骨形成蛋白4对人视网膜微血管内皮细胞糖酵解水平的影响  

Effect of bone morphogenetic protein 4 on glycolysis of human retinal vascular endothelial cells

作　　者：李辉 洪亚茹 刘勃实 东莉洁 滕贺 Li Hui;Hong Yaru;Liu Boshi;Dong Lijie;Teng He(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》2022年第10期840-845,共6页Chinese Journal of Ocular Fundus Diseases

基　　金：天津市教委科研计划一般项目(2021KJ223)。

摘　　要：目的观察骨形成蛋白4(BMP4)对人视网膜微血管内皮细胞(hRMEC)中糖酵解水平的影响。方法实验研究。将体外培养的hRMEC分为正常组、4-羟基壬烯醛(HNE)组(4-HNE组)、4-HNE+BMP4处理组(BMP4组)。4-HNE组细胞培养基中加入10μmmol/L 4-HNE;BMP4组细胞培养基中加入10μmmol/L 4-HNE刺激6 h后,再加入100 ng/ml重组人BMP4。采用流式细胞仪检测各组细胞内活性氧(ROS)水平;噻唑蓝比色法检测4-HNE对细胞生存力的影响;细胞划痕实验、Transwell小室法测定4-HNE对细胞迁移能力的影响。采用实时定量聚合酶链反应、蛋白免疫印迹法检测正常组、4-HNE组细胞中BMP4、SMAD同源物9(SMAD9)mRNA、蛋白相对表达量。采用Seahorse XFe96细胞能量代谢分析仪测定正常组、4-HNE组、BMP4组细胞内糖酵解代谢水平。组间比较采用单因素方差分析。结果正常组、4-HNE组、BMP4组hRMEC内ROS水平分别为21±1、815±5、810±7。与正常组比较,4-HNE组、BMP4组ROS水平显著升高,差异有统计学意义(F=53.40、50.30,P<0.001)。正常组、4-HNE组细胞生存力分别为1.05±0.05、1.28±0.05;迁移率分别为(0.148±0.005)%、(0.376±0.015)%;穿过小孔的细胞数分别为(109.0±9.6)、(318.0±6.4)个。与正常组比较,4-HNE组细胞生存力、细胞迁移率、穿过小孔细胞数量明显提高,差异均有统计学意义(F=54.35、52.84、84.35,P<0.05)。4-HEN组细胞中BMP4、SMAD9 mRNA相对表达量分别为1.680±0.039、1.760±0.011;与正常组比较,差异有统计学意义(F=53.66、83.54,P<0.05)。正常组、4-HEN组细胞中BMP4、SMAD9蛋白相对表达量分别为0.620±0.045、0.860±0.190和0.166±0.049、0.309±0.038;与正常组比较,差异有统计学意义(F=24.87、53.84,P<0.05)。正常组、4-HNE组、BMP4组细胞内糖酵解、糖酵解能力、糖酵解储备水平分别为1.21±0.12、2.84±0.24、1.78±0.36,2.59±0.11、5.34±0.32、2.78±0.45和2.64±0.13、5.20±0.28、2.66±0.33;与正常组比较,差异均有统计�Objective To explore the effect of bone morphogenetic protein 4(BMP4)on the glycolysis level of human retinal microvascular endothelial cells(hRMECs).Methods A experimental study.hRMECs cultured in vitro were divided into normal group,4-hydroxynonenal(HNE)group(4-HNE group)and 4-HNE+BMP4 treatment group(BMP4 group).4-HNE group cell culture medium was added with 10μmmol/L 4-HNE;BMP4 group cell culture medium was added with recombinant human BMP4100 ng/ml after 6 h stimulation with 10μmol/L 4-HNE.The levels of intracellular reactive oxygen species(ROS)were detected by flow cytometry.The effect of 4-HNE on the viability of cells was detected by thiazole blue colorimetry.Cell scratch test and Transwell cell method were used to determine the effect of 4-HNE on cell migration.The relative expression of BMP4 and SMAD9 mRNA and protein in normal group and 4-HNE group were detected by realtime quantitative polymerase chain reaction and Western blot.Seahorse XFe96 cell energy metabolism analyzer was used to determine the level of intracellular glycolysis metabolism in normal group,4-HNE group and BMP4 group.One-way analysis of variance was used for comparison between groups.Results The ROS levels in hRMECs of normal group,4-HNE group and BMP4 group were 21±1,815±5,810±7,respectively.Compared with the normal group,the levels of ROS in the 4-HNE group and the BMP4 group were significantly increased,and the difference was statistically significant(F=53.40,50.30;P<0.001).The cell viability in the normal group and 4-HNE group was 1.05±0.05 and 1.28±0.05,respectively;the migration rates were(0.148±0.005)%,(0.376±0.015)%;the number of cells passing through the pores were 109.0±9.6,318.0±6.4,respectively.Compared with the normal group,the 4-HNE group had significantly higher cell viability,cell migration rate,and the number of cells passing through the pores,and the differences were statistically significant(F=54.35,52.84,84.35;P<0.05).The relative expression levels of BMP4 and SMAD9 mRNA in the cells of the 4-HEN group

关 键 词：骨形态发生蛋白质4 内皮 血管 内皮细胞 糖酵解 细胞增殖 细胞运动 

分 类 号：R774.1[医药卫生—眼科]