应用SELDI—TOF—MS技术筛选输尿管部分梗阻大鼠血清生物学标志物的初步研究

Surface enhanced laser desorption/ionization-time of flight-mass spectrometry for detecting serum protein biomarkers of partial unilateral ureteral obstruction in rats

目的应用表面增强激光解析电离－飞行时间质谱（surface enhanced laser desorption/i-onization-time of flight-mass spectrometry,SELDI-TOF-MS）技术，检测正常大鼠和输尿管部分梗阻（partial unilateral ureteral obstruction,PUUO）肾小管上皮细胞转化前后的大鼠血清，筛选出一组特异性标志物，为先天性肾积水及梗阻性。肾病选择适宜的手术及治疗时机提供依据。方法PUUO组大鼠（n=48）随机数字表法分为梗阻12h、1d、2d、3d、5d、7d等6个时间段组，每组8只；空白对照组（n=30）。制作PUUO模型，于不同时间点分别处死大鼠，分别取双侧肾脏制作石蜡切片、腹主动脉穿刺获取血清，-80℃保存。应用免疫荧光双标方法确定动物模型中肾小管上皮细胞发生上皮间质转化的时间点，选取动物模型肾小管上皮细胞转化前后收集的血清标本，利用SELDI-TOF-MS技术筛选出大鼠血清中肾小管上皮细胞间质转化前后的标志物。结果大鼠PUUO术后3d即有肾小管上皮细胞表达成纤维细胞标志物，发生细胞表型转化，即发生上皮间质转化，随梗阻时间延长，肾脏中发生转化的肾小管上皮细胞逐渐增多。发现PUUO大鼠血清中有27种蛋白质标记物，其中10种蛋白质高表达，17种蛋白质低表达。结论大鼠PUUO模型的手术侧肾脏可用于研究肾间质纤维化。大鼠肾间质纤维化过程中肾小管上皮细胞存在上皮间质转化现象。应用SELDI-TOF-MS技术筛查出PUUO前后大鼠血清的差异蛋白峰可能是PUUO大鼠血清的特异性蛋白质标志物。

Objective To detect the biomarkers in rats with pantial unilateral ureteral obstruction（PUUO） by surgace enhanced laser desorption/ionization time-of-flight mass spectrometry （SELDI-TOF MS）. Methods A total of 78 Sprague-Dawley rats were divided into sham-operated （n = 30） and PUUO （n = 48） groups. All rats were sacrificed at day 1/2, 1, 2, 3, 5, 7 post-operation and renal and serum samples harvested. Immunofluorescent stains of E-cadherin and alpha-small muscle actin （a-SMA） were employed for observing epithelial to mesenchymal transition （EMT） of tubular epithelial cell in kidney. Proteinchip reader PBS Ⅱ C and CM10 chip were employed. And data analyses were performed by ProteinChip Software. Results Compared with the spectra of control group, 27 potential biomarkers were detected in the spectra of PUUO group. The protein expression was high in 10/27 and low in 17/27. Under given conditions, Biomarker Pattern Software automatically selected 2 biomarker proteins （m/z 5901, 2323）for establishing a three-layer decision tree to differentiate two groups. The sensitivity and specificity of decision tree was 100. 0% and 100. 0% respectively. Conclusions The screening may be performed by SELDI-TOF-MS for specific serum protein biomarkers of PUUO in rat. And it may serve as a tool of early diagnosis with EMT of tubular epithelial cell.