锌-α2-糖蛋白对造影剂肾病早期预测及其在碘造影剂诱导大鼠肾小管上皮细胞凋亡中的作用

发布时间：2018-07-03 07:07

本文选题：锌-α2-糖蛋白 + 造影剂肾病　；参考：《大连医科大学》2014年硕士论文

【摘要】：目的：造影剂肾病（contrast-induced nephropathy，CIN）已成为院内获得性急性肾损伤的常见病因，而临床上尚无敏感的检测指标及有效的治疗手段。如何早期诊断对预防CIN极其重要。而血肌酐在CIN诊断的敏感性及特异性方面存在缺陷，故发现一种能在CIN早期即可检测到的、敏感的、特异的生物学标记物对预防CIN的发生具有重要意义。锌-α2-糖蛋白（Zinc-α2-glycoprotein，ZAG）是正常肾小管上皮细胞表达的糖蛋白。本研究通过检测经皮冠状动脉介入治疗（percutaneous coronary intervention，PCI）患者尿中ZAG水平的变化情况，以及含碘造影剂（iodine contrast media，CM）诱导大鼠肾小管上皮细胞（NRK-52E）ZAG表达与细胞凋亡关系，来评价尿中ZAG水平对CIN的早期预测作用，并初步探讨ZAG在CIN发病机制中的作用。方法：选取我院心内科行PCI术患者（包括不稳定型心绞痛、陈旧性及急性心肌梗死的患者）10例，所有患者获知情同意。应用的CM均为低渗非离子型单体造影剂碘佛醇370。造影剂注射方法均为Judkins法行冠脉造影，并根据术中所见冠状动脉狭窄程度行冠脉介入治疗。入选标准：所有患者均行PCI术，术前采取静脉血，检测血肌酐，并根据MDRD公式计算eGFR（estimated glomerularfiltration rate估测肾小球滤过率）≥90ml/min·1.73m2。排除标准：1）慢性肾脏病；2）各种原因所致的急性肾损伤，如近期肾毒性药物的应用、肾前性缺血等；3）合并糖尿病、恶性肿瘤、高脂血症的患者；4）7天内接受过静脉造影的患者。留取患者术前及术后2h、24h尿液标本，-40℃冰箱保存，检测尿肌酐水平，用ELISA法检测尿中ZAG浓度，尿ZAG水平用尿ZAG浓度与尿肌酐比值（mg/g）表示，同时采取造影前静脉血标本，检测生化指标包括：血肌酐、血尿素、胆固醇、低密度脂蛋白、甘油三酯。同时体外培养NRK-52E细胞，用不同碘浓度（25、50、100、140mgI/ml）的碘佛醇作用细胞2h，，用碘浓度为100mgI/ml的碘佛醇作用细胞2h、4h、6h。应用流式Annexin V-FITC/PI双染色法检测不同碘浓度及不同时间诱导下细胞凋亡率，同时分别收集各组细胞总蛋白，应用Western blot法检测各组细胞ZAG蛋白表达情况。结果：10例患者中，男6例，女4例，平均年龄58.40±6.50岁，术前尿素氮5.98±0.67mmol/L，胆固醇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，造影剂用量101.00±15.24ml，血肌酐63.83±9.65umol/L，计算eGFR为104.07±11.08ml/min·1.73m2。术前尿中ZAG水平为22.85±11.10mg/g。术后2h、24h尿中ZAG水平分别为67.44±44.94mg/g、32.24±18.55mg/g，与术前尿中ZAG水平相比，术后2h尿中ZAG水平较前升高，差异有统计学意义（p0.05）。术后24h虽较2h有下降，但仍高于术前，差异无统计学意义（p0.05）。各组尿ZAG水平与各项主要指标均无相关性。体外培养的NRK-52E细胞，用不同碘浓度的碘佛醇作用细胞2h，及碘浓度为100mgI/ml的碘佛醇作用细胞不同时间后，应用流式Annexin V-FITC/PI双染色法检测细胞凋亡率，与对照组相比，细胞凋亡率呈碘浓度(P0.05，n=3)和时间依赖性(P0.05，n=3)增加，应用Western blot法检测ZAG蛋白表达水平亦呈碘浓度(P0.05，n=3)和时间依赖性(P0.05，n=3)增加。线性回归分析提示ZAG蛋白表达水平与细胞凋亡率呈正相关。结论：1、尿中ZAG水平升高可能反应含碘造影剂所致的早期肾损伤。 2、CM诱导的NRK-52E细胞中ZAG蛋白表达与细胞凋亡水平增加相关。
[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.
【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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