【作者】 赵洪岩；

【导师】 高文信； 周延民；

【作者基本信息】 吉林大学 ， 口腔临床医学， 2011， 博士

【摘要】 龋病是牙体硬组织的细菌感染性疾病,是最常见的口腔疾病,变形链球菌是其主要致病菌。氟化物作为目前使用最广泛的防龋制剂,对致龋菌的作用为氟防龋的重要机制之一。近年来,为提高并维持菌斑内较高氟水平,局部应用氟化物的浓度增加,间隔时间缩短,最可能的后果是耐氟菌株的产生。有报道国外学者从应用高浓度氟化钠凝胶防治猛性龋的患者口腔中分离出变形链球耐氟菌株；之后,又有学者从干燥症病人口腔中分离出变形链球菌耐氟菌株,这提示某些特殊人群的口腔中存在变形链球菌耐氟菌株。变形链球菌耐氟菌株产酸能力和耐酸性增强,致龋性较亲代菌株增强,并且耐氟菌株的基因组和蛋白质组均发生改变,但具体基因和蛋白还不能确定。虽然在正常人口腔中尚未分离出变形链球菌耐氟菌株,但氟化物的持续作用,体外耐氟菌株的诱导成功,都提示正常人群中亦可能产生耐氟菌株。耐氟菌株的出现,为氟化物防龋提出了新的问题,受到了国内外学者的密切关注,因而研究其发生、发展因素,对临床上合理应用氟化物及寻找新的防龋药物具有重要意义。本课题组的前期研究发现,变形链球菌耐氟菌株的ffh、dgk和dltc等耐酸相关基因较亲代菌株有不同程度的突变,但对耐酸相关基因编码蛋白的研究还比较缺乏。已有研究表明,仅有约2%的疾病与基因序列有关,而98%的疾病与蛋白表达密切相关。基因是遗传信息的携带者,而执行这些信息所蕴藏的生物学功能的是蛋白质,基因的表达产物蛋白质是生理功能的执行者,我们对蛋白质的研究将直接阐明生命在生理或病理条件下的变化机制。发挥生理功能时蛋白质的表现是多样的、动态的,并不象基因组那样基本固定不变。同一细胞、同一组织、同一器官在不同的生理条件和不同的外界环境影响下,蛋白质的存在状态并不完全相同；病理状态下的细胞蛋白质组与正常生理条件下的也有所不同,这就需要通过差异蛋白质组学技术来进行研究。差异蛋白质组学是针对不同空间、不同时间上动态变化着的蛋白质组的整体进行比较,分析不同蛋白质组之间蛋白质在表达数量、表达水平和修饰状态上的差异,研究差异蛋白质及其功能。由于蛋白质自身具有的多样性、可变性和构象的复杂性等特点,使得蛋白质研究技术难度非常大,蛋白质组学研究能否成功,相当程度上取决于其技术水平的高低。差异蛋白质组学并不要求鉴定“全部”蛋白,而主要是找出有意义的差异蛋白,所以在技术上相对容易实现。对蛋白质在不同状态,不同环境,不同处理等情况下表达量的变化进行检测,这就需要用到蛋白质组学的定量技术。定量技术是整个蛋白质组学的精华部分,这种定量通常不必检测蛋白质在细胞内的绝对含量,而只需对其相对含量进行定量即可。定量蛋白质组学是把一个基因组表达的全部蛋白质或一个复杂的混合体系内所有的蛋白质进行精确定量和鉴定的一门学科,这标志着蛋白质组学研究已从对蛋白质简单的定性向精确的定量方向发展。蛋白质组学研究中应用的定量方法主要有两种,一种是基于传统双向凝胶电泳及染色基础上的定量,另外一种是基于质谱检测技术的定量,包括标记定量技术(Labeling quantitation)和非标记定量技术(Label-free quantitation)两种。基于传统双向凝胶电泳及染色基础上的定量,此方法由于双向凝胶电泳本身的限制,不能对蛋白质实现绝对分离,不能有效检测出具有极端等电点的、分子质量太大和太小的蛋白质以及低丰度的蛋白质和膜蛋白,因而逐渐有被基于质谱的蛋白质组学非标记定量技术代替的趋势。从原理上看,标记定量技术的主要策略是对要比较的样本的蛋白质或者肽段分别进行轻和重的稳定同位素标记,从而使他们的分子量具有一定的差异,酶解后的肽段同时进行质谱分析,根据质谱上成对出现的轻重同位素峰的强度进行相对定量。非标记定量技术,未对研究样本进行标记,样本酶解产物分别进行质谱分析,然后根据峰面积或者肽段总次数对肽段和蛋白质进行相对定量。非标记定量技术(Label-free quantitation)不需要昂贵的同位素标签做内部标准,实验耗费低；对样本的操作也最少,从而使其最接近原始状态；并且不受样品条件的限制,克服了标记定量技术在对多个样本进行定量方面的缺陷,因此它在定量蛋白质组学研究中受到了众多科学工作者的推崇,得到了越来越广泛的应用。目前,已经有一系列配套的非标记定量分析软件,其中包括本研究采用的GE公司开发的DeCyder MSTM,具有很好的定量准确性和可信性。DeCyder MSTM软件对液相色谱串联质谱数据非标记定量分析是将质谱数据由谱峰形式转化为直观的类似双向凝胶的图谱,谱图上每一个点代表一个肽段,而不是蛋白质；再比较不同样本上相应肽段的强度,从而对肽段对应的蛋白质进行相对定量。本研究通过基于质谱的蛋白质组学非标记定量技术,利用液相色谱串联质谱和GE公司开发的DeCyder MSTM软件分析联用研究变形链球菌(UA159)耐氟菌株及其亲代菌株的蛋白质表达差异,发现并鉴定了明显差异蛋白67种,蛋白表达上调的为24种,蛋白表达下调的有43种。这些明显差异蛋白包括部分致龋毒力因子,其中与粘附有关的2种,为葡萄糖基转移酶和细胞表面粘附素PAC；与产酸相关的一种,为乳酸脱氢酶；与耐酸相关的4种,为F-ATP酶β-亚单位、信号识别颗粒蛋白质亚单位Ffh、分子伴侣DnaK和伴侣蛋白GroEL。同时应用GO (Gene Ontology)工具对差异蛋白进行生物学过程、分子功能和细胞定位分析,并构建了变形链球菌耐氟菌株和其亲代菌株的差异蛋白质数据库。本研究的创新性体现在,首次应用蛋白质组学非标记定量技术对变形链球菌(UA159)耐氟菌株及其亲代菌株进行差异蛋白质组学研究,利用GE公司开发的DeCyder MSTM软件,并结合NCBI链球菌蛋白数据库,发现并鉴定了明显差异蛋白67种。同时构建了变形链球菌耐氟菌株和其亲代菌株的差异蛋白质数据库,为以后进一步研究变形链球菌和变形链球菌耐氟菌株致龋毒力因子相关基因及其编码的蛋白的功能打下基础。更多还原

【Abstract】 Caries is a kind of bacteria-infected disease of the tooth hard tissue, which is also the most common disease occurred in oral cavity. The streptococcus mutans is the main pathogenic bacteria of caries. Fluoride is extensively used for caries preventive. Its effect of cariogenic bacteria is among important mechanisms for the caries preventive of fluoride. For the past few years, concentration of fluoride is increased and interval is shorten in order to improve and maintain high fluorin level, then its possible result is appearance of fluoride-resistant strain. From those patients who had used high concentration of fluoride sodium gel for rampant caries prevention, researchers had successfully insolated the fluoride-resistant Strain of streptococcus mutans. Then, some other investigaters had also insolated the fluoride-resistant strain of streptococcus mutans from Xerostomia patients. These researches suggested that the fluoride-resistant strain of streptococcus mutans could live in some special people. The fluoride-resistant strain of streptococcus mutans has stronger cariogenic ability, include its acidogenic ability and acid resistance. The Genome and proteome have been changed, but idiographic gene and protein are unknown. The fluoride-resistant strain of streptococcus mutans has not been separated in normal people now, but the success of inducement of fluoride-resistant strain in vitro and using the fluoride for long-term suggest the possibility of appearance of fluoride-resistant strain in normao people. The fluoride-resistant strain of streptococcus mutans could reduce the anti-caries effect of fluoride and are more difficult to be suppressed, which had given us a new question in this area focussed by many scholars. So the research about the fluoride-resistant strain of streptococcus mutans is very important, for the using fluoride reasonably and looking for new drug to prevent the caries.Our prophase research about the acid-proof-related genes of ffh, fhs, dgk and dltc from the fluoride-resistant strain of the S.M. showed that these genes have some mutations to different extent compared with that from the parental strain, but we are still short of the data about their encoded proteins. About 2% disease is correlative with gene sequence, but 98% disease is closely correlative with expression of protein.Genes are the carriers of hereditary information, and their encoded products, the proteins, are the executors of biological activities. The rsearch about protein will clarity the mechanism which the life changes in physiological or pathological condition. The behave of protein when protein performs physiologic function is variform and dynamic, but genome is basily unchanged. The same cell, tissue and organ express the protein is incompletely alike at different physiologic condition, development stage and environmental influence. The cell proteome is different at pathologic state and normal physiologic condition. These are studied by differential proteomics.Differential proteomics is used to compare the dynamic and changed the whole of proteome at different space and time, analyze the different proteome in expressing quantity, between protein expression level and modified state differences, study differential protein and its function, protein research technique is very difficult because of diversity and variability of the protein itself, and complexity of the conformation of the protein. Proteomics success depends-largely on its level of technical methods. Differential proteomics does not require to capture all protein, which focuses on meaningful differences to identify proteins, and thus has a very high technically achievable.Of proteins in different states, different environments, under different processing conditions to detect changes in expression, which requires use of quantitative proteomics technology. Quantitative techniques are the best parts of the whole proteomics, this quantitative detection of protein in the cells usually do not have the absolute content, but simply to quantify their relative content. Quantitative Proteomics is a subject which precisely quantitate and identify all proteins of a gene group or all proteins of a complex mixture within the system, which marks the proteomics study has been from simple qualitation to precise quantitation direction.There are two main quantitative methods of proteomics research. One is based on the traditional two-dimensional gel electrophoresis and staining, the other is based on mass spectrometry techniques, including labeling quantitation and label-free quantitation. The first method due to two-dimensional gel electrophoresis own limitations, can not achieve absolute separation of proteins, can not effectively detect proteins with extreme isoelectric points, molecular weight too big and too little, low abundance and membrane proteins, so it will be gradually instead by label-free quantitation of proteomics technology based on mass spectrometry.Seen from the principle, the main strategy of labeling quantitation is that proteins or peptides of compared samples are respectively labeled by light and heavy isotope, so that they have certain differences in the molecular weight, at the same time digested peptides are analyzed by mass spectrometry, according to the strength of isotopic peaks in pairs on the MS relative quantitation is in progress. The principle of label-free quantitation is the study sample is not labeled and digested products of the sample are respectively analyzed by mass spectrometry, then peptides and proteins are relatively quantitated according the peak areas or the total numbers of peptides.Label-free quantitation does not need expensive isotope tags as internal standard, so the experiment consumes lowly. The operation of the sample with label-free quantitation is least, so the sample is the most close to its original state. Label-free quantitation overcomes the defect of labeling quantitation to quantitate the multiple samples, so it has been praised highly by many scientists in the quantitative proteomics,and has been more and more widely applied. Now there are already a series of analysis software about label-free quantitation including the DeCyder MSTM by GE company used in this study, with good quantitative accuracy and credibility. The principle of the DeCyder MSTM is the mass spectrometry data has been translated from spectral peaks form intothe direct-viewing similar bidirectional gelatin maps, on the map each dot represents a peptide, not protein. Then compares the intensity of the corresponding peptide from different samples, relatively quantitates the proteins corresponded by peptide.We studied the differences of protein expression between the fluoride-resistant strain of the streptococcus mutans (UA159) and parent strains with label-free quantitation and the DeCyder MSTM by GE company. We found and identified 67 kinds of evident differential proteins, including 24 proteins up-regulation and 43 proteins down-regulation. These differential proteins included some virulence factors for caused caries, the two related adhesion is glucosyltransferase and Major cell-surface adhesin Pac, the one related Producing acid is L-lactate dehydrogenase, the four related acid-resistant is F-ATPase beta-subunit, Ffh, molecular chaperone DnaK and chaperonin GroEL. We analyzed the differential proteins of biological processes, molecular function and cellular localization with Gene Ontology, and we constructed the data base of differential proteins for the fluoride-resistant strain of the streptococcus mutans (UA159) and parent strains.The innovation of our research was that it is the first time to analyse the differential proteomics of the fluoride-resistant strain and parental strain(UA159) of the streptococcus mutans with the technique of label-free quantitation. On basis of the DeCyder MSTN of GE company and related information searched from the NCBI protein data base of the streptococcus, we found 67 kinds evident differential protein.we have preliminarily constructed the differential proteomics data base of the fluoride-resistant strain and parental strain of the streptococcus mutans. The results could provide the basis for analysing the functions of proteins encoded by caries-related genes from the fluoride-resistant strain and parental strain of the streptococcus mutans.更多还原