本文選題：鋅α2糖蛋白 + 原發(fā)性腎小球疾病��； 參考：《大連醫(yī)科大學(xué)》2014年碩士論文

【摘要】：目的：腎間質(zhì)纖維化在原發(fā)性腎小球疾病的演變和轉(zhuǎn)歸中起重要作用。鋅-α2-糖蛋白是一種多功能糖蛋白，在腎小管上皮細(xì)胞中有表達(dá)。本實(shí)驗(yàn)研究鋅-α2-糖蛋白與原發(fā)性腎小球疾病腎間質(zhì)纖維程度的關(guān)系，以尋求診斷原發(fā)性腎小球疾病腎間質(zhì)纖維化新的生物標(biāo)志物。 白蛋白刺激腎小管上皮細(xì)胞可促使其向成纖維細(xì)胞發(fā)生轉(zhuǎn)分化（EMT）。鋅-α2-糖蛋白在腎小管上皮細(xì)胞中有表達(dá)。本實(shí)驗(yàn)同時(shí)探討鋅-α2-糖蛋白與白蛋白誘導(dǎo)大鼠腎小管上皮細(xì)胞（NRK-52E）轉(zhuǎn)分化程度的關(guān)系，，為臨床診斷原發(fā)性腎小球疾病腎間質(zhì)纖維化提供理論和實(shí)驗(yàn)依據(jù)。 方法：選取我院臨床診斷為原發(fā)性腎小球疾病行腎穿刺活檢的患者共16例，清晨抽血并收集中斷尿液，收集一般資料（性別、年齡、血肌酐、尿肌酐、胱抑素C、白蛋白、甘油三酯、膽固醇、24小時(shí)尿蛋白），根據(jù)MDRD公式計(jì)算eGFR。收集的血、尿標(biāo)本用ELISA法檢測(cè)其鋅α2糖蛋白含量。用Masson染色法對(duì)腎臟病理組織進(jìn)行染色，在低倍顯微鏡下觀察腎間質(zhì)纖維化程度。分析血、尿ZAG與上述生化指標(biāo)及腎間質(zhì)纖維化程度的關(guān)系。同時(shí)收集20例健康人的血、尿，檢測(cè)上述生化指標(biāo)，分析其與原發(fā)性腎小球疾病患者上述指標(biāo)間是否存在差異性。 體外用DMEM/F12培養(yǎng)基培養(yǎng)大鼠腎小管上皮細(xì)胞（NRK-52E），當(dāng)細(xì)胞貼壁生長達(dá)60-70%融合時(shí)，給予不同濃度（0、0.5、1、5、10、30mg/ml）的去脂牛血清白蛋白（d-BSA）刺激NRK-52E細(xì)胞48小時(shí)；用同一濃度去脂牛血清白蛋白（d-BSA10mg/ml）刺激NRK-52E細(xì)胞不同時(shí)間（0、2、6、12、24、48小時(shí)）。收集總蛋白，通過Western blot法測(cè)定NRK-52E細(xì)胞ZAG及α-SMA蛋白表達(dá)水平。 結(jié)果：1.原發(fā)性腎小球疾病患者共16例（男/女10/6），健康對(duì)照組共20例（男/女12/8）。原發(fā)性腎小球疾病組與健康對(duì)照組比較，血液、尿液ZAG、膽固醇明顯升高，有顯著差異性（P＜0.01）。與健康對(duì)照組比較，性別、年齡、血尿素氮、血肌酐、甘油三酯均無差異性（P＞0.05）； 2.16例原發(fā)性腎小球疾病患者血ZAG水平與尿ZAG、胱抑素C、血尿素氮、血肌酐、甘油三酯、膽固醇、eGFR無相關(guān)性（P＞0.05）；尿ZAG水平與胱抑素C、血尿素氮、血肌酐、eGFR無相關(guān)性（P＞0.05）。 3.16例原發(fā)性腎小球疾病患者腎間質(zhì)纖維化程度按照Banff病理分級(jí)結(jié)果：0級(jí)（0例）、1級(jí)（9例）、2級(jí)（7例）、3級(jí)（0例）。病理類型主要為：IgA腎�。�7例）、輕度系膜增生性腎小球腎炎（5例）、（Ⅱ期）膜性腎�。�2例）、增生硬化性腎小球腎炎（1例）、紫癜性腎炎（輕度系膜增生型）（1例）。 4.16例原發(fā)性腎小球疾病患者腎間質(zhì)纖維化程度與尿ZAG、血胱抑素C呈正相關(guān)相（P＜0.05）。 5．不同濃度d-BSA刺激NRK-52E細(xì)胞48小時(shí)，隨d-BSA濃度增加，ZAG及α-SAM蛋白水平表達(dá)增加。NRK-52E細(xì)胞ZAG的表達(dá)與d-BSA呈濃度依賴性。 6．同一濃度（10mg/ml）的d-BSA刺激NRK-52E細(xì)胞不同時(shí)間，在0、2、6、12小時(shí)均無ZAG及α-SMA的表達(dá)；在24、48小時(shí)ZAG及α-SMA有表達(dá)，且48小時(shí)ZAG及α-SMA的蛋白表達(dá)水平較24小時(shí)明顯升高。NRK-52E細(xì)胞ZAG的表達(dá)與d-BSA呈時(shí)間依賴性。 7．在白蛋白誘導(dǎo)下NRK-52E細(xì)胞ZAG的表達(dá)量隨α-SMA表達(dá)量的增加而增加。 結(jié)論：1.原發(fā)性腎小球疾病患者較健康對(duì)照組血、尿ZAG均有升高。 2.尿ZAG濃度可能成為原發(fā)性腎小球疾病腎間質(zhì)纖維化的生物標(biāo)志物。 3.在d-BSA刺激的NRK-52E細(xì)胞中，ZAG的表達(dá)水平與d-BSA呈濃度 及時(shí)間依賴性，且ZAG的表達(dá)水平隨α-SMA表達(dá)水平的升高而升高。 4. ZAG蛋白表達(dá)水平可能成為腎小管上皮細(xì)胞EMT程度的生物標(biāo)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.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R692.6

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