摘要
目的探讨成纤维细胞缺氧后葡萄糖转运蛋白1(glucose transporter 1,GLUT-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(n=3):常氧组[普通培养箱(5%CO2,21%O2,37℃)]及缺氧12、24、48 h组[置缺氧培养箱(1%O2,5%CO2,94%N2,37℃)中培养12、24、48 h]。取各组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-1mRNA水平的变化,GOD-PAP测定培养上清葡萄糖含量。结果免疫印迹检测结果显示,缺氧12、24、48 h组GLUT-1的蛋白表达均显著高于常氧组(P<0.05),缺氧48 h组GLUT-1表达显著高于缺氧12、24 h组(P<0.05),缺氧24 h组与缺氧12 h组差异无统计学意义(P>0.05);免疫组化结果与免疫印迹结果相一致,缺氧12、24、48 h组GLUT-1蛋白表达水平均高于常氧组;RT-PCR及琼脂糖凝胶电泳结果显示,GLUT-1 mRNA表达水平随缺氧时间逐渐升高,缺氧12 h组与常氧组相比差异无统计学意义(P>0.05),缺氧24、48 h组均显著高于常氧组(P<0.05),缺氧48 h组显著高于缺氧24 h组(P<0.05);缺氧12、24、48 h组上清中葡萄糖含量均较常氧组显著降低(P<0.05)。结论缺氧上调大鼠肺成纤维细胞GLUT-1的表达,有利于细胞在缺氧状态下对葡萄糖的摄取。
Objective To investigate the expression profile of glucose transporter 1( GLUT-1) in lung fibroblasts under hypoxia exposure. Methods Fibroblasts were isolated from rat lung tissues and cultured in vitro,and then incubated under either normoxia( 5% CO2,21% O2,37 ℃) or hypoxia( 1% O2,5% CO2,94% N2,37 ℃) condition for 12,24 and 48 h,respectively. The expression of GLUT-1 protein was observed by Western blotting and immunohistochemical staining,while the mRNA level was assayed by reverse transcription PCR,and the content of glucose in the supernatant was determined with a GOD-PAP Glucose reagent kit. Results Compared with the normoxic group,the expression of GLUT-1 was significantly higher in 12,24 and 48 h after hypoxia exposure( P < 0. 05). There was no difference in the level between the groups under hypoxia for 24 and 12 h( P > 0. 05),but that of the 48 h hypoxic group was significantly higher than the groups under hypoxia for 24 and 12 h( P < 0. 05). The results of immunohistochemical assay were consistent with those of Western blotting,GLUT-1 protein were increased in the fibroblasts after hypoxic exposure for 12,24 and 48 h. The mRNA expression levels were gradually increased after hypoxia challenge in a time-dependent manner. There was a significant difference in the mRNA level of GLUT-1 between hypoxic24 h and 48 h groups( P < 0. 05),as well as the hypoxic 24 h and 12 h groups( P < 0. 05),but not between the hypoxia 12 h group and the normoxic group( P > 0. 05). Moreover,the level of glucose in the supernatant was significantly decreased in the groups after hypoxia exposure for 12,24 and 48 h than the normoxia group( P < 0. 05). Conclusion Hypoxia up-regulates the expression of GLUT-1,which is beneficial to glucose uptaking in rat lung fibroblasts under hypoxic condition.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2015年第19期1946-1950,共5页
Journal of Third Military Medical University
基金
国家重点基础研究发展计划(973计划
2012CB518201)
国家自然科学基金面上项目(81370150
30973446)~~
