论文部分内容阅读
目的 探讨己糖胺通路 (HBP)在葡萄糖转运蛋白 1(GLUT1)基因转染系膜细胞功能改变中的作用。方法 利用逆转录病毒载体建立GLUT1基因转染的大鼠系膜细胞 ,以 β 半乳糖苷酶转染细胞 (MCLacZ)为对照。用 2 脱氧 3 H 葡萄糖 (2 DG)测定细胞葡萄糖摄入 ,流式细胞仪分析细胞表型和纤维连接蛋白 (FN)的合成 ,采用比色法测定HBP限速酶———谷氨酰胺 :6 磷酸果糖转氨酶(GFAT)的活性 ,RT PCR检测细胞GFAT基因的表达。结果 MCGT1的 2 DG摄入率明显高于MCLacZ〔(741.0± 6 0 .5 )dpm/ μg蛋白质vs (92 .2± 9.0 )dpm/ μg蛋白质 ,P <0 .0 1〕 ,动力学分析发现MCGT1的Vmax是MCLacZ的 3.7倍 ,而两者Km值无明显差别。MCGT1的GFAT活性明显高于MCLacZ〔(3.2 5± 0 .2 5 )OD3 65·μg蛋白质-1·30min-1·10vs (1.15± 0 .16 )OD3 65·μg蛋白质 -1·30min-1·10〕 ,而两者GFAT的mRNA表达差异无显著性。GFAT的抑制剂———重氮丝氨酸能够改善MCGT1的肥大状态和FN合成。结论 GLUT1的过度表达伴随系膜细胞HBP活性的增加 ,该糖代谢通路的活化与系膜细胞表型及其功能改变有关。
Objective To investigate the role of hexosamine pathway (HBP) in the functional alteration of GLUT1 gene transfected mesangial cells. Methods The rat glomerular mesangial cells transfected with GLUT1 gene were established by retroviral vector and transfected by β-galactosidase (MCLacZ). Glucose uptake was measured by 2-deoxy 3 H glucose (2 DG), cell phenotypes and fibronectin (FN) were analyzed by flow cytometry, and the rate-limiting enzyme of HBP, glutamine: 6 phosphofructose aminotransferase (GFAT) activity, RT-PCR detection of cell GFAT gene expression. Results The 2 DG intake of MCGT1 was significantly higher than that of MCLacZ [(741.0 ± 60.5) dpm / μg protein vs (92.2 ± 9.0) dpm / μg protein, P <0.01) The Vmax of MCGT1 is 3.7 times that of MCLacZ, but there is no significant difference between Km values of MCGT1. The GFAT activity of MCGT1 was significantly higher than that of MCLacZ [(3.2 5 ± 0.25) OD3 65 · μg protein -1 · 30 min -1 · 10 vs (1.15 ± 0.16) · OD3 65 · μg protein -1 · 30 min -1 · 10〕, but there was no significant difference in GFAT mRNA expression between the two groups. Diazoxide, an inhibitor of GFAT, improves hypertrophy and FN synthesis of MCGT1. Conclusion The overexpression of GLUT1 is associated with the increase of HBP activity in mesangial cells. The activation of glucose metabolism pathway is related to the phenotype and function of mesangial cells.