背景 视网膜血管内皮细胞(RVECs)是视网膜微血管的主要成分,通过增生、迁移以及血管发生等生物行为在糖尿病视网膜病变(DR)的发生和发展中发挥关键作用.载脂蛋白A-Ⅰ (ApoA-Ⅰ)是高密度脂蛋白中主要的载脂蛋白,既往研究表明ApoA-Ⅰ在糖尿病患者视网膜组织内呈高表达,且在不同微环境中对RVECs发挥不同作用,但其在高糖环境下与RVECs生物学行为的关系仍不清楚. 目的 研究ApoA-Ⅰ对高糖环境下培养的人RVECs(hRVECs)生物行为及血管内皮生长因子(VEGF)表达的抑制作用. 方法 用含体积分数10％胎牛血清的DMEM体外培养hRVECs并传代,取3～6代细胞用于实验.将培养的细胞分为低糖组、低糖+ApoA-Ⅰ组、高糖组和高糖+ApoA-Ⅰ组,按照分组分别在DMEM中添加葡萄糖和ApoA-Ⅰ,低糖浓度为5 mmol/L,高糖浓度为25 mmol/L,ApoA-Ⅰ终质量浓度为30 μg/ml.采用细胞计数试剂盒-8、CCK-8法检测细胞增生能力(吸光度,A值);采用细胞划痕法检测细胞的迁移率;采用管腔形成实验检测培养细胞的体外成管能力;分别采用实时荧光定量PCR法和Western blot法检测各组细胞中VEGF mRNA及其蛋白的相对表达量.结果 高糖组细胞增生值(A值)和细胞迁移率明显高于低糖组,高糖+ApoA-Ⅰ组细胞增生值和细胞迁移率明显低于高糖组,差异均有统计学意义(A值:P=0.001、0.033;迁移率:P=0.001、0.010).低糖组、低糖+ApoA-Ⅰ组、高糖组和高糖+ApoA-Ⅰ组管腔数分别为(7.250±2.217)、(9.250±2.630)、(19.000±3.916)和(11.500±3.697)个,组间总体比较差异有统计学意义(F=10.335,P=0.001).高糖组管腔数较低糖组显著增加,高糖+ApoA-Ⅰ组较高糖组管腔数显著减少,差异均有统计学意义(P=0.001、0.037).低糖组、低糖+ApoA-Ⅰ组、高糖组和高糖+ApoA-Ⅰ组细胞中VEGF mRNA相对表达量分别为0.944±0.083、1.117±0.204、1.768±0.164和1.301±0.077,VEGF蛋白相对表达量分别为1.000±0.130、1.217±0.152、1.871+0.101和1.609±0.087,组间总体比较差异均有统计学意义(mRNA:F=18.640,P=0.001;蛋白:F=10.335,P=0.001),其中高糖组细胞中VEGF mRNA及其蛋白的相对表达量均明显高于低糖组和高糖+ApoA-Ⅰ组,差异均有统计学意义(mRNA:P=0.000、0.004;蛋白:P=0.000、0.029). 结论 ApoA-Ⅰ对高糖环境下hRVECs的增生、迁移及管腔形成具有抑制作用,可能与其下调细胞VEGF的表达有关.%Background As a main cellular component of retinal microvascular,retinal vascular endothelial cells (RVECs) play critical roles in the occurrence and development of diabetic retinopathy (DR) by proliferating,migrating and angiogenesis.Apolipoprotein A-Ⅰ (ApoA-Ⅰ) is the major apolipoprotein of high density lipoprotein.ApoA-Ⅰ is overexpressed in the retina of diabetic patients and plays different effects on RVECs upon different microenvironments,but its relationship with RVECs in high glucose environment is still not elucidated.Objective This study was to investigate the effects of ApoA-Ⅰ on proliferation,migration,tubulogenesis and vascular endothelial growth factor (VEGF) expression in human RVECs (hRVECs) in high glucose environment.Methods hRVECs were cultured with DMEM containing 10％ fetal bovine serum and passaged,and the cells at generation 3 to 6 were used in the study.The cells were divided into low-glucose group,low-glucose+ApoA-Ⅰ group,high-glucose group and high-glucose+ApoA-Ⅰ group,and the low concentration glucose (5 mmol/L),high contration glucose (25 mmol/L)and ApoA-Ⅰ (30 μg/ml) was added separately according to grouping.The proliferation and migration rate of the cells were evaluated by cell counting kit-8 (CCK-8) assay and scratch wound test respectively.The tubulogenesis of the cells was examined by tube formation test.The mRNA and protein expression of VEGF in the cells was detected by using real-time fluorescence quantitative PCR and Western blot.Results The prolifative value (absorbancy) and migration rate of the cells in the high-glucose group were significantly higher than those in the low-glucose group,and those in the high-glucose+ApoA-Ⅰ group were significantly reduced in comparion with the high-glucose group (A value:P =0.001,0.033;migration rate:P =0.001,0.010).The number of tubes in the low-glucose group,low-glucose+ ApoA-Ⅰ group,high-glucose group and high-glucose+ApoA-Ⅰ group was 7.250±2.217,9.250±2.630,19.000± 3.916 and 11.500±3.697,showing a significant difference among the groups (F=10.335,P=0.001).The number of tubes in the high-glucose group was more than that in the low-glucose group,and the number of tubes in the highglucose+ApoA-Ⅰ group was less that that in the high-glucose group (P=0.001,0.037).The relative expression levels of VEGF mRNA were 0.944 ± 0.083,1.117 ± 0.204,1.768 ± 0.164 and 1.301 ± 0.077,and those of V EGF protein were 1.000±0.130,1.217±0.152,1.871 ±0.101 and 1.609±0.087 in the low-glucose group,low-glucose+ ApoA-Ⅰ group,high-glucose group and high-glucose+ApoA-Ⅰ group,respectively,with significant differences among the groups (mRNA:F =18.640,P =0.001;protein:F =10.335,P =0.001),and the expressions of VEGF mRNA and protein in the high-glucose group were significantly higher than those in the low-glucose group and high glucose+ ApoA-Ⅰ group (mRNA:P=0.000,0.004;protein:P=0.000,0.029).Conclusions ApoA-Ⅰ plays inhibitory effects on the proliferation,migration and tubulogenesis of hRVECs in high glucose environment,which may be associated with the downregulation of VEGF expression.
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