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目的通过检测肥胖相关性肾小球病(ORG)小鼠血清中TNF-α水平及肾小球中TNF-αmRNA及蛋白表达,探讨ORG的发病机制。方法选取40只清洁级健康35日龄C57BL/6雄性小鼠,按体质量随机分为肥胖组和对照组各20只。肥胖组予高脂高能量饲料喂养,对照组予普通饲料喂养。2组分别于8周末留取尿液,ELISA法检测其尿微量清蛋白(Alb)、转铁蛋白(TRF)、尿视黄醇结合蛋白(RBP)、β2-微球蛋白(β2-MG)等尿微量系列蛋白;留取静脉血,ELISA法检测其血清TNF-α水平;游离其肾脏组织,固定、切片、染色,分别于光镜、电镜下观察肾脏组织病理学改变;提取肾组织RNA,实时定量(RT)-PCR检测TNF-αmRNA表达;提取肾组织,采用Western blot检测TNF-α蛋白表达。结果采用SPSS 13.0软件进行统计学处理。结果与对照组比较,肥胖组尿Alb、TRF、RBP、β2-MG及血清TNF-α水平均明显增高,差异均有统计学意义(Pa<0.01)。肥胖组肾组织TNF-αmRNA及蛋白表达明显增高(Pa<0.01)。肾组织病理学检查发现肥胖组均出现肾小球肥大;电镜下见上皮细胞足突融合,部分肾小管上皮细胞胞质内见较多脂滴,基膜增厚,三层结构消失,节段系膜插入。结论 ORG小鼠早期可出现尿微量蛋白异常;血清及肾组织中TNF-α表达异常可能在ORG的发生发展中扮演重要角色。
Objective To investigate the pathogenesis of ORG by detecting the level of TNF-α in sera and the expression of TNF-αmRNA and protein in glomeruli of obesity-associated glomerulopathy (ORG) mice. Methods Forty clean healthy 35-day-old C57BL / 6 male mice were randomly divided into obesity group and control group according to body weight. The obese group was fed with high fat and high energy feed, while the control group was fed with normal feed. Urine was collected from the two groups at the end of the 8th week. Urinary albumin (Alb), transferrin (TRF), urinary retinol binding protein (RBP) and β2-microglobulin (β2-MG) The urine samples were collected for venous blood and serum TNF-α levels by ELISA. The renal tissues were removed, fixed, sectioned and stained. The pathological changes of kidney tissues were observed under light microscope and electron microscope. The mRNA expression of TNF-α was detected by real-time quantitative RT-PCR. The renal tissue was extracted and the protein expression of TNF-α was detected by Western blot. Results SPSS 13.0 software for statistical analysis. Results Compared with the control group, the levels of urinary Alb, TRF, RBP, β2-MG and serum TNF-α in obese group were significantly increased, the differences were statistically significant (Pa <0.01). The expression of TNF-αmRNA and protein in obese group were significantly increased (Pa <0.01). Renal histopathological examination revealed glomerular hypertrophy in the obese group. Electron microscopy showed fusion of the foot process of epithelial cells. Some lipid droplets were seen in the cytoplasm of the tubular epithelial cells, thickening of the basement membrane and disappearance of the three-layer structure. Mesangial insertion. Conclusions Urine microalbumin abnormalities may occur in early stage of ORG mice. Abnormal expression of TNF-α in serum and kidney tissues may play an important role in the development of ORG.