摘要
吉兰-巴雷综合征（Guillain-Barré syndrome，GBS）是目前急性弛缓性瘫痪的最主要原因，临床上主要表现为对称性进行性弛缓性瘫痪, 伴有或不伴有呼吸肌麻痹、运动颅神经麻痹。随着近年来神经生理和病理学的研究进展，将GBS重新划分为急性炎症性脱髓鞘性多发神经病（acute inflammatory demyelinating polyneuropathy，AIDP）、急性运动轴索性神经病（acute motor axonal neuropathy，AMAN）、急性运动感觉性轴索性神经病（acute motor and sensory axonal neuropathy，AMSAN）、Miller Fisher 综合征(Miller Fisher syndrome, MFS)等几种亚型。约2/3的GBS患者发病前1～3周有前驱感染史，多为呼吸道感染或胃肠道感染。空肠弯曲菌（Campylobacter jejuni，Cj）是引起急性肠炎最常见的一种肠道病原菌，近年来发现，Cj感染与部分GBS发病有密切关系。国内李春岩等成功地在GBS病人粪便中分离出多株Cj，并率先在国际上用Cj活菌感染或纯化的Cj脂多糖免疫动物，诱发出GBS的动物模型，明确了Cj感染是GBS的一个重要病因。我国人群以及家禽、家畜中Cj的感染率都很高，而只有某些特殊血清型Cj菌株的感染可引起GBS。与其它几种类型相比，AMAN病情重，致残率高，预后差，严重者可导致死亡。因此，确定致病菌型、进行致病菌型的分布调查并进行有效的人群干预以减少发病率显得尤为重要。
虽然目前有学者倾向于认为Cj的脂多糖与人周围神经的神经节苷脂之间的分子模拟机制在GBS发病中起重要作用，但Cj感染导致GBS的确切发病机制尚不十分明确。许多学者在基因水平上对GBS相关Cj的分子特征及其特异性标志物进行了研究，但尚未得出一致的结论。由于与GBS相关的Cj的分子生物学特征目前不很清楚，使得致病菌株的基因水平鉴定、疫苗研制及人群干预等措施的实施都受到一定限制。
近年来基因组研究发展迅猛，但面对大量的遗传信息，人们对其功能却不清楚，因此提出“功能基因组学”，即后基因组学这个概念。蛋白质作为基因的表达产物、基因功能的最终体现者，蛋白质组学(proteomics)
已成为功能基因组学的主要研究内容之一。蛋白质双向电泳(two-dimensional polyacrylamide gel electrophoresis，2-DE)和质谱(mass spectrometry)是目前蛋白质组学研究的两大支柱技术。
蛋白质组学已越来越多地应用于疾病相关的差异表达蛋白的研究。细菌在不同的条件下或由于不同株系间的差别, 其所表达的蛋白质组会发生变化。找到与正常细胞差异表达的蛋白质, 将有助于发现与疾病相关的蛋白质、基因以及与疾病相关的生物标记分子，有助于了解细菌的致病机制。
2000年完成的Cj全基因序列、蛋白质双向电泳和质谱分析技术近年来的发展为我们从表型和基因水平高通量地分析GBS相关Cj的分子生物学特征提供了可能性。
本实验从蛋白质组学的角度出发，分析了GBS相关Cj的特征性蛋白。由于2DE系统的稳定性和重复性的好坏关系到实验数据的可靠性、可比性及不同实验室间的数据的相互交流，2DE的可重复性一直为人们所关注。因此，本研究首先对双向电泳技术的稳定性和可靠性进行了评价，在此基础上，利用2-DE技术对GBS相关Cj和非GBS相关Cj的差异表达蛋白进行分析，应用质谱对这些蛋白质进行鉴定，寻找GBS相关Cj的特征性蛋白，并对差异蛋白的编码基因的序列特征进行分析。本研究将为在分子水平了解Cj致GBS的发病机制，并为进一步研究GBS相关Cj的分子生物学特征及对该病的防治等奠定基础。现将各部分内容概述如下：
一、空肠弯曲菌双向电泳系统及其稳定性评价
目的：为验证2DE系统的可重复性，本实验建立了空肠弯曲菌（Campylobacter jejuni，Cj）的2DE系统，并对其稳定性和重复性进行了评价，为以后进行的实验结果的可靠性提供保障。
方法：选取一株从GBS病人粪便中分离的Cj，在37℃微需氧环境中培养48小时，收获后制备全菌蛋白样品，Bradford法测定蛋白质浓度，采用固相pH 梯度胶条进行第一向等电聚焦、12.5%的SDS-PAGE进行第二向SDS-PAGE电泳、染色、图像扫描及分析。为比较不同上样量和不同批次对实验结果的影响，共进行两次实验，第一次采用上样量100ug和上样量200ug的两块胶同时进行。 第二次实验采用上样量100ug单独进行。
结果：不同批次和不同上样量的胶进行比较，结果表明1. 蛋白质的匹配率均可达到90.89%；2. 未匹配蛋白主要是一些低丰度蛋白；3. 增加上样量可增加低丰度蛋白检出，但可产生蛋白质的共迁移，因此选择合适的上样量很重要；4. 不同批次蛋白质点在等电点方向的位置偏移为0.62±0.37mm，在SDS-PAGE方向的位置偏差为0.72±0.51mm，相对含量的相关系数为0.973。
结论：本系统具有很好的稳定性及很高的可重复性，符合蛋白质组学的要求，实验结果具有可比性。
二、吉兰-巴雷综合征相关空肠弯曲菌的蛋白质谱特征分析
目的：虽然目前认为分子模拟机制在发病中起重要作用，但Cj感染导致GBS的发病机制尚不十分清楚。本研究利用2-DE和质谱两种技术对GBS相关Cj和非GBS相关Cj的蛋白质图谱进行分析，寻找GBS相关Cj的特征性蛋白，以在分子水平揭示Cj致GBS的发病机制、进一步了解GBS相关Cj的分子生物学特征。
方法：选取分离自
Guillain-Barré syndrome(GBS) is now the most common cause of acute flaccid paralysis worldwide in the post-polio era, it is clinically characterized by the progressive weakness, usually symmetrical, with or without paralysis of respiratory muscle and cranial nerves. Recent neurophysiological and pathological studies have led to a reclassification of GBS into acute inflammatory demyelinating polyneuropathy(AIDP)、acute motor axonal neuropathy（AMAN）、acute motor and sensory axonal neuropathy（AMSAN）、Miller Fisher syndrome(MFS).About two-thirds of GBS are preceded by infections within 1-3 weeks of GBS onset, often suggestive of gastrointestinal enteritis or upper respiratory tract infection. Campylobacter jejuni(C.jejuni), which is a leading cause of acute enteritis, has become recognized as the most frequent antecedent pathogen for part of the GBS cases in recent years. Professor Li in our department isolated several strains of C.jejuni from GBS patients and chickens, and established the first animal model of AMAN worldwide by feeding or immunization with live C.jejuni strains and the purified lipopolysaccharide(LPS) fraction, therefore demonstrated that C.jejuni infection is an important pathogeny of GBS. The ratio of C.jejuni infection is high in the population, poultry and domestic animals in our country, but only certain C.jejuni strains with specific serotype are associated with development of GBS. There is a high incidence of GBS in northern China, the most frequently encountered pattern of GBS in northern China is AMAN with a relatively severe clinical course and a poor recovery. Therefore, it is very important to determine the strains capable of triggering GBS in susceptible hosts, and perform the risk-assessment studies and intervention of the pathogenic C.jejuni to reduce the incidence. The exact mechnism by which C.jejuni infection develops to GBS is still unclear , although the molecular
mimicry between the core of LPS of certain strains of C.jejuni and gangliosides in peripheral nerves of human body is mostly considered to play an important role in the pathogenesis of GBS. Several investigators attempted to find the molecular characterization or specific markers of GBS-related C.jejuni at genic level, but did not draw a consistent conclusion. The lack of the molecular characterization of GBS-related C.jejuni hampers the performance of identification of the pathogenic C.jejuni strains, vacculation development and intervention.
Following the rapid progress of genome, the functional genome, or post-genome is put forward. Protein is the production of gene expression and the final indicator of the function of gene, so proteomics has become one of the main contents of functional genome. Two-dimensional polyacrylamide gel electrophoresis (2DE) and mass spectrometry are the two key techniques of proteomics .
Proteomics has increasingly been used to the studies of differentially expressed proteins related to diseases. Bacteria in different conditions and different strains can express different proteome. Finding the differentially expressed proteins between the pathogenic and normal cells helps to find the gene, proteins or the molecular marker related to diseases, thus helpful to the diagnosis, treatment and prevention for the diseases.
Completion of C.jejuni genome and the development of the techniques in 2DE and mass spectrometry provide us with possibility of studying the molecular characterization of GBS-related C.jejuni highthroughputly at both phenotypic and genic level.
The marker proteins of GBS-associated C.jejuni were analyzed by proteomics in this study. Since the stability and reproducibility of 2DE concern the reliability and inter-laboratory comparison of the data, we first established the 2DE system for C.jejuni and evaluated its stability and reproducibility, then analyzed the differentially expressed proteins between GBS-associated C.jejuni and non-GBS-associated C.jejuni strains and identified them by mass spectroscopy to find the marker proteins of
GBS-associated C.
引文
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