缺氧上调成纤维细胞葡萄糖转运蛋白1的表达.doc

缺氧上调成纤维细胞葡萄糖转运蛋白1的表达 李晓栩1,黄瑊1,徐刚3,杨诚忠1,谭小玲1,高钰琪3,官立彬1* （400038重庆, 第三军医大学高原军事医学系：高原生理学与高原生物学教研室1，高原军事医学系高原特殊药物与装备研究室3） ［摘要］ 目的: 探讨成纤维细胞缺氧后葡萄糖转运蛋白1表达特点与规律。方法:分离培养大鼠肺成纤维细胞，将细胞分成4组：常氧组，缺氧组（12h、24h、48h）。常氧组置普通培养箱(5 % CO2, 21 %O2, 37 ℃) 中培养，各缺氧组置缺氧培养箱中培养(1 %O2, 5 %CO2, 94 %N2, 37 ℃) 不同时间(12h、24h、48h)后，取细胞进行免疫荧光和免疫印迹分析蛋白水平的表达变化，RT-PCR分析细胞GLUT-1 mRNA水平的变化，GOD-PAP法测定培养上清葡萄糖含量。结果: 免疫印迹灰度分析结果显示缺氧各组GLUT-1表达量显著高于常氧组( p﹤0.05)，缺氧24 h组GLUT-1表达量显著高于缺氧12 h组(p﹤0.05)，缺氧48 h较缺氧12 h组灰度值显著降低(p﹤0.05)；缺氧24h、48 h组mRNA水平均显著高于常氧组(p﹤0.05)，缺氧12 h组与常氧组相比无差异；免疫荧光显示各缺氧组GLUT-1染色强度均高于常氧组；缺氧12h、24h、48 h组上清中葡萄糖含量较常氧组显著下降（p＜0.05），缺氧24 h，48h组较缺氧12 h组葡萄糖含量显著减少（p＜0.05）。 结论: 缺氧上调大鼠肺成纤维细胞GLUT-1的表达，这有利于细胞在缺氧状态下对葡萄糖的摄取。 ［关键词］：缺氧；成纤维细胞；葡萄糖转运蛋白1； ［中图法分类号］R363.1; R363.2-1 ［文献标识码］ A Hypoxia upregulates the expression of Glucose transporter 1 in Fibroblast Li Xiaoxu1, Huang Jian1,Xu Gang,Yang Chengzhong1,Tan Xiaoling1, Gao Yuqi, Guan Libin1* Objective: To explore the characteristics of Glucose transporter 1 expression in lung fibroblast under hypoxia exposure. Methods: Lung fibroblasts cultured in vitro were incubated under either normoxia (5% CO2, 21% O2, 37℃) or hypoxia (1% O2, 5% CO2, 94% N2, 37℃) condition for 12 h, 24 h, 48 h. The expression of GLUT-1 protein was observed by western blotting and immunohistochemisty staining, while the mRNA level was assayed by reverse transcription PCR, and the level of Glucose was determined by the method of GOD-PAP. Results: Compared with normoxic group, western blotting showed significantly stronger expression of protein of GLUT-1 in the hypoxic groups than in the normoxic group (p0.05), the hypoxic 24-hour group was significantly higher than the hypoxic 12-hour group (p0.05) , immunofluorescence showed GLUT-1 protein in all hypoxic groups was enhanced. The result of reverse transcription PCR indicated the mRNA of GLUT-1 exposed to hypoxia for 12-hour was of no significance (p﹥0.05) and that for 24 and 48-hour was of statistical significance (p 0.05). The le