摘要
目的 研究 2型糖尿病肾病系膜细胞表型和功能的改变 ,进一步探讨糖尿病肾病的发病机理。方法 经肾活检从 2型糖尿病肾病患者获取肾组织 ,体外培养系膜细胞。采用流式细胞术、3 H 胸腺嘧啶掺入及细胞倍增时间观察细胞表型改变。系膜细胞α 平滑肌肌动蛋白 (α SMA)、层粘连蛋白、纤维连接蛋白的表达用免疫荧光染色及流式细胞仪检测。用 2 脱氧 3 H 葡萄糖 (2 DG)测定细胞葡萄糖摄入。Northern杂交和流式细胞仪检测葡萄糖转运蛋白 1(GLUT1)的表达。细胞谷氨酰胺 :6 磷酸果糖转氨酶 (GFAT)的活性采用比色法测定。结果 2型糖尿病肾病来源的系膜细胞较正常对照表现出细胞体积增大、RNA/DNA比值增加并伴细胞增殖加快 ,细胞骨架蛋白α SMA和细胞外基质合成增加。糖尿病肾病系膜细胞的葡萄糖摄入率高于对照 (15 92cpm·10 5cell-1与 12 75cpm·10 5cell-1,P <0 .0 5 ) ,同时伴GLUT1mRNA及蛋白质表达增加。此外 ,糖尿病肾病系膜细胞的GFAT活性明显增高。结论 2型糖尿病肾病系膜细胞具有明显的表型与功能改变。此外 ,糖尿病肾病患者系膜细胞还表现出细胞糖摄入及己糖胺通路活性增加。上述表型及功能改变可能是糖尿病肾病系膜细胞病变形成的基础。
Objective To explore the phenotypic and functional changes of glomerular mesangial cells (MC) in the development of diabetic nephropathy (DN). Methods Renal biopsy specimens were obtained from patients with type II DN. MC from microdissected glomeruli were cultured in vitro. Both the cells deriving from minute piece of renal biopsy specimen of type 2 DN with overt proteinuria and from transplanted donors' kidneys were investigated. Cell volume, RNA/DNA ratio and the production of fibronectin and glucose transporter 1(GLUT1) was analyzed by flow ctrometry. Cell proliferation was determined by 3?H-Tdr incorporation assay and doubling time. Immunofluorescence staining and flow ctrometry were used to examine the expression of α-smooth muscle actin and extracellular matrix. The mRNA expression of GLUT1 was determined by Northern blotting and flow cytometry. Glucose uptake rate by MC was detected using the -2-DG. The activity of glutamine: fructose-6-P aminotransferase (GFAT), the key enzyme of hexosamine pathway, was measured by spetrophotometry method. Specimens from transplanted kidneys were used a s controls. Results In specimens from patients with type II DN, increase in volume of MC (in arbitrary units) and RNA/DNA ratio (0.29±0.05 vs. 0.17±0.03, P<0.01) accompanied with high 3?H-Tdr incorporation rate (1 898±421 vs. 1 221±262, P<0.05) and shorter doubling time (31.6±1.84 h vs. 35.0±5.37 h, P<0.05) was found in comparison with the control. The synthesis of α-smooth muscle actin and extracellular matrix, including fibronectin and laminin, increased. Enhanced mRNA and protein expression of GLUT1 were verified in MC from DN. 2-DG uptake assay showed increased glucose uptake rate in MC from DN compared to the control(1 592cpm·10 5 cell -1 vs. 1 275cpm·10 5 cell -1 , P<0.05). Furthermore, MC from DN demonstrated a higher GFAT activity as compared to control. Conclusion Dramatic phenotypic and functional changes, including cell hypertrophy, increased cell turnover, excessive formation of ECM, enhanced expression of GLUT1 as well as alterration in cell glucose uptake, excessive flux of glucose metabolism through the hexosamine pathway, occur in MC from type II DN. The above mentioned phenotypic and functional changes may be the basis of pathogenesis of MC in DN.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2001年第22期1369-1373,共5页
National Medical Journal of China
基金
国家自然科学基金资助项目 ( 39870 2 88)
