【摘要】 目的分离纯化表现为肾病综合征(NS)的微小病变型(MCD)及膜性肾病(MN)患者尿IgG,比较它们对人近端小管上皮细胞(HK-2)表达巨噬细胞移动抑制因子(MIF)的影响。方法采用硫酸铵沉淀、蛋白G亲和层析纯化尿中IgG,并经SDS-PAGE、Western印迹分析鉴定。用不同浓度(0、0.5、1.0、2.5、5.0、10.0mg/ml)的上述两种患者的尿IgG分别刺激HK-2细胞6h,应用RT-PCR检测细胞表达。MIFmRNA的变化:应用Western印迹检测细胞中MIF的蛋白水平。结果纯化的尿IgG经SDS—PAGE分析显示其分解为4个片段,以兔抗人IgG抗体进行免疫印迹鉴定,证实这些蛋白条带均为IgG成分。两种不同病理类型NS患者的尿IgG均可上调HK-2细胞MIF的基因及蛋白表达,并呈剂量依赖性。MN患者的尿IgG0.1mg/ml即可明显上调HK-2细胞.MIFmRNA和蛋白表达(P<0.01);而MCD患者的尿IgG需达到2.5mg/ml才具有显著上调效应。结论呈NS的MCD和MN患者尿IgG可上调HK-2细胞表达MIF。MN患者尿IgG的作用强于MCD患者,提示这两种不同病理类型患者尿IgG可能存在结构或功能上的差异。更多还原

【Abstract】 Objective To investigate the effect of purified urinary IgG from patients with minimal change nephrotic syndrome and membranous nephropathy on the expression of macrophage migration inhibitory factor (MIF) in human proximal tubular epithelial cells (HK-2). Methods Proteins were precipitated from urine with (NH4)2SO4, and urinary IgG was purified by protein G column chromatography. SDS-PAGE and Western blotting were performed to analyze the urinary IgG. HK-2 cells were incubated with urinary IgG (0,0.5,1.0,2.5,5.0,10.0 mg/ml) from either minimal change nephrotic or membranous nephropathy patients. The expression of MIF mRNA was assessed by RT-PCR, and MIF protein was assessed by Western blotting. Results SDS-PAGE showed that there were four bands, which were all IgG fractions confirmed by Western blotting. The urinary IgG up-regulated the expression of MIF mRNA and protein in HK-2 cells. The expression increased in a dose-dependent manner. The expression of MIF mRNA and protein in HK-2 cells increased significantly when they were incubated with urinary IgG from membranous nephropathy patients at 1mg/ml (P<0.01). However, a dose of 2.5 mg/ml of urinary IgG from minimal change nephrotic patients was required to increase MIF expression. Conclusions The urinary IgG from nephrotic patients with minimal change and membranous nephropathy can up regulate expression of MIF in HK-2 cells. Compared to the urinary IgG from minimal change nephrotic syndrome, the urinary IgG from membranous nephropathy is more potent to stimulate MIF synthesis in HK-2 cells. These data suggest that structural and functional discrepancies exist between the urinary IgG from minimal change and the one from membranous nephropathy.更多还原