幽门螺杆菌动物模型菌株SS1定植因素分析

发布时间：2018-01-13 16:02

本文关键词：幽门螺杆菌动物模型菌株SS1定植因素分析　出处：《中国疾病预防控制中心》2009年博士论文　论文类型：学位论文

【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。
[Abstract]:Helicobacter pylori (Helicobacter pylori, H.pylori or HP) by Marshall BJ and WarrenJR in 1983 from human gastric mucosa was first successfully isolated were gram negative microaerophilic bacteria infection in the world. The average rate in the general population more than half, in the development of China infection rate is higher, and once the infection, if for eradication treatment medication, most will carry life.H.pylori the host and tissue specificity is very strong, so for many clinical isolates of animal experiments often manifested as a transient infection, can be a good simulation of H.pylori in the human body caused by infection and disease.
In 1996, Andrew Lee and colleagues screened from 120 isolates to H.pylori can migrate to C57BL/6 mice by a strain, named Sydney strain 1 (Sydney Strain 1 strain, referred to as SS1). The experimental data of this strain are in line with the Lausanne meeting proposed can be used for the animal model of strains, and get good colonization ability of strain.SS1 with repeated 10~7cfu/g mice in other laboratories, and the strain was the starting strain mice domestication before 10700 also has 10~6cfu/g higher than other clinical strains colonization ability, except change before and after acclimation gene level is small, because the strain itself or the domestication process can cause not much change very clear. In addition, due to the SS1 strain without whole genome sequencing directly from the genome level analysis is not feasible.
This experiment uses the H.pylori strain for purchase in the United States Preservation Center (ATCC) strain 700392/26695, strain isolated from mice after generation 7 times within SS1 and SS1 10700. SS1 and SS1 plans from 10700, compared with 700392 in two aspects, analysis of SS1 during the acclimation to changes in protein expression levels. SS1 and colonization ability is not good 700392 and interaction between mouse stomach, in order to understand the cause of SS1 is better than that of general bacterial colonization ability.
In vitro experiment by SS1 and 10700 of total bacterial protein extraction, three trichloroacetic acid acetone method on two-dimensional electrophoresis (two-dimensional, electrophoresis, 2-DE) separation of whole bacterial proteins, and compare the SS1 and protein expression in 10700 strains of different level, the differences of tandem mass spectrometry with matrix assisted laser desorption ionization time of flight (matrix-assisted laser desorption ionization time of flight-tandemmass spectrometry, MALDI-TOF-MS/MS) identification of.SS1 compared with 10700, there is no obvious 2-DE expression level visible type change, but found the expression of 11 protein decreased, 4 of which belong to the redox system, 5 energy metabolism, 1 function unknown.
H.pylori colonization in mice stomach is a long process, and the strain of mice and rat stomach environment have a relationship, this study will be SS1 and 700392 with gastric tissue cells in mice may have short-term effect in the process of interaction as a target, periodate lysine paraformaldehyde fixed by liquid before (Periodate-Lysine-paraform-alde-hyde fixative, PLP) treated C57BL/6 mice gastric mucosa as the solidus surface with SS1 and 700392 of the total bacterial protein were incubated with buffer after washing many times with twelve sodium dodecyl sulfate (SDS) H.pylori component solution enriched in mice gastric mucosa and mucosa in the elution, concentration of eluent and to salt treatment with 2-DE after separation, all 2-DE glue visible protein enzyme and identified by MALDI-TOF-MS/MS and SS1. And the common component 700392 incubated in mice gastric mucosa enriched 8 proteins: HP0175 HP1286,48, kDa antigen (HP0599), gamma glutamyltransferase (Chain A, Gamma-Glutamyltranspeptidase A chain), gamma glutamyltransferase (Chain B, Gamma-Glutamyltranspeptidase B chain), alkyl hydroperoxide reductase (alkyl hydroperoxide reductase), catalase, isocitrate dehydrogenase (isocitrate dehydrogenase) and, most of the pro-inflammatory.HP0175 and gamma glutamine enzyme have pro apoptotic function, and HP0175 has confirmed the existence of the interaction between AGS and TLR-4 on the cell surface. The eluent only existed in the mouse stomach Yu and SS1 and the protein was incubated in a hypothetical protein - keto aldehyde reductase (Putative aldo-keto reductase 3-) and ketoacyl reductase (3-ketoacyl- (acyl-carrier-protein) reductase), and the putative protein aldehyde keto reductase in SS1 and 10700 expression levels are much higher than 700392, the high expression of the protein It is beneficial to the colonization of SS1.
The common group HP1286 of unknown function in this experiment, in Escherichia coli (E.coli) expression of the signal peptide removed HP0175 and HP1286 protein in BL21 by cloning, vector His tag fusion protein was purified, and the removal of salt ions and filtration, and the fusion protein to plate culture medium of AGS cells, detected by AGS cells in the induction of HP0175 protein occurred with reported similar levels of apoptosis, and the HP1286 has similar capabilities.
The experimental results show that the SS1 acclimation modulation of redox system and energy metabolism in the process of protein expression decreased slightly; the domestication process did not make SS1 change, SS1 high colonization ability may be its own unique characteristics, strengthen the domestication of this feature. In addition, a total of HP1286 protein has the ability of AGS cell apoptosis in vitro, may help to explain the phenomenon of macro H.pylori infection leads to gastric tissue apoptosis of host.

【学位授予单位】：中国疾病预防控制中心
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R378

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