SELDI技术对开放性脊柱裂胎鼠羊水蛋白质组学的初步分析  

Amniotic Fluid Proteomic Analysis of Fetal Rat with Spina Bifida Using Surface Enhanced Laser Desorption/Ionization Time-of-flight Mass Spectrometry

作　　者：刘振江[1] 袁正伟[2] 赵群[1] 

机构地区：[1]中国医科大学附属盛京医院小儿外科,沈阳110004 [2]中国医科大学附属盛京医院卫生部小儿先天畸形重点实验室,沈阳110004

出　　处：《中国医科大学学报》2014年第1期26-30,33,共6页Journal of China Medical University

基　　金：国家自然科学基金青年基金(81000254)

摘　　要：目的利用表面增强激光解析/离子化飞行时间质谱(SELDI-TOF-MS)技术对脊柱裂胎鼠模型的羊水进行初步的蛋白质组学分析。方法将25只雌性Wistar大鼠随机分为脊柱裂组(n=15)和对照组(n=10)。于妊娠第10日(E10)8时,脊柱裂组孕鼠经胃管注入全反式维甲酸与矿物油的混合物(140 mg/kg);对照组孕鼠给予等量的橄榄油。于妊娠第17日(E17)取胎鼠,立体显微镜下抽取羊水(0.2 mL/只)并确定开放性脊柱裂胎鼠。选取60只脊柱裂胎鼠和55只对照组胎鼠的羊水离心取上清。采用PBSⅡC型蛋白质芯片阅读机读取数据。采用Ciphergen protein chip 3.1.1软件对数据进行统计分析。SELDI数据结果采用分类与回归树分析建立诊断模型,用于区分脊柱裂组胎鼠与对照组胎鼠。结果脊柱裂胎鼠羊水中共检测到55个差异蛋白峰,有统计学意义的差异蛋白峰有9个。包括5种蛋白峰高表达,优化相对分子质量(m/z)分别为11 658,27 387,7 898,11 603,13 831 Da;4种蛋白峰低表达,m/z分别为5 124,14 702,5 403,13 626 Da。选取m/z 13 831 Da和m/z 17 798 Da用于建立决策分类树诊断模型,诊断灵敏度93.33%,特异度96.36%,阳性预测值96.55%,阴性预测值96.36%。结论 SELDI技术可初步筛选出脊柱裂胎鼠羊水中的差异蛋白峰,这些蛋白可能是脊柱裂胎鼠羊水中的特异性羊水标志物。Objective Neural tube defects （NTDs） are usually identified by ultrasonography and confirmed by alpha-fetoprotein （AFP） assay and acetylcholinesterase （AchE） electrophoresis in amniotic fluid. Both of these biomarkers can be found positive in other etiologies. Amniotic fluid pro- teomic analysis of fetal rat with spina bifida using surface enhanced laser desorption/ionization time-of-flight mass spectrometry （SELDI-TOF-MS）. Methods 25 female Wistar white rats （ 250 - 280 g） were purchased from the animal center of China Medical University. The animals were main- tained in an environment controlled for temperature （ 20-25 ~C ） and humidity （60%-70%） with a 12-h light/dark cycle. Adult female rats were mat- ed with males of the same strain. The appearance of vaginal plugs in the female rat the morning after mating was determined E0. All animal experi- ments were approved by the local ethics committee. Spina bifida fetuses were induced with ATRA administration on El0. Twenty-five pregnant rats were divided into two groups, incluiding spina bifida groups （ n =15 ） and contrd groups （n =10）. Fifteen pregnant rats in spina bifida groups were subjected to intragastric administration of ATRA [ Sigma, 4% （wt/vol） in olive oil; 140 mg/kg body weight ] by a single gavage feeding. The same amount of olive oil was given to the other ten pregnant rats, which were the controls. All pregnant rats were killed on El7, and the fetuses and amniot- ic fluid were hmvested. The fetuses were examined for spina bifida aperta, and the amniotic fluid of each fetal sac was aspirated under a stereomicro- scope. We obtained 60 amniotic fluid from the fetal sacs of embryoswith spina bifida aperta exposed to ATRA treatment and 50 amniotic fluid from fe- tal sacs of normal controls. Amniotic fluid was immediately centrifuged at 4 000 r/min and 4 ~C for 10 minutes, aliquoted as 50 microliters, and stored at -80 ~C until analysis. Protein chip biosystem （ PBS II C） and CM10 chip were purchased from Ciphe

关 键 词：神经管缺陷 脊柱裂 表面增强激光解析 离子化飞行时间质谱 羊水 蛋白标志物 

分 类 号：R714.53[医药卫生—妇产科学]