骨形成蛋白4调控视网膜色素上皮细胞迁移  

Experimental study on the regulation of migration of retinal pigment epithelial cells by bone morphogenetic protein 4

作　　者：李文博 曹靖靖 庄彤彤 王晴 东莉洁 Li Wenbo;Cao Jingjing;Zhuang Tongtong;Wang Qing;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》2024年第3期208-214,共7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)。

摘　　要：目的观察氧化应激条件下骨形成蛋白4(BMP4)对人视网膜色素上皮细胞(RPE)增殖和迁移的影响,初步探讨其对RPE细胞上皮-间充质转化(EMT)的作用。方法将体外培养的人RPE细胞分为正常组、单纯4-羟基壬烯醛(HNE)组(4-HNE组)、4-HNE+空白对照组(4-HNE+NC组)、4-HNE+小干扰BMP4组(4-HNE+siBMP4组)。噻唑蓝比色法检测4-HNE对RPE细胞增殖的影响;细胞划痕实验测定4-HNE、BMP4对细胞迁移能力的影响;免疫荧光染色、蛋白质免疫印迹法、实时定量聚合酶链反应检测细胞中BMP4的表达;荧光显微镜拍照观察siBMP4转染效率;流式细胞术检测细胞线粒体活性氧(MitoSOX)水平;免疫荧光实验检测细胞内EMT标志物钙粘蛋白E(E-cadherin)和纤维连接蛋白(Fibronection)的表达。两组间比较采用t检验,三组间比较采用单因素方差分析。结果与正常组比较,4-HNE组细胞增殖、迁移能力明显增强,差异有统计学意义(t=21.619、24.469,P<0.05);细胞内BMP4表达明显升高,差异有统计学意义(t=19.441,P<0.05);BMP4 mRNA、蛋白相对表达量亦显著升高,差异均有统计学意义(t=26.163、37.163,P<0.05)。转染siBMP424 h后,RPE细胞中BMP4转染效率>90%。与4-HNE组、4-HNE+NC组比较,正常组、4-HNE+siBMP4组细胞内BMP4蛋白(F=27.241)、mRNA(F=36.943)相对表达量、细胞迁移率(F=46.723)、MitoSOX水平(F=39.721)显著降低,差异均有统计学意义(P<0.05);上皮标志物E-cadherin表达显著增多,间充质标志物Fibronection表达显著降低,差异均有统计学意义(F=51.722、45.153,P<0.05)。结论氧化应激条件下BMP4抑制RPE增殖和迁移;BMP4参与诱导RPE细胞的EMT过程。Objective To observe the effect of bone forming protein 4(BMP4)on the proliferation and migration of human retinal pigment epithelium(RPE)cells under oxidative stress,and to preliminarily explore its effect on epithelial-mesenchymal transition(EMT)of RPE cells.Methods Human RPE cells cultured in vitro were divided into normal group,pure 4-hydroxynonenal(HNE)group(4-HNE group),4-HNE+NC group and 4-HNE+small interfering BMP(siBMP4)group.The effect of 4-HNE on the proliferation of RPE cells was detected by thiazole blue colorimetry.The effects of 4-HNE and BMP4 on cell migration were determined by cell scratch test.The expression of BMP4 was detected by immunofluorescence staining,Western blot and realtime quantitative polymerase chain reaction.The transfection efficiency of siBMP4 was observed by fluorescence microscopy.Mitochondrial reactive oxygen species(MitoSOX)were detected by flow cytometry. The expression of EMT markers E-cadherin and Fibronection were detected by immunofluorescence assay. t-testwas used for comparison between the two groups, and one-way analysis of variance was used for comparisonbetween the three groups. Results Compared with normal group, cell proliferation and migration ability of4-HNE group were significantly enhanced, with statistical significance (t=21.619, 24.469;P<0.05). Theexpression of BMP4 in cells was significantly increased, and the difference was statistically significant(t=19.441, P<0.05). The relative expression levels of BMP4 mRNA and protein were also significantlyincreased, with statistical significance (t=26.163, 37.163;P<0.05). After transfection with siBMP4 for 24 h, thetransfection efficiency of BMP4 in RPE cells was>90%. Compared with 4-HNE group and 4-HNE+NC group,the relative expression levels of BMP4 protein (F=27.241), mRNA (F=36.943), cell mobility (F=46.723) andMitoSOX expression levels (F=39.721) in normal group and 4-HNE+siBMP4 group were significantlydecreased. The differences were statistically significant (P<0.05). The epithelial marker E-cadherin increase

关 键 词：增生性玻璃体视网膜病变 骨形成蛋白4 上皮-间充质转化 视网膜色素上皮细胞 细胞增殖 细胞迁移 活性氧 

分 类 号：R774.1[医药卫生—眼科]