摘要
目的通过建立葡萄糖转运蛋白 １（ＧＬＵＴ１）的基因转染系统，观察 ＧＬＵＴ１的过度表达对系膜细胞功能的影响。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（ＭＣＧＴ１），以β－半乳糖苷酶转染细胞（ＭＣＬａｃＺ）为对照。用２－脱氧－３Ｈ－萄萄糖（２－ＤＧ）测定细胞葡萄糖摄入，流式细胞仪分析细胞表型，３Ｈ－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，逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）检测细胞胶原ＩＶ、ＦＮ的表达。结果ＭＣＧＴ１的２－ＤＧ摄入率明显高于ＭＣＬａｃＺ［（７４１．０±６０．５）ｄｐｍ·μｇ ｐｒｏｔ－１比（９２．２±９．０）ｄｐｍ·μｇ ｐｒｏｔ－１，Ｐ ＜０．０１］。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。在相同的葡萄糖浓度下，ＭＣＧＴ１的乳酸生成增多，表现出细胞体积增大、ＲＮＡ／ＤＮＡ和蛋白质／ＤＮＡ比值增高等细胞肥大表型。与对照相比，ＭＣＧＴ１的胶原和ＦＮ合成与表达明显增加。结论ＧＬＵＴ１的过度表达使系膜细胞具有糖尿病肾病（ＤＮ）的细胞表型。ＧＬＵＴ１的功能状态直接影响系膜细胞的糖代谢及功能变化。
Objective To investigate the effects of glucose transporter 1 (GLUT1) gene overexpression on mesangial cells. Methods Rat mesangial cells were transduced with the human GLUT1 gene(MCGT1) by retrovirus vector. Mesangial cells transduced with bacterial β - glactosidase (MC LacZ ) were used as control. Glucose uptake was detected by 2 - deoxyglucose (2 - DG ). Lactate was measured with a spectrophotometry method. Cell volume. RNA/DNA ratio and protein/DNA ratio were evaluated by flow cytometry analysis. The syntheses of collagen and fibronectin were measured by 3 H-proline incorporation and flow cytometry. The expression of collagenIV . fibronectin mRNA were analyzed by RT-PCR. Results Northern blot and RT-PCR showed that the exogenous GLUT1 gene was expressed in MCGT1. MCGT1 demonstrated a higher 2-DG uptake than MCLacZ [ (741. 0 ± 60. 5)dpm·μg prot-1 vs (92. 2 ± 9. 0) dpm·μg prot-1, P < 0. 01 ]. Kinetic analysis revealed a 3. 7-fold higher Vmax in MCGT1 vs MCLacZ with no difference in Km values. Lactate release into the media as well as that associated with the cell layer were 2. 4, 1. 9-fold greater, respectively, in MCGT1 than in MCLacZ. MCGT1 showed an increase in cell volume.RNA/ DNA ratio and protein/DNA ratio. In addition, MCGT1 exhibited greater synthesis of extracellular matrix than MCLacZ. Conclusions Glucose transport activity is an important modulator of cellular glucose metabolism in mesangial cells. GLUT1 may play an important role in the pathogenesis of diabetic nephropathy.
出处
《中华肾脏病杂志》
CAS
CSCD
北大核心
2000年第6期357-362,共6页
Chinese Journal of Nephrology
基金
国家自然科学基金资助项目!(39870288)
