYPT)were examined by Western blotting and Immunocytochemical staining.The filamentous actin(F-actin)was detected by Phalloidin method.t test was used to compare the relative expression of each protein between the two groups.ResultsWestern blotting and Immunocytochemical staining showed that compared with the NC group,the expression level of E-cad was down-regulated,while the expression levels ofα-SMA,Rho GDIα,RhoA,ROCK,p-MYPT were increased,and F-actin expression was enhanced in the over-Ocstamp group.The differences were statistically significant(P<0.05).There were no significant differences in E-cad andα-SMA protein expression in si-Ocstamp group compared with si-NC group(P>0.05).Compared with over-Ocstamp group,the expression level of E-cad protein in over-Ocstamp+Fasudil group was up-regulated,the expression levels ofα-SMA,Rho GDIα,RhoA,ROCK and p-MYPT protein were decreased,and F-actin expression was weakened,with statistical significance(P<0.05).ConclusionOC-STAMP overexpression in alveolar typeⅡepithelial cells may induce actin cytoskeleton remodeling through activation of Rho GDIα/RhoA/ROCK signaling pathway,thus promoting EMT.

关 键 词：肌动蛋白 上皮细胞-间充质细胞转换 破骨细胞刺激性转膜蛋白 矽肺 

分 类 号：R135[医药卫生—劳动卫生]