|Comparative proteomics from serum and epidemiology of cleft lip and/or palate
|Place of Conferral
|目的：1. 建立血清中低分子量蛋白质组双向凝胶电泳体系。2. 寻找唇腭裂患者血清中差异表达蛋白质以及唇腭裂相关血清生物标记物。3. 制作唇腭裂动物模型，发现致畸胎鼠的孕鼠血清中差异表达蛋白质。4.了解甘肃省唇腭裂的患病状况与特点，分析与唇腭裂发生有关的影响因素，为该地区唇腭裂的预防提供参考。方法：1. 用C18填料富集血清中低分子量蛋白质，进行等电聚焦（isoelectric focusing, IEF）电泳和15%十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（15% SDS-PAGE）。2. 采用C18填料富集蛋白质法和丙酮沉淀蛋白法，提取CLP患者和对照组血清中蛋白质，分别进行双向凝胶电泳（2-DE），图像分析，识别差异表达的蛋白质点，基质辅助激光解吸飞行时间质谱（MALDI-TOF-MS）鉴定差异表达的蛋白质点。3. SPF级C57BL/6J受孕小鼠72只，随机分为维甲酸组、植物油组、空白对照组，维甲酸组和植物油组于GD10分别采用维甲酸植物油和植物油灌胃（维甲酸100 mg/kg，植物油10 ml/kg），三组均在GD14、GD17时分别实施眼球采血，离心取血清。2-DE和基质辅助激光解吸飞行时间质谱（MALDI-TOF-MS）分离和鉴定差异表达的蛋白质点。4. 对2000年1月-2009年3月住院的1010例先天性唇腭裂患者进行回顾性统计分析。结果：1. C18填料有效富集了血清中低分子量蛋白质，对2-DE各项条件进行优化、调整，2-DE图谱中低分子量蛋白质分辨率显著提高。2. 初步发现血清转铁蛋白在唇腭裂组血清中表达上调。3. 成功建立C57BL/6J小鼠唇腭裂动物模型，鉴定了两种差异表达蛋白-触珠蛋白和血清淀粉样蛋白P成分。4. 流行病学研究，CLP患者总体男性多于女性（1.89:1），有遗传史的病例数为64例，占总例数的6.34%；唇腭裂患者母亲妊娠前3个月接触风险因素者有356例（35.25%）；患者母亲生育年龄20岁以下者占总例数20%。结论：1. 建立了相对稳定的人血清低分子量蛋白质2-DE技术，为进一步开展疾病的血清蛋白质组学的研究奠定基础。2. CLP患者血清中转铁蛋白表达上调，与临床上唇腭裂患儿贫血症状相符。3. 触珠蛋白和血清淀粉样蛋白P物质有可能成为小鼠CLP的血清生物标记物。4. CLP病因复杂，可能与孕早期感染、重度呕吐、生育年龄低有关。
|Objective:1. To establish two dimensional gel electrophoresis (2-DE) system of low molecular weight proteins in serum.2. To identify differential proteins in serum of cleft lip and/or cleft palate (CLP) patients and CLP-related biomarkers in their serum.3. To establish CLP animal model and identify differential proteins in serum of pregnant mice whose fetuses had been in malformation. 4. To understand the clinical characteristics and deformity conditions in patients with CLP in Gansu Province, analyze specific factors related to CLP, and furnish evidence for etiology of CLP which prevention can be based.Methods:1. After low molecular weight proteins in serum were concentrated by the C18 absorbent, isoelectric focusing (IEF) and 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were employed to two dimensional electrophoresis.2. Proteins in serum of CLP patients and control group were extracted by C18 absorbent and aceton, respectively. 2-DE and matrix-assisted laser desorption mass/ionization spectrometry-time of flight (MALDI-TOF MS) were used to identify differential proteins in comparative serum.3. 72 pregnant C57BL/6J mice in SPF rank were randomly divided into all-trans retinoic acid group, vegetable oil group and control group. All-trans retinoic acid (100 mg/kg) and vegetable oil (10ml/kg) were injected into stomach of mice in all-trans retinoic acid group and vegetable oil group, respectively, in the 10th day of their gestation (GD10). Blood was drawn from eyeball of the three groups of mice in GD 14 and GD17. 2-DE and MALDI-TOF MS were used to separate and identify different proteins.4. 1010 samples of patients with CLP taken from January 2000 to March 2009 were analyzed by the retrospective evaluation survey.Results:1. Low molecular weight proteins in serum were concentrated effectively by the C18 absorbent. With a steady 2-DE technique procedure established under optimized conditions, low molecular weight protein dots in the 2-DE map were visualized in higher resolution.2. Preliminary study shows that serum transferrin in CLP group was more intensively expressed than that in the control group. 3. CLP animal model of C57BL/6J mice were established. Haptoglobin and serum amyloid P component were more intensively expressed than those in the control group.4. There were more male than female (1.89:1) in all patients with CLP. There were 64 cases (6.34%) whose family members also suffered from CLP; there were 356 cases (35.2...
马红霞. 唇腭裂血清比较蛋白质组学及流行病学研究[D]. 兰州. 兰州大学,2010.
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