【作者】 周海涛；

【导师】 孙开来；

【作者基本信息】 中国医科大学 ， 遗传学， 2004， 博士

【摘要】 目的 先天性马蹄内翻足是一种较常见的先天畸形，患儿一出生即表现为跟骨内翻，前足内收，跖骨旋后，踝关节下垂形成马蹄，以致行走时足外侧缘着地。畸形不影响患儿生存和生育，但严重影响其生活质量。马蹄内翻足的病理改变在神经、肌肉、骨骼、软骨及软组织方面均有表现。关于马蹄内翻足的发病机制存在宫内压迫、发育停滞以及遗传学说，但都缺少足够明确的证据。遗传流行病学研究表明遗传因素是马蹄内翻足发病重要因素。由于患儿标本难以取得，且存在加重患儿损伤的风险，建立马蹄内翻足动物模型对深入研究该病具有重要意义。 一切生命活动和生命现象最终要和蛋白质相联系，蛋白质才是生命的直接体现者。蛋白质组学是一个新兴的研究领域，研究特定病理生理条件下某种组织细胞的全部蛋白质。本实验拟从蛋白质入手，首先查找相关蛋白，然后据此寻找马蹄内翻足有关基因。 蛋白质组分析的首要步骤是将来自于全细胞、组织或生物体中多达数千种混合蛋白质进行分离。双向电泳技术是目前分辩率最高也最常用的工具，在寻找未知蛋白质、蛋白质组的全景分析、表达特性分析等方面有独特的功效。通过双向电泳对蛋白质多肽点进行高度分离纯化，辅之以质谱等鉴定技术，并结合生物信息学技术，可用于规模化未知蛋白质的筛查和鉴定。 因此，本研究首先利用全反式维甲酸(All-trans retinoic acid，ATRA)诱导孕鼠，探讨ATRA诱导马蹄内翻足样畸形的最佳剂量，建立稳定的马蹄内翻足动物模型，为深入研究马蹄内翻足发病机制提供物质基础；同时选择正常Wistar大鼠胚胎肌组织蛋白质组进行双向电泳分析，建立胚胎肌组织双向电泳技术平台；随后利用双向电泳分离模型鼠及正常鼠肌肉蛋白质组，比较模型与正常对照蛋白质点的差异，通过质谱测定结合生物信息学手段鉴定差异蛋白，为马蹄内翻足的研究提供基础资料。实验方法 1.马蹄内翻足大鼠胚胎模型建立 (1)将孕10d的Wistar近交系大鼠随机分为对照组和4个实验组，ArrRA(40m扩nil)用前混悬于矿物油中，用灌胃法按体重分别给予实验组大鼠一Zom含‘kg、13om岁kg、135m扩kg、x40 mg/kg，对照组(0 mg/吨组)给予140m扩kg组相应体积矿物油。 (2)孕21d剖腹取出胚胎，通过胎鼠畸形情况的检查和结构畸形病理分析确定内翻足样畸形(足前部内收内旋，足跟内翻，棵关节下垂，棵角大于130”)及其他畸形的发生情况，并记录死胎，吸收胎数和活胎体重身长，经数据统计处理确定维甲酸最佳诱导剂量。 (3)分别制作马蹄内翻足畸形鼠和正常对照鼠脊髓、椎骨和胫骨后肌群石蜡切片。参照试剂盒DeadEndT“而orometric TuNEL衍stem说明，对切片TuNEL法标记作凋亡分析。 2.胎鼠肌组织蛋白质组双向电泳，每份样品重复进行三次。 (l)取正常孕Zld胎鼠，实体解剖镜下胫骨后肌群，吸除液体，冰浴中捣碎，加少量裂解液研磨成匀浆，补加裂解液至lmF50mg组织，混匀，室温放置l小时，离心取上清，定量。 (2)等电、聚焦使用PROTEAN IEF等电聚焦仪。上样量pH3一10 IpG胶条为0.5 mg，pHS一IpG胶条为1.omg，上样体积为350诅。SOv，13h;250v，30min;IO00v，lh;最后10 000v聚焦60(X心vh。 (3)平衡与SDS电泳经两步平衡后SDS电泳，30v，30分钟后加压至120v，直到嗅酚蓝泳出玻璃板前缘为止。 (4)银染及考玛斯亮蓝染色，图像利用PDQuest7.0进行分析并标识白点分子量和等电点。 (5)选清晰匹配点手工切取并进行质谱分析。数据查询使用Peptldent搜索Swiss~Prot，同时利用Maseot或MS一Fit辅助搜索NCBInr。 3.模型鼠与正常鼠肌肉蛋白质组差异分析 (l)分离模型胎鼠及正常对照胎鼠胫骨后肌群，提取蛋白，等电聚焦，SDS电泳，考马斯亮蓝R一50染色，利用PDQuest软件对图像进行分析。 (2)差异分析重点分析高差异蛋白，包括未匹配斑点(仅在正常鼠出现，或仅在模型鼠出现)和上调10倍以上，上调5一10倍，下调5一10倍和下调10倍以上斑点。 (3)选取8个差异点作质谱测定，选取原则是:可重复，非边缘化，斑点清楚，匹配关系肯定，肉眼可见。所获质谱用Peptldent，Masoot，MS一Fit三个不同的查询软件搜索蛋白质序列数据库。搜索结果为同一蛋白时，再综合表观分子量、等电点判定结果。实验结果 1.维甲酸实验组可见包括马蹄内翻足样畸形在内的多种畸形。畸形发生率随剂量增高而增加。135m岁kg组马蹄内翻足样畸形发生率为60.98%，14Dm留kg组发生率为68.18%。140m扩kg组发生马蹄内翻足样畸形的胎鼠多数伴发其它畸形且表现胚胎毒性。135m留kg组单纯马蹄内翻足样畸形的发生率最高，达29.27%，胚胎毒性轻微。120m扩kg组与130m扩kg组马蹄内翻足畸形及其它畸形发生率很低。 足大体组织切片显示，典型畸形足与对照相比在横截面上足前部明显向内倾斜，距骨变小呈楔形位于跟骨后内侧;距骨前后轴缩短，呈菱形;胫骨和附骨趋向同一解剖面;距骨、跟骨前部均向内偏。胫骨躁趋向于距骨的掌面。排骨跺位于较后的位置，甚者与跟骨形成更多还原

【Abstract】 Idiopathic talipes equinovarus (ITEV, also called clubfoot) is a common type of congenital deformity of the foot and lower leg with an estimated incidence of 1/1,000 live births. The malformation typically manifests fixed in adduction, supination, and varus, in addition with soft tissue abnormalities. Previous epi-demiological studies have attempted to reveal the aetiology of ITEV but none has supported a significant association with any socioeconomic factors or teratogenic exposures. On the other hand, complex segregation analyses of ITEV pedigrees from different populations have strongly suggested a genetic etiology with Mende-lian and/or multifactorial inheritance. Although it may not influence the survival and reproductivity of patients, the ITEV deformity can severely affect their quality of life. Exploring its pathogenetic mechanisms may.provide important clues for its prediction, prevention and treatment.Proteins play a dominant role in all life activities and proteomics research has attracted much attention in recent years. Since it is difficult to perform biopsy for talipes equinovarus in human subjects, to construct a rat model for clut-foot-like deformity and analyze the variations in normal and abnormal rat pro-teomes by 2D gel electrophoresis and mass spectrometry, in combination with bioinformatics, may help to delineate roles of particular proteins in the pathogen-esis of clubfoot-like deformities. The objectives of this research include:(1) To construct a rat model for isolated-type clubfoot-like deformities with all-trans retinoic acid ( ATRA) and to determine the optimum dosage of ATRA.(2) To explore the method for analyzing the proteomes obtained from tibia-fibulae musculature of rat fetus.(3 ) To compare the disease and normal proteomes by 2D gel electrophore-sis and mass spectrometry, and to obtain some clue for further study of the basic molecular mechanisms for talipse equinovarus.MethodsPregnant Wistar rats were randomly assigned to one control and four experimental groups. On day 10 of pregnancy, variable doses (120 mg/kg, 130 mg/ kg, 135 mg/kg and 140 mg/kg, respectively) of ATRA in mineral oil suspension were given intragestically to the experimental groups, a volume of mineral oil equivalent to the 140 mg/kg group was given to the controls.On day 21 of gestation, rat fetus was removed through laparotomy and carefully examined under a stereomicroscope for structural abnormalities including clubfoot-like deformities ( Adduction/inner rotation of anterior portion of the feet, introversion of heels, dropping of ankle, with the angle between longitude axis of the foot and leg being greater than 130). Optimum dosage of ATRA was determined through statistical analysis.Hind legs of model rat fetus and normal controls were removed and made into serial sec tions along three anatomical planes of the foot, and analyzed under a light microscope.Sections of spinal cords, vertebra and musculatures from lower hind legs of model rat fetus and normal controls were made for apoptosis analysis by TUNEL labelled with DeadEnd?Fluorometric TUNEL System kit .Musculatures from hind lower legs of model rat fetus and normal controls were removed and dried, and then thoroughly mashed within ice-bath following addition of lysis solution (1 ml/50mg). The mash was left at room temperature for approximately 1 hour and then centrifuged. The supernatant was retained, with concentration determined.For 2D gel electrophoresis, immobilized pH gradient gel (IPG, pH 3-10 and pH 5-8) isoelectric focusing was set as the first dimension, and 12% SDS polyacrylamide gel electrophoresis was set as the second dimension. For isoelectric focusing, PROTEAN IEF cell was used. Loaded sample sizes were: 0. 5 mg (350ul) for pH 3-10 and 1.0 mg for pH 5-8, respectively. Isoelectric focus wasperformed at 50v for 13 hour, 250v for 30 min, 1000v for 1 hour,and finally focused under 10 000v for 60 000 vh.The IPG was bathed in balance solution I and II 15 minutes, respectively, then transferred onto更多还原