骨形成蛋白4促进视网膜血管内皮细胞增生和迁移  

Bone morphogenetic protein 4 promotes the proliferation and migration of retinal vascular endothelial cells

作　　者：刘巨平 洪亚茹 姚旭阳 张哲 步绍翀 李辉 曹靖靖 白小妹 李筱荣 东莉洁 Liu Juping;Hong Yaru;Yao Xuyang;Zhang Zhe;Bu Shaochong;Li Hui;Cao Jingjing;Bai Xiaomei;Li Xiaorong;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》2022年第4期304-309,共6页Chinese Journal of Ocular Fundus Diseases

基　　金：天津市教委科研计划一般项目(2017KJ214)。

摘　　要：目的观察氧化应激条件下骨形成蛋白4(BMP4)对人视网膜微血管内皮细胞(hRMEC)的增生和迁移影响。方法将体外培养的hRMEC分为对照组、4-羟基壬烯醛(HNE)处理组(4-HNE组)、4-HNE+BMP4处理组(BMP4组)。4-HNE组细胞培养基加入10μmmol/L 4-HNE;BMP4组细胞培养基加入10μmmol/L 4-HNE刺激6 h后,再加入100 ng/ml重组人BMP4。噻唑蓝比色法检测4-HNE、BMP4对hRMEC生存力的影响。细胞划痕实验测定4-HNE、BMP4对细胞迁移能力的影响。实时定量聚合酶链反应(qRT-PCR)检测对照组、4-HNE组细胞中BMP4 mRNA相对表达量以及对照组、BMP4组细胞中纤维连接蛋白(FN)、层粘连蛋白(Laminin)、α-平滑肌收缩蛋白(α-SMA)、胶原蛋白Ⅰ型(CollagenⅠ)、血管内皮生长因子(VEGF)、结缔组织生长因子(CTGF)mRNA相对表达量。蛋白免疫印迹法(Western blot)检测对照组、4-HNE组细胞中BMP4蛋白相对表达量。两组间比较采用t检验。结果与对照组比较,4-HNE组、BMP4组细胞生存力(t=12.73、16.26,P=0.0002、<0.0001)、细胞迁移率(t=28.17、37.48,P<0.0001、<0.0001)均明显提高,差异有统计学意义;4-HNE组细胞中BMP4 mRNA、蛋白相对表达量明显升高,差异有统计学意义(t=16.36、69.35,P=0.0001、<0.0001)。qRT-PCR检测结果显示,与对照组比较,BMP4组细胞中VEGF、FN、Laminin、α-SMA、CollagenⅠ、CTGF mRNA相对表达量明显升高,差异有统计学意义(t=10.61、17.00、14.85、7.78、12.02、10.61,P=0.0004、<0.0001、0.0001、0.0015、0.0001、0.0004)。结论BMP4可诱导hRMEC的增生和迁移,并可调控hRMEC中血管生成因子和纤维化相关因子表达。Objective To observe the effect of bone morphogenetic protein 4(BMP4)on the proliferation and migration of human retinal microvascular endothelial cells(hRMEC)under oxidative stress.Methods The hRMEC cultured in vitro were divided into control group,4-hydroxynonenal(HNE)treatment group(4-HNE group),4-HNE+BMP4 group(BMP4 group).Cell culture medium of 4-HNE treatment group was added with 10μmmol/L 4-HNE;cell culture of BMP4 group was cultured with 10μmmol/L 4-HNE,and after stimulation for 6 h,100 ng/ml recombinant human BMP4 was added.The effects of 4-HNE and BMP4 on hRMEC viability was detected by thiazole blue colorimetric method.The effects of 4-HNE and BMP4 on cell migration was determined by cell scratch test.The relative expression of BMP4 mRNA in the cells of the control group and 4-HNE treatment group and the mRNA expression of the control group,the fibronectin(FN)of BMP4 group,laminin(Laminin),α-smooth muscle contractile protein(α-SMA),and collagen typeⅠ(CollagenⅠ),vascular endothelial growth factor(VEGF),and connective tissue growth factor(CTGF)were detected by real-time quantitative polymerase chain reaction(qRT-PCR).Western blot was used to detect the relative expression of BMP4 protein in the control group and 4-HNE group.The control group and 4-HNE group were compared by t test.Results Compared with the control group,cell viability(t=12.73,16.26,P=0.0002,<0.0001),cell migration rate(t=28.17,37.48,P<0.0001,<0.0001)in 4-HNE group and BMP4 group were significantly increased,and the difference was statistically significant;the relative expression of BMP4 mRNA and protein in the 4-HNE group was significantly increased,and the difference was statistically significant(t=16.36,69.35,P=0.0001,<0.0001).The qRT-PCR test results showed that compared with the control group,the relative expression of VEGF,FN,Laminin,α-SMA,CollagenⅠ,and CTGF mRNA in the cells of the BMP4 group was significantly increased,and the difference was statistically significant(t=10.61,17.00,14.85,7.78,12.02,10.61,P=0.0004,<0.0001

关 键 词：糖尿病视网膜病变 骨形态发生蛋白质4 内皮 血管 细胞增殖 细胞运动 

分 类 号：R587.2[医药卫生—内分泌] R774.1[医药卫生—内科学]