【作者】 李文豪；

【导师】 袁正伟；

【作者基本信息】 中国医科大学 ， 儿科学， 2008， 硕士

【摘要】 目的先天性脊柱裂、先天性肛门直肠畸形均为常见的小儿先天性畸形,人群发生率分别为1‰-5‰、0.2‰-0.7‰,中国发生率明显高于西方国家。根据目前研究结果普遍认为先天性脊柱裂和先天性肛门直肠畸形均为多基因遗传病,而且是内在因素(遗传因素)和环境因素(致畸因素)共同作用的结果。但是,这些畸形的发病机制尚不清楚,病理改变复杂,而且没有良好的预防和治疗措施。因此,研究这些畸形的胚胎发生规律和早期诊断的方法,可以为畸形的早期治疗和预防提供必要的前期基础,对减少出生缺陷、提高人口素质具有极其重要意义。经过系列研究,我们证实先天性肛门直肠畸形大多数存在明显的腰骶部脊髓神经发育异常。利用药物致畸制作动物模型过程中我们也发现维甲酸和乙烯硫脲均可同时产生先天性脊柱裂、先天性肛门直肠畸形这两种畸形。目前发现的畸形相关基因中Hox基因与这二种畸形均有关,SHH和Gli3基因是先天性脊柱裂和先天性肛门直肠畸形的共同相关基因。许多研究已经证实口服叶酸可以预防神经管畸形的发生,同时也能减少肛门直肠畸形和四肢畸形的发生。因此,我们提出一个新的假设:先天性脊柱裂、先天性肛门直肠畸形可能存在共同的发生机制,它们的发生可能都是与胚胎期脊髓神经发育异常有关。蛋白质组学是一个新兴的研究领域,其核心在于大规模地对蛋白质进行综合分析,通过对某物种、个体、器官、组织或细胞的全部蛋白质性质包括表达水平、结构、翻译后修饰、细胞内定位、蛋白质相互作用等的研究,对蛋白质所执行的生理性、病理性生命活动做出最精细、最准确、最本质的阐述。蛋白质本身的存在形式、活动规律仍依赖于直接对蛋白质的研究来解决。因此,直接研究蛋白质的变化具有不可替代的意义。而且,随着蛋白质组学的研究进展,基于固相pH梯度等电聚焦(IPG)的双向凝胶电泳和各种质谱技术结合,对蛋白质进行分离和鉴定,已经成为蛋白质组研究中的核心技术。以固相pH梯度等点聚焦为第一向的双向电泳技术是当前分辨率最高、信息量最大的电泳技术。因此本研究将采用双向电泳技术,研究两种畸形动物模型在胚胎发育阶段的脊髓神经组织蛋白质组的表达情况和变化规律,在蛋白质水平探讨两种畸形之间的共同发生规律,为畸形的胚胎早期诊断和治疗提供基础资料。方法1、建立脊柱裂、肛门直肠畸形大鼠胚胎模型(1)将孕10d大鼠随机分为对照组和实验组,按160mg/kg体重用灌胃法给予实验组大鼠,对照组给予相应体积橄榄油。(2)孕21d剖腹取出胚胎,取出脊髓组织(范围:第12胸椎下缘至脊髓末端)。2、模型胎鼠与对照胎鼠脊髓组织蛋白质双向电泳图谱差异分析(1)提取胎鼠脊髓组织的总蛋白质,Bradford法进行蛋白定量。(2)等电聚焦使用Ettan IPGphorⅡ等电聚焦仪。聚焦后的胶条经两步平衡后进行SDS-PAGE电泳,银染、扫描后用ImageMaster 2D Platinum 6.0软件进行图像分析,寻找差异蛋白点。结果1、成功制作了脊柱裂、肛门直肠畸形大鼠胚胎模型孕21d剖检共获取胎鼠127只。对照组20只,维甲酸组中剖出存活胎鼠96只,死胎及吸收胎11只,其中给药无畸形组19只,单纯肛门直肠畸形组29只,肛门直肠畸形合并腰骶部显性脊柱裂组28只。2、维甲酸组胎鼠和对照组胎鼠脊髓组织双向电泳图谱差异分析结果使用pH3～10,24cm胶条获得分离较好的蛋白表达图谱,对照组获得可识别平均斑点数为1355±30个,给药无畸形组1425±48个,单纯肛门直肠畸形组1340±55个,肛门直肠畸形合并腰骶部显性脊柱裂组1056±68个。肛门直肠畸形组与肛门直肠畸形合并脊柱裂组间差异蛋白点有164个,87个在肛门直肠畸形组中高表达,77个蛋白低表达。有3个点仅在肛门直肠畸形组中表达,70个点在肛门直肠畸形合并脊柱裂组表达。结论初步获得了脊柱裂和肛门直肠畸形模型胎鼠脊髓蛋白质双向电泳图谱,发现脊柱裂、肛门直肠畸形模型胎鼠与对照组胎鼠脊髓组织之间存在一些差异表达的蛋白点,为进一步研究脊柱裂和肛门直肠畸形发病机制奠定了基础。更多还原

【Abstract】 ObjectiveCongenital anorectal malformation and spina bifida are both common congenital disorders which greatly influence life quality,and incidence are 1‰-5‰、0.2‰-0.7‰, respectively.The causes of these disorders are generally considered as the result from synergistic action of internal agent（genetic factor）and environmental factor （teratogenic factor）.However,the aetiology and pathology of these disorders are still unclear,and accordingly no good measures for prevention and therapy.Therefore, studying embryogenesis pattern and early diagnosis of these disorders is necessary to provide message for early treatment and prevention,event-rally to reduce birth defect and raise population diathesis.Though series investigations,we confirmed most cases of congenital anorectal malformation had lumbosacral spinal cord neural developmental anomaly.Among the process of making animal model by medicine’s teratogenic effects,we found Retinoid acid and ethylene thiourea both could induce congenital anorectal malformation and spina bifida.At present,we have found Hox gene was involved in the aetiology of these disorders,and SHH、Gli3 were common related genes of these disorders.Several investigations have confirmed folic acid supplementation could prevent the development of neural tube defects,and decrease the number of anorectal malformation and dysmelia.Therefore,we propose a new hypothesis:there may be some common developmental mechanism between congenital anorectal malformation and spina bifida, and spinal nerves maldevelopment may be involved in the development of these two disorders.Proteomics is the science and methodology with which to simultaneously study the expression of all proteins（the protome,rather than individual protein）in a cell, tissue,or organ.Proteomic analysis of identified global proteins can provide expression profiles of the proteins and their PTMs in cell.So,direct investigation on protein profiles is not substitutional.Despite altemative technologies that have emerged, 2-dimensional（2-D）electrophoresis is currently the only technique that can be routinely applied for parallel quantitative expression profiling of large sets of complex protein mixtures.Furthermore,it delivers a map of intact proteins that reflects changes in protein expression level,isoforms,or post-translational modifications.Last but not least,today’s 2-D electrophoresis technology with immobilized pH gradients（IPGs） has overcome the former limitations of carrier-ampholyte-based 2-D electrophoresis with respect to reproducibility,handling,resolution.Here,we employ a 2-D gel-based proteomic technique to detect the global changes of the spinal cord protein expression in rats that have developed animal models, and approach the common mechanism between congenital anorectal malformation and spina bifida at protein level,to provide elementary datas for early diagnosis and treatment during embryonic period.Methods1.Construct rat embryonic models for spina bifida and anorectal malformation.（1）Pregnant Wistar rats were randomly assigned to either control（n=2）or RA （n=19）groups.On E10,RA group received intragastrically all-trans-retinoic acid （160mg/Kg dissolved in olive oil at a concentration of 40mg/ml）,whereas control animals received 1 ml of olive oil.（2）On E21,Cesarean section was carried out,and the pups were removed,then spinal cord tissue was removed(form inferior border of the 12^thvertebrae thoracales to the end of spinal cord).2.Differential protein expression analysis of spinal cord between control group and RA group in the rat fetus.（1）Total protein of spinal cord was extracted from control and RA rat fetus, protein concentrations were determined by Bradford.（2）For isoelectric focusing,Ettan IPGphorⅡwas used.Samples were applied to pH 3-10,24cm non-linear IPG strips and two-dimensional electrophoresis was performed,using 12.5%second dimension gels.Analytical gels were silver stained and scanned.Protein patterns were analyzed using ImageMaster 2D Platinum 6.0 software to find differential spots.Results1.Construct the rat model for spina bifida and anorectal malformation.127 rat fetus were obtained totally on E21.20 for control group,and 107 for RA group.Among the RA group,there were 19 fetus for undefect group with RA exposure,29 for isolated anorectal malformation,and 28 for anorectal malformation coincidence with spina bifida in lumbosacral area.2.Analyze the differential protein expression between RA group and control group.The 2D gel protein spot patterns were gained through pH3～10、24cm strips,on average,1355±30 protein spots were detected in control group,and 1425±48、1340±55、1056±68 spots in undefect group with RA exposure、isolated anorectal malformation group and anorectal malformation coincidence with spina bifida group, respectively.In total,we found that 164 protein spots that showed significant differences between the isolated anorectal malformation and anorectal malformation coincidence with spina bifida groups,87 spots from isolated anorectal malformation group were up-regulated,and 77 spots were down-regulated.There were 3 spots which only expressed in isolated anorectal malformation group,and 70 spots in anorectal malformation coincidence with spina bifida group.ConclusionSpinal cord protein profiles of spina bifida and anorecotal malformations were established,and some differential protein spots were found.The data obtained in this study may be helpful for further studies on pathogenesy of spina bifida and anorectal malformation.更多还原