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目的 通过观察大黄酸对葡萄糖转运蛋白 1(GLUT 1)基因转染系膜细胞 (MCGT 1)功能的影响 ,探讨大黄酸治疗糖尿病肾病 (DN)的作用机制。方法 利用逆转录病毒载体建立GLUT 1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 ,3 H 脯氨酸掺入和流式细胞仪分别检测细胞胶原和纤维连接蛋白 (FN)的合成 ,采用比色法测定谷氨酰胺 :6 磷酸果糖转氨酶 (GFAT)的活性 ,RT PCR检测细胞胶原Ⅳ、FN和GFAT的表达。结果 MCGT 1的 2 DG摄入率明显高于MCLacZ[(741± 6 0 5 )dpm·μgprot 1比 (92 2± 9)dpm·μgprot-1],同时表现出细胞大小、RNA/DNA和蛋白 /DNA明显增加的细胞肥大表型 ,细胞外基质合成增加 ,MCGT 1的3 H 脯氨酸掺入量明显高于MCLacZ[(7 0± 0 4)dpm·cell 1比 (4 6± 0 6 )dpm·cell 1],GFAT活性明显增强 (约增加 1 8倍 )。大黄酸能够减少MCGT 1的糖摄取 [(5 6 0± 6 4)dpm·μgprot 1比 (741± 6 0 5 )dpm·μgprot 1) ,纠正MCGT 1的肥大状态 ,并抑制MCGT 1细胞外基质的合成和表达 ,降低MCGT 1的GFAT活性。结论 GLUT 1的过度表达会明显改变系膜细胞的功能 ,大黄酸能逆转GLUT 1基因转染所致系膜?
Objective To investigate the effect of rhein on the function of glucose transporter 1 (GLUT 1) transfected mesangial cells (MCGT 1) and to explore the mechanism of action of rhein on diabetic nephropathy (DN). Methods Retroviral vectors were used to establish rat mesangial cells transfected with GLUT 1 gene and transfected with β-galactosidase (MCLacZ) as control. Glucose uptake was measured with 2 deoxygenated 3 H glucose (2 DG). Cell phenotypes were analyzed by flow cytometry. 3 H proline incorporation and flow cytometry were used to detect the synthesis of collagen and fibronectin (FN), respectively. The activity of glutamine 6-phosphofruct transaminase (GFAT) was measured by colorimetric method, and the expression of collagen IV, FN and GFAT was detected by RT PCR. Results The 2 DG uptake rate of MCGT 1 was significantly higher than that of MCLacZ [(741± 6 0 5 ) dpm·μgprot 1 ratio (92 2± 9) dpm·μgprot-1], while showing cell size, RNA/DNA and protein /DNA significantly increased the cell hypertrophy phenotype and increased extracellular matrix synthesis. The 3 H proline incorporation of MCGT 1 was significantly higher than that of MCLacZ [(7 0 ± 0 4) dpm·cell 1 ratio (4 6± 0 6). ) dpm·cell 1], GFAT activity was significantly enhanced (approximately 1 8 fold increase). Rhein can reduce the sugar uptake of MCGT 1 [(5 6 0 6 4) dpm·μgprot 1 ratio (741± 6 5 5) dpm·μgprot 1), correct hypertrophy of MCGT 1, and inhibit MCGT 1 extracellular matrix The synthesis and expression of GTAT reduced GFAT activity. Conclusion The overexpression of GLUT 1 can significantly change the function of mesangial cells. Rhein can reverse the transmembrane-induced GLUT-1 gene transfection.