锌-α2-糖蛋白与原发性肾小球疾病肾间质纤维化及白蛋白诱导NRK-52E细胞转分化程度的关系

发布时间：2018-06-04 16:03

本文选题：锌α2糖蛋白 + 原发性肾小球疾病　；参考：《大连医科大学》2014年硕士论文

【摘要】：目的：肾间质纤维化在原发性肾小球疾病的演变和转归中起重要作用。锌-α2-糖蛋白是一种多功能糖蛋白，在肾小管上皮细胞中有表达。本实验研究锌-α2-糖蛋白与原发性肾小球疾病肾间质纤维程度的关系，以寻求诊断原发性肾小球疾病肾间质纤维化新的生物标志物。白蛋白刺激肾小管上皮细胞可促使其向成纤维细胞发生转分化（EMT）。锌-α2-糖蛋白在肾小管上皮细胞中有表达。本实验同时探讨锌-α2-糖蛋白与白蛋白诱导大鼠肾小管上皮细胞（NRK-52E）转分化程度的关系，，为临床诊断原发性肾小球疾病肾间质纤维化提供理论和实验依据。方法：选取我院临床诊断为原发性肾小球疾病行肾穿刺活检的患者共16例，清晨抽血并收集中断尿液，收集一般资料（性别、年龄、血肌酐、尿肌酐、胱抑素C、白蛋白、甘油三酯、胆固醇、24小时尿蛋白），根据MDRD公式计算eGFR。收集的血、尿标本用ELISA法检测其锌α2糖蛋白含量。用Masson染色法对肾脏病理组织进行染色，在低倍显微镜下观察肾间质纤维化程度。分析血、尿ZAG与上述生化指标及肾间质纤维化程度的关系。同时收集20例健康人的血、尿，检测上述生化指标，分析其与原发性肾小球疾病患者上述指标间是否存在差异性。体外用DMEM/F12培养基培养大鼠肾小管上皮细胞（NRK-52E），当细胞贴壁生长达60-70%融合时，给予不同浓度（0、0.5、1、5、10、30mg/ml）的去脂牛血清白蛋白（d-BSA）刺激NRK-52E细胞48小时；用同一浓度去脂牛血清白蛋白（d-BSA10mg/ml）刺激NRK-52E细胞不同时间（0、2、6、12、24、48小时）。收集总蛋白，通过Western blot法测定NRK-52E细胞ZAG及α-SMA蛋白表达水平。结果：1.原发性肾小球疾病患者共16例（男/女10/6），健康对照组共20例（男/女12/8）。原发性肾小球疾病组与健康对照组比较，血液、尿液ZAG、胆固醇明显升高，有显著差异性（P＜0.01）。与健康对照组比较，性别、年龄、血尿素氮、血肌酐、甘油三酯均无差异性（P＞0.05）； 2.16例原发性肾小球疾病患者血ZAG水平与尿ZAG、胱抑素C、血尿素氮、血肌酐、甘油三酯、胆固醇、eGFR无相关性（P＞0.05）；尿ZAG水平与胱抑素C、血尿素氮、血肌酐、eGFR无相关性（P＞0.05）。 3.16例原发性肾小球疾病患者肾间质纤维化程度按照Banff病理分级结果：0级（0例）、1级（9例）、2级（7例）、3级（0例）。病理类型主要为：IgA肾病（7例）、轻度系膜增生性肾小球肾炎（5例）、（Ⅱ期）膜性肾病（2例）、增生硬化性肾小球肾炎（1例）、紫癜性肾炎（轻度系膜增生型）（1例）。 4.16例原发性肾小球疾病患者肾间质纤维化程度与尿ZAG、血胱抑素C呈正相关相（P＜0.05）。 5．不同浓度d-BSA刺激NRK-52E细胞48小时，随d-BSA浓度增加，ZAG及α-SAM蛋白水平表达增加。NRK-52E细胞ZAG的表达与d-BSA呈浓度依赖性。 6．同一浓度（10mg/ml）的d-BSA刺激NRK-52E细胞不同时间，在0、2、6、12小时均无ZAG及α-SMA的表达；在24、48小时ZAG及α-SMA有表达，且48小时ZAG及α-SMA的蛋白表达水平较24小时明显升高。NRK-52E细胞ZAG的表达与d-BSA呈时间依赖性。 7．在白蛋白诱导下NRK-52E细胞ZAG的表达量随α-SMA表达量的增加而增加。结论：1.原发性肾小球疾病患者较健康对照组血、尿ZAG均有升高。 2.尿ZAG浓度可能成为原发性肾小球疾病肾间质纤维化的生物标志物。 3.在d-BSA刺激的NRK-52E细胞中，ZAG的表达水平与d-BSA呈浓度及时间依赖性，且ZAG的表达水平随α-SMA表达水平的升高而升高。 4. ZAG蛋白表达水平可能成为肾小管上皮细胞EMT程度的生物标志物。
[Abstract]:Objective: renal interstitial fibrosis plays an important role in the evolution and prognosis of primary glomerular disease. Zinc alpha 2- glycoprotein is a kind of multifunctional glycoprotein that is expressed in renal tubular epithelial cells. This experiment studies the relationship between zinc alpha 2- glycoprotein and the degree of renal interstitial fibrosis in primary glomerular disease in order to find the diagnosis of primary glomerular disease. A new biomarker for the disease of renal interstitial fibrosis.
Albumin stimulates renal tubular epithelial cells to induce transdifferentiation to fibroblasts (EMT). Zinc alpha 2- glycoprotein is expressed in renal tubular epithelial cells. The relationship between zinc alpha 2- glycoprotein and albumin induced renal tubular epithelial cells (NRK-52E) in rats was also discussed in order to diagnose primary glomerular disease in clinical practice. The theoretical and experimental basis of renal interstitial fibrosis is provided.
Methods: a total of 16 patients with primary glomerular disease diagnosed as renal biopsy were selected in our hospital. The blood was collected in the morning and urine was collected in the early morning. General data (sex, age, serum creatinine, creatinine, Cystatin C, albumin, triglyceride, cholesterol, 24 small time urine protein) were collected, and the blood collected by eGFR. was calculated according to the MDRD formula, and the urine specimen was used for E LISA method was used to detect the content of zinc alpha 2 glycoprotein. The renal pathological tissue was stained by Masson staining and the degree of renal interstitial fibrosis was observed under low power microscope. The relationship between blood, urine ZAG and the biochemical indexes and the degree of renal interstitial fibrosis was analyzed. At the same time, the blood and urine of 20 healthy people were collected and the above biochemical indexes were detected. Is there any difference between these indicators in patients with glomerular diseases?
Rat renal tubular epithelial cells (NRK-52E) were cultured with DMEM/F12 medium in vitro. When cell adherent growth reached 60-70% fusion, NRK-52E cells with different concentrations (0,0.5,1,5,10,30mg/ml) were given to stimulate NRK-52E cells for 48 hours, and NRK-52E cells were stimulated with the same concentration of defatted bovine serum albumin (d-BSA10mg/ml) at a different time (0). The total protein was collected and the expression level of ZAG and alpha -SMA protein in NRK-52E cells was determined by Western blot method. The expression level of NRK-52E protein was detected by 2,6,12,24,48.
Results: 1. there were 16 cases of primary glomerular disease (male / female 10/6), and 20 cases (male / female 12/8) in the healthy control group. Compared with the healthy control group, the primary glomerular disease group was significantly higher in blood, urine ZAG and cholesterol (P < 0.01). Compared with the healthy group, the sex, age, blood urea nitrogen, blood creatinine, triglyceride were all compared with the healthy group. No difference (P > 0.05);
Blood ZAG levels in 2.16 patients with primary glomerular disease were not associated with urinary ZAG, Cystatin C, blood urea nitrogen, serum creatinine, triglyceride, cholesterol, eGFR (P > 0.05); urine ZAG levels were not associated with cystatin C, blood urea nitrogen, serum creatinine, eGFR (P > 0.05).
The degree of renal interstitial fibrosis in 3.16 patients with primary glomerular disease: grade 0 (0 cases), grade 1 (9 cases), 2 grade (7 cases), 3 (0 cases). The main pathological types were IgA nephropathy (7 cases), mild mesangial glomerulonephritis (5 cases), (2 cases), hyperplasia sclerosing glomerulonephritis (1 cases), purpura nephritis (mild mesangial hyperplasia) (1 cases).
The level of renal interstitial fibrosis in 4.16 patients with Primary Glomerulopathy was positively correlated with urinary ZAG and serum cystatin C (P < 0.05).
5. different concentrations of d-BSA stimulated NRK-52E cells for 48 hours, and increased with the concentration of d-BSA. The expression of ZAG and alpha -SAM protein increased the expression of ZAG in.NRK-52E cells and was dependent on the concentration of d-BSA.
6. the same concentration (10mg/ml) d-BSA stimulated NRK-52E cells at different time, and there was no ZAG and alpha -SMA expression at 0,2,6,12 hours, and expressed in 24,48 hours ZAG and alpha -SMA, and the expression level of 48 hours ZAG and alpha -SMA increased significantly in the time dependence of the expression of.NRK-52E cells.
7. the expression of ZAG in NRK-52E cells increased with the increase of -SMA expression induced by albumin.
Conclusion: 1. patients with primary glomerular disease have higher blood and urine ZAG than healthy controls.
2. urinary ZAG concentration may be a biomarker of renal interstitial fibrosis in primary glomerulonephritis.
3. in d-BSA stimulated NRK-52E cells, the expression level of ZAG was d-BSA.
The expression level of ZAG increased with the increase of the expression level of -SMA.
4. the level of ZAG protein expression may be a biomarker of EMT level in renal tubular epithelial cells.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R692.6

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