高糖诱导牛视网膜微血管周细胞凋亡及与整合蛋白连接酶表达的关系

Overexpression of integrin-linked kinase in bovine retinal microvascular pericytes induced by glucose

目的研究高糖培养的牛视网膜微血管周细胞的凋亡和整合蛋白连接酶(ILK)表达的变化及二者的关系。方法原代培养周细胞,用流式细胞Annexin V检测周细胞的凋亡,用免疫细胞化学和Western blot检测牛视网膜微血管周细胞ILK的表达。结果用30mmol/LD-葡萄糖培养24、48、72h后,牛视网膜微血管周细胞凋亡显著增加,分别为47.6%、58.4%和68.7%且具有时间依赖性。用5mmol/LD-葡萄糖培养72h后未见周细胞ILK表达。在30mmol/LD-葡萄糖或5μmol/LH2O2条件下培养72h后,周细胞形态发生改变,ILK表达显著增强,主要分布在细胞质;Westernblot检测发现30mmol/LD-葡萄糖培养的条件下周细胞ILK蛋白水平随时间延长显著增加。结论高糖可以诱导视网膜微血管周细胞凋亡,高糖条件下周细胞ILK过表达可能参与周细胞的损伤。

Objective As a kinase,integrin-linked kinase（ILK） plays a potential role in regulating cellular activities in human. However,there still is the assessing report about effect of ILK on diabetic retinopathy. The aim of this paper was to study the apoptosis and expression of ILK in bovine retinal microvascular pericytes cultured by high glucose and the possible relationship of ILK and hyperglycemia-induced damage of pericytes. Methods Bovine retinal microvascular pericytes were primary incubated and passeged in DMEM containing 20% fetal bovine serum. The apoptosis of cultivated pericytes was detected by flow cytometry. ILK expression was detected in bovine retinal microvascular pericytes cultured in low glucose （5 mmol/L）, 30 mmol/L mannitol（24. 5 mmol/L mannitol ＋ 5.5 mmol/L glucose） , high glucose （ 30 mmol/L） and H2O2 （ 5 μmol/L） using immunocytochemistry and Western blot analysis. Results Cultured pericytes showed the positive response to et-SMA and absent response to von Willebrand factor. The apoptosis rate of pericytes in high glucose group was 47.6% ,58.4% ,68.7% in 24,48 and 72 hours respecyively,showed a statistically significant increase in comparison with low glucose group（ 1.68% ）. The retinal microvascular pericytes cultured in low glucose for 72 hours group were absent responsive to ILK. However, ILK immunoreactivity was significantly enhanced in retinal microvascular pericytes in high glucose and H2O2 for 72 hours groups, exhibiting a brown staining mainly in the cytoplasm of pericytes. Western blot analysis indicated that ILK protein expression levels in the cells of high glucose group presented increasing tendency with prolong of time. Conclusion Hyperglucose induces pericyte apoptosis and upregulates expression of ILK in the retinal microvascular pericytes in time-dependent manner,indicating that ILK may be involved in a glucose-induced damage of pericytes.