神经管缺陷胎儿和神经管缺陷胎鼠羊水蛋白质标记物的比较研究

Amniotic fluid proteomic analysis of neural tube defects using surface enhanced laser desorption/ionization time-of-flight mass spectrometry

目的利用表面增强激光解析／离子化飞行时间质谱（SELDI—TOF-MS）技术研究神经管缺陷胎儿羊水和神经管缺陷胎鼠羊水的蛋白质谱变化规律。方法根据实验对象不同分为①经超声确诊为胎儿神经管缺陷的孕妇11例为畸形组，包括脊柱裂5例，无脑儿5例，脑积水1例，病例组孕母均于引产前经腹抽取羊水5ml，另选产前行羊膜腔穿刺取羊水做染色体核型检测，结果正常且孕周数相匹配的正常孕妇9例为对照组；②45只雌性Wistar大鼠（体重220-260g），随机分为畸形组（n=30）和对照组（n=15）。在标准条件下饲养，午夜雌：雄=4：1合笼。次日清晨8时阴道涂片，镜检精子，确定其妊娠日期，有精子者计为孕0日（E0）。在孕10日（E10）晨8时，畸形组雌鼠称体重，按120mg／kg称取维甲酸与矿物油混合均匀（维甲酸浓度为40mg／ml），经胃管一次注入孕鼠胃内。对照组仅将与畸形组同量且不含维甲酸的矿物油经胃管一次注入。在孕17d剖腹，每只胎鼠抽取羊水约0．1～0．3ml，选取11只脊柱裂畸形胎鼠的羊水，15只正常对照胎鼠的羊水。选用CM10蛋白质芯片，在PBSⅡC型蛋白质芯片阅读机读取数据。采用Ciphergen protein chip3．1．1软件对数据进行统计分析。结果在设定的优化相对分子量1～30×10^3范围内，神经管缺陷胎儿的孕母羊水中共检测到35个差异蛋白峰，其中7个有统计学意义，有6种蛋白质／多肽高表达，相对分子质量分别为14700，7995，15891，16027，13776，11040；1种蛋白质／多肽低表达，相对分子质量为23417。神经管缺陷胎鼠羊水中共检测到55个羊水差异蛋白峰，有统计学意义的差异蛋白峰有9个。脊柱裂畸形组有5种蛋白质／多肽高表达，相对分子质量分别为11658，27387，7898，11603，13829；4种蛋白质／多肽低表达，相对分子质量分别为5124，14702，5403，13626。将神经管缺陷胎鼠和神经管缺陷胎儿羊水蛋白质谱进行比较分析，发现二者羊水中共同蛋白质／多肽：4138（4145），7947（7941），8588（8588），9385（9390），14702（14700）。其中14700（P=0．006）差异具有统计学意义，很可能是神经管缺陷胎儿和神经管缺陷胎鼠羊水共同蛋白质／多肽标志物。结论神经管缺陷胎儿和胎鼠羊水中可能存在相同的特异性蛋白质／多肽标志物，相对分子质量14700的蛋白质／多肽对神经管缺陷诊断具有更重要的意义。

Objective Using SELDI-TOF：MS technology to detect specific protein biomarkers in maternal amniotic fluid of fetus with neural tube defects（NTDs）and pregnant rat amniotic fluid of fetal rat with NTDs. Methods Of the 20 maternal amniotic fluid samples obtained, 11 were from pregnant women with neural tube defects fetuses and 9 from healthy volunteers undergoing routine pregnancy pdtrasonography examination. Amniotic fluid from five spina bifida, five anencephaly and one hydrencephaly pregnancies was retrieved by transabdominal amniocentesis. 45 female Wistar White rats were randomly assigned to deformity groups （n = 30） and control groups（n = 15）. Pregnant Wistar rats in deformity groups were given a suspension of all-trans retinoic acid （40 mg/ml） and liquid paraffin and pregnant Wistar rats in control group were only given liquid paraffin on gestational days 10. About 0. 1 ml to 0. 3 ml Amniotic fluid of each pregnant rat was aspirated by a needle on gestational days 17. Eleven amniotic fluids of fetal rats with spina bifida and 15 normal controls were selected as experi- ment group for further SELDI. Protein chip biosystem （PBS Ⅱ C） and CM10 chip were purchased from Ciphergen Biosystems （USA）. The data were processed with the ProteinChip Software version 3. 1.1. Results A total of 35 qualified mass peaks （signal-to-noise ratio〉5） were detected in maternal amniotic fluid between 1,000 and 30,000 Da. Compared with the spectra of control groups, there were 7 potential maternal amniotic fluid biomarkers detected in the spectra of the neural tube defects patients. The protein expression was high in 6 of 7 （m/a 14 700, 7 995, 15 891 16 027, 13 776, 11 040） and low in 1 of 7 （m/z 23 417）. A total of 55 qualified mass peaks （signal-to-noise ratio〉5） were detected in pregnant rat amniotic fluid between 1,000 and 30,000. Compared with the spectra of control groups, there were 9 potential rat amniotic fluid biomarkers detected in the spectra of the neural tube defects rats. The protein expression was high in 5 of 9 （m/z 11 658, 27 387, 7 898, 11 603, 13 829） and low in 4 of 9 （m/z 5 124, 14 702, 5 403, 13 626）. Compared those protein biomarkers between rat amniotic fluid and maternal amniotic fluid, five mass peaks, including 4 138（4 145）, 7 947（7 941 ）, 8 588（8 588）, 9 385 （9 390）, 14 702 （14 700）, especially m/z 14 700 （P = 0. 006） may be the same protein/peptide of them. Conelusiom The biomarker proteins/peptides in maternal amniotic fluid of fetus with NTDs and pregnant rat amniotic fluid of fetal rat with NTDs were similar. This may be clinically pseful in diagnosing NTDs.