本文關(guān)鍵詞：幽門螺桿菌動物模型菌株SS1定植因素分析　出處：《中國疾病預(yù)防控制中心》2009年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 幽門螺桿菌 蛋白質(zhì)組 相互作用 凋亡 基質(zhì)輔助激光解析離子化飛行時間質(zhì)譜

【摘要】： 幽門螺桿菌(Helicobacter pylori,H.pylori或HP)由Marshall BJ和WarrenJR于1983年自人胃竇黏膜組織中首次分離成功,為革蘭氏染色陰性微需氧細(xì)菌。在世界范圍內(nèi)普通人群中的平均感染率過半,在發(fā)展中國家感染率更高,并且一旦感染,若不用藥進(jìn)行根除治療,大多數(shù)會攜帶終身。H.pylori具有很強(qiáng)的宿主和組織特異性,因此用于動物實(shí)驗(yàn)的許多臨床分離菌株常常表現(xiàn)為一過性感染,不能很好地模擬H.pylori在人體內(nèi)的感染及其所致病變。 1996年,Andrew Lee及其同事從120株臨床菌株中篩選到一株能定植于C57BL/6品系小鼠的H.pylori,命名為悉尼株1(Sydney Strain 1株,簡稱SS1)。該菌株的各項(xiàng)實(shí)驗(yàn)數(shù)據(jù)都符合洛桑會議提出的能用于動物模型的菌株的各項(xiàng)要求,并且在其他實(shí)驗(yàn)室得到重復(fù)。SS1菌株擁有10~7cfu/g小鼠胃的良好定植能力,并且該菌株經(jīng)小鼠體內(nèi)馴化前的出發(fā)菌株10700也具有10~6cfu/g的高于其他臨床菌株的定植能力,除馴化前后基因水平變化較小外,菌株自身的原因或者馴化過程能引起多大的變化不很明確。另外,由于SS1菌株未經(jīng)過全基因組測序,直接從基因組水平分析不可行。 本實(shí)驗(yàn)所用H.pylori菌株為購買于美國菌株保藏中心(ATCC)的700392/26695、分離自小鼠體內(nèi)后傳代次數(shù)7次以內(nèi)的SS1和SS1的出發(fā)菌株10700。擬從SS1與10700、SS1與700392兩個方面進(jìn)行比較,分析SS1在馴化過程中蛋白表達(dá)水平的變化、SS1與定植能力不好的700392與小鼠胃相互作用間的差別,以便了解SS1具有優(yōu)于一般菌株定植能力的原因。 實(shí)驗(yàn)通過體外培養(yǎng)SS1和10700,丙酮三氯醋酸法提取全菌蛋白,以二維電泳(two-dimensional electrophoresis,2-DE)分離全菌蛋白,并比較SS1和出發(fā)菌株10700在蛋白表達(dá)水平上的差異,對差異點(diǎn)用基質(zhì)輔助激光解析離子化飛行時間串聯(lián)質(zhì)譜(matrix-assisted laser desorption ionization time of flight-tandemmass spectrometry,MALDI-TOF-MS/MS)鑒定。SS1與10700相比,沒有明顯的2-DE水平可見的蛋白表達(dá)種類的增減,但發(fā)現(xiàn)有11個蛋白表達(dá)量降低,其中4個屬于氧化還原系統(tǒng),5個有能量代謝功能,1個功能未知。 H.pylori定植于小鼠胃內(nèi)是一個長期的進(jìn)程,與菌株、小鼠及鼠胃內(nèi)環(huán)境都有關(guān)系,本實(shí)驗(yàn)擬將SS1和700392與小鼠胃組織細(xì)胞短期作用過程中可能有相互作用關(guān)系研究作為目標(biāo),利用過碘酸鹽-賴氨酸-多聚甲醛固定液(Periodate-Lysine-paraform-alde-hyde fixative,PLP)處理的C57BL/6小鼠胃黏膜作為固相面與SS1和700392的全菌蛋白共同孵育,緩沖液洗滌多次后用含有十二烷基硫酸鈉(SDS)的溶液將富集在小鼠胃黏膜及粘膜內(nèi)的H.pylori成分洗脫,濃縮洗脫液并作去鹽處理后以2-DE分離,將所有2-DE膠圖上可見的蛋白點(diǎn)酶解并用MALDI-TOF-MS/MS鑒定。與SS1和700392分別孵育的小鼠胃黏膜富集的共同成分有8個蛋白:HP0175、HP1286、48 kDa抗原(HP0599)、γ-谷氨酰轉(zhuǎn)肽酶A鏈(Chain A,Gamma-Glutamyltranspeptidase)、γ-谷氨酰轉(zhuǎn)肽酶B鏈(Chain B,Gamma-Glutamyltranspeptidase)、烷基過氧化氫還原酶(alkyl hydroperoxide reductase)、過氧化氫酶、異檸檬酸脫氫酶(isocitrate dehydrogenase),大部分與促炎癥反應(yīng)有關(guān)。HP0175和γ-谷氨酰胺酶都有促細(xì)胞凋亡功能,且HP0175已確認(rèn)與AGS細(xì)胞表面的TLR-4存在相互作用關(guān)系。只存在于與SS1全菌蛋白孵育過的小鼠胃的洗脫液中的有假想蛋白醛基-酮基還原酶(Putative aldo-keto reductase)和3-酮脂酰還原酶(3-ketoacyl-(acyl-carrier-protein)reductase),且假想蛋白醛基-酮基還原酶在SS1及10700中表達(dá)量都遠(yuǎn)高于700392,可能該蛋白高表達(dá)對SS1的定植有利。 共同組分中的HP1286功能未知,本實(shí)驗(yàn)在大腸桿菌(E.coli)BL21中克隆表達(dá)去除信號肽的HP0175和HP1286蛋白,借助載體上的His標(biāo)簽純化融合蛋白,并去除鹽離子和過濾除菌,加融合蛋白到平板培養(yǎng)的AGS細(xì)胞培養(yǎng)液中,檢測到AGS細(xì)胞在HP0175蛋白的誘導(dǎo)下發(fā)生了與文獻(xiàn)報道類似水平的凋亡現(xiàn)象,且HP1286具有類似的能力。 本實(shí)驗(yàn)結(jié)果顯示:SS1馴化過程中調(diào)節(jié)氧化還原系統(tǒng)和能量代謝的蛋白的表達(dá)量輕度降低;馴化過程并未使SS1發(fā)生明顯改變,SS1的高定植能力可能是其自身獨(dú)有的特點(diǎn),馴化強(qiáng)化了這一特性。另外,共有蛋白HP1286具有體外誘導(dǎo)AGS細(xì)胞凋亡的能力,可能有利于解釋H.pylori感染導(dǎo)致宿主胃組織部分細(xì)胞凋亡的宏觀現(xiàn)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.

【學(xué)位授予單位】：中國疾病預(yù)防控制中心
【學(xué)位級別】：博士
【學(xué)位授予年份】：2009
【分類號】：R378

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