【作者】 李晶；

【导师】 张建中；

【作者基本信息】 中国疾病预防控制中心 ， 病原生物学， 2009， 博士

【摘要】 幽门螺杆菌(Helicobacter pylori,H.pylori或HP)由Marshall BJ和WarrenJR于1983年自人胃窦黏膜组织中首次分离成功,为革兰氏染色阴性微需氧细菌。在世界范围内普通人群中的平均感染率过半,在发展中国家感染率更高,并且一旦感染,若不用药进行根除治疗,大多数会携带终身。H.pylori具有很强的宿主和组织特异性,因此用于动物实验的许多临床分离菌株常常表现为一过性感染,不能很好地模拟H.pylori在人体内的感染及其所致病变。1996年,Andrew Lee及其同事从120株临床菌株中筛选到一株能定植于C57BL/6品系小鼠的H.pylori,命名为悉尼株1(Sydney Strain 1株,简称SS1)。该菌株的各项实验数据都符合洛桑会议提出的能用于动物模型的菌株的各项要求,并且在其他实验室得到重复。SS1菌株拥有10~7cfu/g小鼠胃的良好定植能力,并且该菌株经小鼠体内驯化前的出发菌株10700也具有10~6cfu/g的高于其他临床菌株的定植能力,除驯化前后基因水平变化较小外,菌株自身的原因或者驯化过程能引起多大的变化不很明确。另外,由于SS1菌株未经过全基因组测序,直接从基因组水平分析不可行。本实验所用H.pylori菌株为购买于美国菌株保藏中心(ATCC)的700392/26695、分离自小鼠体内后传代次数7次以内的SS1和SS1的出发菌株10700。拟从SS1与10700、SS1与700392两个方面进行比较,分析SS1在驯化过程中蛋白表达水平的变化、SS1与定植能力不好的700392与小鼠胃相互作用间的差别,以便了解SS1具有优于一般菌株定植能力的原因。实验通过体外培养SS1和10700,丙酮三氯醋酸法提取全菌蛋白,以二维电泳(two-dimensional electrophoresis,2-DE)分离全菌蛋白,并比较SS1和出发菌株10700在蛋白表达水平上的差异,对差异点用基质辅助激光解析离子化飞行时间串联质谱(matrix-assisted laser desorption ionization time of flight-tandemmass spectrometry,MALDI-TOF-MS/MS)鉴定。SS1与10700相比,没有明显的2-DE水平可见的蛋白表达种类的增减,但发现有11个蛋白表达量降低,其中4个属于氧化还原系统,5个有能量代谢功能,1个功能未知。H.pylori定植于小鼠胃内是一个长期的进程,与菌株、小鼠及鼠胃内环境都有关系,本实验拟将SS1和700392与小鼠胃组织细胞短期作用过程中可能有相互作用关系研究作为目标,利用过碘酸盐-赖氨酸-多聚甲醛固定液(Periodate-Lysine-paraform-alde-hyde fixative,PLP)处理的C57BL/6小鼠胃黏膜作为固相面与SS1和700392的全菌蛋白共同孵育,缓冲液洗涤多次后用含有十二烷基硫酸钠(SDS)的溶液将富集在小鼠胃黏膜及粘膜内的H.pylori成分洗脱,浓缩洗脱液并作去盐处理后以2-DE分离,将所有2-DE胶图上可见的蛋白点酶解并用MALDI-TOF-MS/MS鉴定。与SS1和700392分别孵育的小鼠胃黏膜富集的共同成分有8个蛋白:HP0175、HP1286、48 kDa抗原(HP0599)、γ-谷氨酰转肽酶A链(Chain A,Gamma-Glutamyltranspeptidase)、γ-谷氨酰转肽酶B链(Chain B,Gamma-Glutamyltranspeptidase)、烷基过氧化氢还原酶(alkyl hydroperoxide reductase)、过氧化氢酶、异柠檬酸脱氢酶(isocitrate dehydrogenase),大部分与促炎症反应有关。HP0175和γ-谷氨酰胺酶都有促细胞凋亡功能,且HP0175已确认与AGS细胞表面的TLR-4存在相互作用关系。只存在于与SS1全菌蛋白孵育过的小鼠胃的洗脱液中的有假想蛋白醛基-酮基还原酶(Putative aldo-keto reductase)和3-酮脂酰还原酶(3-ketoacyl-(acyl-carrier-protein)reductase),且假想蛋白醛基-酮基还原酶在SS1及10700中表达量都远高于700392,可能该蛋白高表达对SS1的定植有利。共同组分中的HP1286功能未知,本实验在大肠杆菌(E.coli)BL21中克隆表达去除信号肽的HP0175和HP1286蛋白,借助载体上的His标签纯化融合蛋白,并去除盐离子和过滤除菌,加融合蛋白到平板培养的AGS细胞培养液中,检测到AGS细胞在HP0175蛋白的诱导下发生了与文献报道类似水平的凋亡现象,且HP1286具有类似的能力。本实验结果显示:SS1驯化过程中调节氧化还原系统和能量代谢的蛋白的表达量轻度降低;驯化过程并未使SS1发生明显改变,SS1的高定植能力可能是其自身独有的特点,驯化强化了这一特性。另外,共有蛋白HP1286具有体外诱导AGS细胞凋亡的能力,可能有利于解释H.pylori感染导致宿主胃组织部分细胞凋亡的宏观现象。更多还原

【Abstract】 Helicobacter pylori (H. pylori) is a Gram-negative, microaerobic bacterium. This main pathogenic agent of upper digestive diseases chronically infects the gastric mucosa of more than half of all people worldwide, and the infection rate is much higher in developing countries. Besides that, once infected, most people will carry this bacterium for decades until it is been eradicated. H. pylori strains have host and tissue tropism. So most clinical strains were emptied in short period of time after inoculating and could not induce inflammatory reaction and other diseases.In 1996, Andrew Lee and his colleagues got a H.pylori strain with good colonizing ability after screening a range of fresh clinical isolates and named it Sydney Strain 1(SS1). Experiment data of this strain fulfills the Lausanne criteria and can be repeated in other laboratories. SS1 and its parent strain 10700 colonized in C57BL/6 with high level of 10~7 and 10~6 cfu/g animal stomachs respectively. Genomic typing revealed few modifications of genes during the process of mouse adaptation. We know little about the genome of SS1 itself and the changes during the course of adaptation.H.pylori strains used in this study is 700392 bought from ATCC, pre- and postmouse strains( 10700 and SS1) cultured in vitro less than seven times. SS1 and 10700 were compared for searching changes during adaptation, while 700392 was used as control for poor colonization.SS1 and 10700 were cultured on plates. Whole cell proteins were prepared by precipitation with TCA and acetone and separated using 2-DE. The spots of the differential expressed proteins between SS1 and 10700 were cut for zymohydrolysis and identified using MALDI-TOF-MS/MS. No proteins were found only in SS1 or 10700. Eleven down-regulated spots presented ten proteins. Four were related to anti-oxidation, five were enzymes associated with metabolism, and the last one is a putative protein HPAG0942.Colonization ability is determined by many factors, such as the characteristic of strains, the kind of mouse, and the stomach circumstances. Components involved in interaction between H.pylori strains and mouse gastric mucous were studied. Gastric mucous fixed with PLP fixative was incubated with whole cell proteins of SS1 and 700392 respectively. Proteins enrichment on C57BL/6 mouse gastric mucous and epithelium were eluted by buffer containing SDS. The eluent were separated with 2-DE and all eyeable spots were cut for identification. Eight proteins were identified in both eluents: HP0175, HP1286, 48 kDa antigen(HP0599), Chain A ofγ-GGT, Chain B ofγ-GGT, alkyl hydroperoxide reductase, Catalase, and isocitrate dehydrogenase. Most of these were related with inflammation. Among them, HP0175 and GTT can induce apoptosis of AGS cell and HP0175 interacts with TLR-4 on surface of AGS. Proteins identified only in eluent from SS1 were putative aldo-keto reductase and 3-ketoacyl-(acyl-carrier-protein) reductase. Much more putative aldo-keto reductase expressed in SS1 than in 700392 may be helpful for colonization.Function of common component HP1286 is unknown. In this study, HP0175 and HP1286 without signal peptide were cloned into pET32a(+) and expressed in E.coli BL21. Recombinant proteins were purified by nickel-affinity resin. Before adding proteins into culture medium of adherent AGS cells in 24-well plates, all proteins were desalting and sterilized by filtration. By detecting apoptosis of AGS cell, it is obvious that His-HP0175 and His-HP1286 can induce much more serious cell death in a time-dependent manner than tag protein. In current study, there were not many changes between SS1 and its primary strain 10700, just a few proteins related to anti-oxidation and metabolism. Maybe some high expressed proteins were beneficial for colonization, such as putative aldo-keto reductase. We supposed that no obvious changes happened in the course of adaptation and the good colonizing ability is inborn to some extent, then the mouse-adaptation strengthened the ability. HP1286 which induced apoptosis of AGS cell in vitro was one of many microstructures of H.pylori, and many microstructures lead to the fact that H.pylori infection can induce apoptosis of gastric epithelium cell.更多还原