本文選題：鋅-α2-糖蛋白 + 造影劑腎病　； 參考：《大連醫(yī)科大學》2014年碩士論文

【摘要】：目的：造影劑腎�。╟ontrast-induced nephropathy，CIN）已成為院內(nèi)獲得性急性腎損傷的常見病因，而臨床上尚無敏感的檢測指標及有效的治療手段。如何早期診斷對預防CIN極其重要。而血肌酐在CIN診斷的敏感性及特異性方面存在缺陷，故發(fā)現(xiàn)一種能在CIN早期即可檢測到的、敏感的、特異的生物學標記物對預防CIN的發(fā)生具有重要意義。鋅-α2-糖蛋白（Zinc-α2-glycoprotein，ZAG）是正常腎小管上皮細胞表達的糖蛋白。本研究通過檢測經(jīng)皮冠狀動脈介入治療（percutaneous coronary intervention，PCI）患者尿中ZAG水平的變化情況，以及含碘造影劑（iodine contrast media，CM）誘導大鼠腎小管上皮細胞（NRK-52E）ZAG表達與細胞凋亡關系，來評價尿中ZAG水平對CIN的早期預測作用，并初步探討ZAG在CIN發(fā)病機制中的作用。 方法：選取我院心內(nèi)科行PCI術患者（包括不穩(wěn)定型心絞痛、陳舊性及急性心肌梗死的患者）10例，所有患者獲知情同意。應用的CM均為低滲非離子型單體造影劑碘佛醇370。造影劑注射方法均為Judkins法行冠脈造影，并根據(jù)術中所見冠狀動脈狹窄程度行冠脈介入治療。入選標準：所有患者均行PCI術，術前采取靜脈血，檢測血肌酐，并根據(jù)MDRD公式計算eGFR（estimated glomerularfiltration rate估測腎小球濾過率）≥90ml/min·1.73m2。排除標準：1）慢性腎臟��；2）各種原因所致的急性腎損傷，如近期腎毒性藥物的應用、腎前性缺血等；3）合并糖尿病、惡性腫瘤、高脂血癥的患者；4）7天內(nèi)接受過靜脈造影的患者。留取患者術前及術后2h、24h尿液標本，-40℃冰箱保存，檢測尿肌酐水平，用ELISA法檢測尿中ZAG濃度，尿ZAG水平用尿ZAG濃度與尿肌酐比值（mg/g）表示，同時采取造影前靜脈血標本，檢測生化指標包括：血肌酐、血尿素、膽固醇、低密度脂蛋白、甘油三酯。同時體外培養(yǎng)NRK-52E細胞，用不同碘濃度（25、50、100、140mgI/ml）的碘佛醇作用細胞2h，，用碘濃度為100mgI/ml的碘佛醇作用細胞2h、4h、6h。應用流式Annexin V-FITC/PI雙染色法檢測不同碘濃度及不同時間誘導下細胞凋亡率，同時分別收集各組細胞總蛋白，應用Western blot法檢測各組細胞ZAG蛋白表達情況。 結果：10例患者中，男6例，女4例，平均年齡58.40±6.50歲，術前尿素氮5.98±0.67mmol/L，膽固醇3.97±0.76mmol/L，甘油三酯1.39±0.34mmol/L，低密度脂蛋白2.09±0.57mmol/L，造影劑用量101.00±15.24ml，血肌酐63.83±9.65umol/L，計算eGFR為104.07±11.08ml/min·1.73m2。術前尿中ZAG水平為22.85±11.10mg/g。術后2h、24h尿中ZAG水平分別為67.44±44.94mg/g、32.24±18.55mg/g，與術前尿中ZAG水平相比，術后2h尿中ZAG水平較前升高，差異有統(tǒng)計學意義（p0.05）。術后24h雖較2h有下降，但仍高于術前，差異無統(tǒng)計學意義（p0.05）。各組尿ZAG水平與各項主要指標均無相關性。體外培養(yǎng)的NRK-52E細胞，用不同碘濃度的碘佛醇作用細胞2h，及碘濃度為100mgI/ml的碘佛醇作用細胞不同時間后，應用流式Annexin V-FITC/PI雙染色法檢測細胞凋亡率，與對照組相比，細胞凋亡率呈碘濃度(P0.05，n=3)和時間依賴性(P0.05，n=3)增加，應用Western blot法檢測ZAG蛋白表達水平亦呈碘濃度(P0.05，n=3)和時間依賴性(P0.05，n=3)增加。線性回歸分析提示ZAG蛋白表達水平與細胞凋亡率呈正相關。 結論：1、尿中ZAG水平升高可能反應含碘造影劑所致的早期腎損傷。 2、CM誘導的NRK-52E細胞中ZAG蛋白表達與細胞凋亡水平增加相關。
[Abstract]:Objective: contrast-induced nephropathy (CIN) has become a common cause of nosocomial acute renal injury, but there is no sensitive detection index and effective treatment. The early diagnosis is very important for the prevention of CIN. However, the sensitivity and specificity of serum creatinine in the diagnosis of CIN have been found to be defective. The sensitive, specific biological markers that can be detected at the early stage of CIN are important for the prevention of the occurrence of CIN. Zinc alpha 2- glycoprotein (Zinc- alpha 2-glycoprotein, ZAG) is a glycoprotein expressed in normal tubular epithelial cells. This study was conducted by detecting percutaneous coronary artery intervention (percutaneous coronary intervention, PCI). The changes in the level of ZAG in the urine and the iodine contrast media (CM) induced the relationship between the expression of NRK-52E ZAG and apoptosis in rat renal tubular epithelial cells (NRK-52E), to evaluate the early prediction of the level of ZAG in urine on CIN, and to explore the role of ZAG in CIN pathogenesis.
Methods: 10 patients (including unstable angina pectoris, obsolete and acute myocardial infarction) were selected in the Department of Cardiology of our hospital. All patients received informed consent. All of the patients were used in the CM of the low permeability nonionic single contrast agent iormol 370. contrast agent. The coronary angiography was performed by the Judkins method, and the coronary artery was observed according to the coronary artery in the operation. The degree of stenosis was treated with coronary intervention. Admission criteria: all patients underwent PCI surgery, preoperative venous blood was taken, serum creatinine was detected, and eGFR (estimated GlomerularFiltration Rate estimated glomerular filtration rate) > 90ml/min. 1.73m2. exclusion criteria were calculated according to the MDRD formula: 1) slow kidney disease; 2) acute renal injury caused by various causes, such as Recent use of nephrotoxic drugs, prerenal ischemia, etc.; 3) patients with diabetes, malignant tumor, hyperlipidemia, and hyperlipidemia; 4) patients who had received intravenous angiography within 7 days. 2h, 24h urine specimens, -40 centigrade refrigerators were preserved, urine creatinine levels were detected, ZAG concentration in urine was detected by ELISA, ZAG concentration in urine ZAG level and urinary ZAG concentration and urine ZAG level were measured. The urine creatinine ratio (mg/g) indicated that the serum creatinine, blood urea, cholesterol, low density lipoprotein, triglyceride, and NRK-52E cells were cultured in vitro, and the cell 2H was treated with iodopol with different iodine concentrations (25,50100140mgI/ml), and the action of iodopol with iodine concentration of 100mgI/ml was fine. Cell 2h, 4h, and 6h. were used to detect the cell apoptosis rate under different iodine concentration and different time by flow Annexin V-FITC/PI double staining. The total protein of each cell was collected and the expression of ZAG protein in each group was detected by Western blot.
Results: of the 10 cases, there were 6 males and 4 females, the average age was 58.40 + 6.50 years. The preoperative urea nitrogen 5.98 + 0.67mmol/L, cholesterol 3.97 + 0.76mmol/L, triglyceride 1.39 + 0.34mmol/L, low density lipoprotein 2.09 + 0.57mmol/L, contrast agent dosage 101 15.24ml, and serum creatinine 63.83 + 9.65umol/L, calculated eGFR as 104.07 + 11.08ml/min 1.73m2. preoperation urine. The level of middle ZAG was 22.85 + 11.10mg/g. after operation 2h, and the level of ZAG in 24h urine was 67.44 + 44.94mg/g, 32.24 + 18.55mg/g respectively. Compared with the level of ZAG in the urine before operation, the ZAG level of 2H urine was higher than that before the operation. The difference was statistically significant (P0.05). There was no correlation with the main indexes. The apoptosis rate of NRK-52E cells cultured in vitro was detected by flow type Annexin V-FITC/PI double staining method with iodopol acting cells with different iodine concentration and iodine concentration of 100mgI/ml. Compared with the control group, the apoptosis rate was iodine concentration (P0.05, n=3). The time dependence (P0.05, n=3) increased, and the expression level of ZAG protein was also detected by Western blot (P0.05, n=3) and time dependence (P0.05, n=3). The linear regression analysis showed that the expression level of ZAG protein was positively correlated with the rate of apoptosis.
Conclusion: 1. Elevated ZAG levels in urine may reflect early renal injury induced by iodinated contrast media.
2, the expression of ZAG protein in CM induced NRK-52E cells is associated with an increased level of apoptosis.
【學位授予單位】：大連醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R692.6

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