高糖通过PI3K-Akt信号途径抑制内皮细胞迁移和增殖及血管发生改变  

High D-glucose alters PI3K and Akt signaling and leads to endothelial cell migration, proliferation and angiogenesis dysfunction

作　　者：于珮[1] 于德民[1] 齐建成[2] 王静[2] 张秋梅[1] 张景云[1] 汤云昭[1] 邢秋玲[1] 李明珍[1] 

机构地区：[1]天津医科大学代谢病医院,300070 [2]澳大利亚新南威尔士大学圣乔治院免疫教研室

出　　处：《中华医学杂志》2006年第48期3425-3430,共6页National Medical Journal of China

基　　金：天津高等学校科技发展基金项目(200326)

摘　　要：目的观察高糖对内皮细胞迁移、增殖和血管发生改变及PI3K-Akt信号途径的影响。方法不同浓度(5、15、30mmol/L)D-葡萄糖处理后,用体外“伤口愈合”实验、细胞增殖试剂盒和缺乏生长因子的Matrigel胶观察内皮细胞迁移、增殖和血管发生情况,并用免疫沉淀和Western印迹检测p85/PI3K、磷酸化PI3K、Akt、磷酸化Akt(苏氨酸308)和磷酸化GSK3β的表达变化。观察加入PI3K抑制剂——LY294002后,内皮细胞的迁移、增殖和血管发生的变化。结果与5mmol/LD-葡萄糖组(细胞迁移数为100个/μm2±23个/μm2,增殖细胞数为59128个/孔±7415个/孔)相比,15mmol/L和30mmol/LD-葡萄糖组内皮细胞迁移明显降低(77个/μm2±18个/μm2和46个/μm2±18个/μm2,均P<0·01)、细胞增殖(33144个/孔±9082个/孔和11625个/孔±4196个/孔,均P<0·01)和血管发生改变(包括血管床面积、平均血管长度、毛细血管数和血管分支点数)及磷酸化PI3K、磷酸化Akt(苏氨酸308)和磷酸化GSK3β的表达(均P<0·05或P<0·01),且随葡萄糖浓度增高抑制作用增强(均P<0·05或P<0·01)。0·1、1和10μmol/L的LY294002抑制内皮细胞迁移(细胞迁移率分别为68μm2±16μm2、36μm2±12μm2和13μm2±3μm2,均P<0·01)、增殖(增殖细胞数分别为42560个/孔±4213个/孔、17688个/孔±7198个/孔和5704个/孔±558个/孔,均P<0·01)和血管发生水平(均P<0·05或P<0·01),且随LY294002浓度增高抑制作用增强(均P<0·05)。结论高糖可能通过PI3K-Akt信号途径抑制内皮细胞的迁移、增殖和血管发生变化。Objective To investigate the effects of high D-glucose on migration, proliferation, and angiogenesis of endothelial cells and to make sure whether PI3K and Akt signaling pathway plays an important role in the pathogencsis of diabetic vascular eomplications. Methods Human umbilical vein endothelial cells （HUVECs） were cultured. D-glucose of the concentrations of 5 mmol/L, 15 mmol/L, and 30 mmol/L and mannitol of the concentrations of 5 mmol/L, 15 mmol/L, and 30 mmol/L were added in to the medium, the migration rate of the cells was measured by wound healing test and the cell proliferation was examined with CellTiter 96 AQueous, One Solution cell proliferation assay . Matrigel was spread on 96-well plate, and culture of HUVECs with D-glucose and mannitol of different concentrations were added. Microscopic photography was used to calculate the total area of vascular bed, average vessel length, vessel number, branch points, so as to observe the angiogenesis. Immuno-precipitation was used to detect the expression of p85/PI3K. Western blotting was used to detect the protein expression of p85/PI3K, p-PI3K, GSK3β （downstream kinase of Akt）, p-Akt （Threonine^308） and p-GSK3β. LY294002, a PI3 K inhibitor, of the concentrations of 0. 1 μmoL/L, 1 μmoL/L, and 10 μmoL/L was added into the culture fluid with 5 mmoL/ L D- glucose, then the endothelial cell migration, proliferation number, total area of blood bed, etc were observed. Results The migration rate of the 5 mmoL/L D-glucose group was 100 ± 23/μm^2, and D-glucose dose-dependently decreased the migration rate, e.g. the migration rates of the 15 mmoL/L and 30 mmoL/L D-glucose groups were 77 ± 18/μm^2 and 46 ± 18/μm^2 respectively, both significantly lower than that of the 5 mmol/L D-glucose group （ both P 〈 0.01 ）. LY294002 of the concentrations of 0. 1 μmol/L, 1 μmol/L, and 10 μmoL/L dose-dependently decrease the endothelial cells migration rates to 68 ± 16/μm^2, 36 ± 12/μm^2, and 13 ±3/μm^2 respectively （all P 〈0.01 ） The cell

关 键 词：糖尿病 内皮细胞 信号传递 

分 类 号：R363[医药卫生—病理学]