骨形成蛋白4靶向调控SMAD9表达影响Miller细胞迁移和活性氧产生  

Effects of bone morphogenetic protein 4 on migration and reactive oxygen species production of Miller cells by regulating SMAD9 expression

作　　者：王勇 曹靖靖 寇振宇 韩菲菲 刘爱华 东莉洁 Wang Yong;Cao Jingjing;Kou Zhenyu;Han Feifei;Liu Aihua;Dong Lijie(Tianjin Key Laboratory of Retinal Functions and Diseases,Tianjin Branch of National Clinical Research Center for Ocular Disease,Eye Institute and School of Optometry,Tianjin Medical University Eye Hospital,Tianjin 300384,China)

机构地区：[1]天津医科大学眼科医院、眼视光学院、眼科研究所、国家眼耳鼻喉疾病临床医学研究中心天津市分中心,天津市视网膜功能与疾病重点实验室,天津300384

出　　处：《中华眼底病杂志》2023年第9期754-759,共6页Chinese Journal of Ocular Fundus Diseases

基　　金：天津市高等教育委员会科技发展基金项目(2022ZD057);天津市滨海新区卫生健康委员会科技项目(2022BWKZ003);天津市视网膜功能与疾病重点实验室开放项目(2021tjswmm002);天津市卫生健康科研项目(TJWJ2023ZD002);天津市医学重点学科(专科)建设项目(TJYXZDXK-037A);白求恩·朗沐中青年眼科科研基金(BJ-LM2019006J)。

摘　　要：目的观察并初步探讨骨形成蛋白4(BMP4)靶向调控SMAD9表达对Muiller细胞迁移、活性氧(ROS)生成以及血管内皮生长因子(VEGF)表达的影响。方法体外培养的Muller细胞分为正常对照组、BMP4组、BMP4+空载质粒组(BMP4+NC组)、BMP4+SMAD9小干扰质粒组(BMP4+siSMAD9)。BMP4组、BMP4+NC组、BMP4+siSMAD9组细胞培养基加人100ng/mlBMP4诱导细胞24h。其后,BMP4+NC组转染空载质粒;BMP4+siSMAD9组转染SMAD9小干扰质粒48h。细胞划痕实验测定BMP4对Miller细胞迁移的影响;流式细胞仪检测BMP4对Muller细胞内ROS生成的影响;蛋白质免疫印迹法(Westerm blots)、实时荧光定量聚合酶链反应(qPCR)检测Muller细胞内谷氨酰胺合成酶(GS)、胶质纤维酸性蛋白(GFAP)蛋白、mRNA相对表达量;免疫荧光检测Miller细胞内VEGF的表达。多组间比较采用单因素方差分析。结果细胞划痕实验检测结果显示,BMP4+siSMAD9组细胞迁移率较BMP4组、BMP4+NC组显著降低,差异有统计学意义(F=68.319,P<0.001)。流式细胞仪检测结果显示,BMP4+siSMAD9组细胞内ROS水平较BMP4组、BMP4+NC组显著降低,差异有统计学意义(F=52.158,P<0.001)。Westernblot、qPCR检查结果显示,BMP4+siSMAD9组细胞内GS、GFAP蛋白(F=42.715、36.618)、mRNA相对表达量(F=45.164、43.165)较BMP4组、BMP4+NC组显著降低,差异有统计学意义(P<0.01)。免疫荧光检测结果显示,BMP4组、BMP4+NC组细胞内VEGF荧光强度较BMP4+siSMAD9组显著升高,差异有统计学意义(F=46.384,P<0.05)。结论BMP4靶向调控SMAD9表达,可上调VEGF表达,进而促进Miller细胞迁移及ROS生成。Objective To investigate the effects of targeted regulation of SMAD9 expression by bone morphogenetic protein 4(BMP4)on Muller cell migration,reactive oxygen species(ROS)generation and vascular endothelial growth factor(VEGF)expression.Methods Muller cells cultured in vitro were divided into normal control group,BMP4 group,BMP4+no-load plasmid group(BMP4+NC group)and BMP4+SMAD9 small interference plasmid group(BMP4+siSMAD9).Cells in BMP4 group,BMP4+NC group and BMP4+siSMAD9 group were induced by adding 100 ng/ml BMP4 into cell medium for 24 h.Subsequently,BMP4+NC group was transfected with empty plasmid.BMP4+siSMAD9 group was transfected with SMAD9 small interference plasmid for 48 h.The effect of BMP4 on Muiller cell migration was determined by cell scratch test.The effect of BMP4 on the production of ROS in Muller cells was detected by flow cytometry.Western blots and real-time quantitative fluorescence polymerase chain reaction(qPCR)were used to detect the relative mRNA expression levels of glutamine synthetase(GS)and glial fibrinoacidic protein(GFAP)in Muller cells.VEGF expression in Muller cells was detected by immunofluorescence.One-way analysis of variance was used to compare groups.ResultsThe results of cell scratch test showed that the cell mobility of BMP4+siSMAD9 group was significantly lower than that of BMP4 and BMP4+NC group,and the difference was statistically significant(F=68.319,P<0.001).Flow cytomethods showed that the level of ROS in BMP4+siSMAD9 group was significantly lower than that in BMP4 and BMP4+NC group,and the difference was statistically significant(F=52.158,P<0.001).Western blot and qPCR results showed that the protein levels of GS and GFAP(F=42.715,36.618)and mRNA relative expression levels(F=45.164,43.165)in BMP4+siSMAD9 group were significantly lower than those in BMP4 and BMP4+NC group.The difference was statistically significant(P<0.01).The results of immunofluorescence detection showed that the intracellular VEGF fluorescence intensity in BMP4 group and BMP4+NC group was signif

关 键 词：骨形成蛋白4 SMAD9 洁活性氧 血管内皮生长因子 Miller细胞 细胞迁移 细胞实验 

分 类 号：R77[医药卫生—眼科]