【摘要】 目的 明确大鼠肾脏缺血/再灌注(ischemia/reperfusion, I/R)损伤后不同时间中性粒细胞明胶酶相关脂质运载蛋白(neutrophil gelatinase-associated lipocalin, NGAL)表达水平及肾组织自噬状态的变化情况及意义。方法 建立大鼠肾脏I/R损伤模型，将30只Wistar雄性大鼠按随机数字表法分为假手术组(Sham组)和I/R组，按照再灌注时间，将I/R组分为2h组、6h组、24h组和48h组。在上述不同时间点分别留取大鼠血、尿及肾组织标本。采用酶联免疫吸附试验(enzyme-linked immunoadsordent assay, ELISA)检测血和尿中NGAL水平；全自动生化分析仪检测血尿素氮(blood urea nitrogen, BU)和血肌酐(serum creatinine, SCr)水平；苏木精-伊红染色法检测肾组织损伤程度并进行损伤程度积分；应用原位末端标记法(TdT-mediated dUTP nick end labeling, TUNEL)法检测肾小管上皮细胞凋亡情况；实时荧光定量聚合酶链反应(real-time quantitative polymerase chain reaction, RT-qPCR)检测自噬相关基因LC3及Beclin-1的表达。结果 血和尿中NGAL表达水平在I/R损伤后2h即开始升高，6h达到高峰，24h后呈下降趋势。BU及Scr在I/R损伤后6h开始升高，24h达到高峰。TUNEL阳性细胞于I/R损伤后6h开始增加，24h数量达峰值。苏木精-伊红染色显示，I/R损伤后各组均有不同程度的肾小管上皮细胞肿胀、坏死。从I/R损伤后6h开始，LC3和Beclin-1基因表达呈上升趋势，24h后LC3和Beclin-1基因表达量达高峰。结论 大鼠肾脏I/R损伤早期NGAL表达水平即升高，早于BU、SCr水平的变化，可以作为早期诊断肾脏I/R损伤的分子指标。大鼠肾脏I/R损伤加重过程中NGAL表达呈增加趋势，自噬被激活，提示NGAL蛋白及自噬均在I/R损伤过程中发挥一定的作用。更多还原

【Abstract】 Objective To investigate the changes and clinical significance of the expression of neutrophil gelatinase-associated lipocalin(NGAL) and autophagy during different time of renal ischemia/reperfusion(I/R) injury in rats.Methods The rat renal I/R injury model was established, thirty male Wistar rats were divided by random number table method into sham operation group(Sham group) and I/R group, according to the different time of reperfusion after I/R injury, the I/R group was divided into 4subgroups: 2h group, 6h group, 24h group, 48h group. Blood, urine and renal tissue samples were collected at different time points, NGAL levels of blood and urine were detected by enzyme-linked immunoadsordent assay(ELISA) method. Blood urea nitrogen(BU) and serum creatinine(SCr) were detected by automatic biochemical analyzer. Hematoxylin-eosin staining was used to detect the degree of histopathological injury and score the degree of injury. TdT-mediated dUTP nick end labeling(TUNEL) method was used to detect the apoptosis of renal tubular epithelial cells; the expression of autophagy related genes LC3 and Beclin-1gene was detected by real-time quantitative polymerase chain reaction(RT-qPCR) method. Results The expression levels of NGAL in blood and urine were elevated in the 2h after I/R injury, and peaked at 6h of reperfusion, and showed a downward trend at 24h. BU and Scr values began to increase after the 6h reperfusion of I/R injury and peaked at 24h of reperfusion. TUNEL positive cells began to increase at 6h after I/R injury, the number of the highest was at 24h reperfusion of I/R injury. Hematoxylin-eosin staining showed that there were different degrees of swelling and necrosis of renal tubular epithelial tissue in all groups after I/R injury. The expression of LC3 and Beclin-1 gene began to increased after the 6h reperfusion of I/R injury and peaked at 24h of reperfusion. Conclusion The expression level of NGAL increased at the early stage of renal I/R injury in rats, which was earlier than the changes of BU and SCr levels, and could be used as a molecular indicator for early diagnosis of renal I/R injury. During the aggravation of renal I/R injury, the expression of NGAL was increased and autophagy was activated, which indicated that NGAL and autophagy played a certain role in the process of renal I/R injury.更多还原