糖尿病小鼠视网膜p21活化激酶4表达上调及其对视网膜血管内皮细胞行为和线粒体功能的影响  

Up-regulation of p21 activated kinase 4 expression in the retina of diabetes mice and its effects on the behavior and mitochondrial function in retinal vascular endothelial cells

作　　者：焦明菲 李辉 曹靖靖 寇振宇 吴桂佳 刘爱华 东莉洁 Jiao Mingfei;Li Hui;Cao Jingjing;Kou Zhenyu;Wu Guijia;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5期401-407,共7页Chinese Journal of Ocular Fundus Diseases

基　　金：新疆维吾尔自治区自然科学基金面上项目(2020D01A06);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(2019BWKY021,2022BWKZ003);天津市视网膜功能与疾病重点实验室开放项目(2021tjswmm002);天津市医学重点学科(专科)建设项目(TJLCZDXKQ010);白求恩·朗沐中青年眼科科研基金(BJ-LM202003)。

摘　　要：目的观察p21活化激酶4(PAK4)对视网膜血管内皮细胞行为和线粒体功能的影响。方法实验研究,分为体内动物实验和体外细胞实验两部分。体内动物实验:健康C57BL/6J雄性小鼠12只,随机分为正常对照组、糖尿病组,每组各6只小鼠。糖尿病组小鼠经链脲佐菌素诱导建立糖尿病模型。建模后8周,采用蛋白质免疫印迹法、实时荧光定量聚合酶链反应检测正常对照组、糖尿病组小鼠视网膜PAK4表达情况。体外细胞实验:人视网膜微血管内皮细胞(hRMEC)分为常规培养细胞组(N组)、空载体组(Vector组)、PAK4过表达组(PAK4组)。各组加人150μg/ml糖基化终末产物诱导细胞。细胞划痕实验检测各组hRMEC内细胞迁移情况;流式细胞仪检测各组白细胞粘附于hRMEC数量。SeahorseXFe96细胞能量代谢分析仪测定细胞内线粒体基础呼吸、三磷酸腺苷(ATP)生成、最大呼吸与备用呼吸能力。两组间比较采用检验;三组间比较采用单因素方差分析。结果体内动物实验:与正常对照组小鼠比较,糖尿病组小鼠视网膜中PAK4mRNA、蛋白相对表达量显著升高,差异均有统计学意义(t=25.372、22.419、25.372,P<0.05)。体外细胞实验:与N组、Vector组比较,PAK4组hRMEC中PAK4蛋白、mRNA相对表达量和细胞迁移率均显著升高,差异有统计学意义(F=36.821、38.692、29.421,P<0.05)。流式细胞仪检测结果显示,PAK4组hRMEC上白细胞粘附数量明显升高,差异有统计学意义(F=39.649,P<0.01)。线粒体压力测量结果显示,PAK4组hRMEC内线粒体基础呼吸、ATP生成、最大呼吸与备用呼吸能力明显减弱,差异有统计学意义(F=27.472、22.315、31.147、27.472,P<0.05)。结论PAK4高表达损伤线粒体功能并显著促进白细胞粘附和内皮细胞迁移。ObjectiveTo observe the effects of p21 activated kinase 4(PAK4)on the mitochondrial function and biological behavior in retinal vascular endothelial cells.Methods The experimental study was divided into two parts:in vivo animal experiment and in vitro cell experiment.In vivo animal experiments:12 healthy C57BL/6J male mice were randomly divided into normal control group and diabetes group,with 6 mice in each group.Diabetes mice were induced by streptozotocin to establish diabetes model.Eight weeks after modeling,quantitative real-time polymerase chain reaction and Western blots were performed to detect the expression of PAK4 in diabetic retinas.In vitro cell experiments:the human retinal microvascular endothelial cells(hRMEC)were divided into three groups:conventional cultured cells group(N group),empty vector transfected(Vector group);pcDNA-PAK4 eukaryotic expression plasmid transfected group(PAK4 group).WB and qPCR were used to detect transfection efficiency,while scratching assay,cell scratch test was used to detect cell migration in hRMEC of each group.In vitro white blood cell adhesion experiment combined with 4',6-diamino-2-phenylindole staining was used to detect the number of white blood cells adhering to hRMEC in each group.The Seahorse XFe96 cell energy metabolism analyzer measures intracellular mitochondrial basal respiration,adenosine triphosphate(ATP)production,maximum respiration,and reserve respiration capacity.The t-test was used for comparison between the two groups.Single factor analysis of variance was used for comparison among the three groups.Results In vivo animal experiments:compared with normal control group,the relative expression levels of PAK4 mRNA and protein in retina of diabetic mice were significantly increased,with statistical significance(t=25.372,22.419,25.372;P<0.05).In vitro cell experiment:compared with the N group and Vector group,the PAK4 protein,mRNA relative expression and cell mobility in the hRMEC of PAK4 group were significantly increased,with statistical significance(F

关 键 词：糖尿病视网膜病变 p21活化激酶4 补视网膜血管内皮细胞 线粒体功能稳态 细胞迁移 白细胞粘附 动物实验 细胞实验 

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