【作者】 黄增荣；

【导师】 王建华； 兰春慧；

【作者基本信息】 西北农林科技大学 ， 预防兽医学， 2010， 博士

【摘要】 幽门螺杆菌(Helicopter pylori,Hp)是人体内最常见的病原微生物之一,其感染率约占世界人口的50%。目前我国约有7亿人已感染Hp,未感染人群也普遍易感。流行病学研究提示Hp与胃癌的发生关系密切,世界卫生组织(WHO)已把Hp列为胃癌的首要致病因子。Hp感染引起的胃上皮细胞凋亡异常在胃癌的发生发展中起主要作用,但是其具体机制尚不清楚。Hp空泡细胞毒素(Vacuolating cytotoxin,VacA)与Hp产生的其它已知细菌毒素无明显的同源性,因其可致真核上皮细胞发生空泡变性而得名,是目前倍受关注的细胞毒力因子之一。胃癌患者所感染的Hp大多含有VacA,推测其在胃癌发生过程中可能起着较为重要的作用。VacA作为Hp的主要毒力因子,诱导上皮细胞凋亡是引发细胞癌变的关键分子,VacA诱导凋亡的作用与致空泡的作用虽有关联但不互为因果,但均与其膜通道活性改变有关。进一步研究发现VacA进入上皮细胞,能够靶向线粒体,引起线粒体膜通透性发生转换(mitochondrial permeability transition,MPT),造成线粒体跨膜电位降低、细胞色素c释放、活化凋亡相关因子等一系列变化,诱导细胞发生凋亡, VacA导致MPT的机理尚不明确。腺嘌呤核苷酸转移酶(adenine nucleotide translocase,ANT)是线粒体内膜上含量最丰富的蛋白。它是将细胞的产能和耗能过程耦联起来的主要载体,也是组成线粒体膜通透性转换孔(mitochondrial permeability transition pore,MPTP,或简称PT孔)的关键组分,参与调节线粒体膜通透性转换孔的开放,与细胞凋亡紧密相关。线粒体凋亡途径是细胞的内源性凋亡途径,线粒体凋亡的发生最终都归结于MPT,而MPT的发生又是由于PT孔的开放所致。PT孔的分子构成尚不完全清楚,但目前已明确ANT是其位于线粒体内膜的关键蛋白,它在线粒体内、外膜的交接点处可形成“非特异性孔道”,通过它的开放、关闭以及与一些凋亡调控因子(如Bax/bcl-2家族)的互动,参与组织细胞凋亡的调控。VacA可以迅速转运至线粒体内膜,其导致的线粒体内膜通透性转换明显早于外膜通透性转换。其它配体或蛋白质可与ANT发生相互作用改变其构象来控制PT孔的开放与关闭,通过调节膜通透性的改变来达到调控细胞凋亡的目的,ANT在MPT中处于核心地位。研究VacA与ANT的相互作用关系,将有助于进一步明确VacA致MPT的机理。本研究通过酵母双杂交和免疫共沉淀来探索VacA与ANT的相互作用关系,为阐明Hp通过线粒体凋亡途径诱发胃癌的分子机制提供科学依据。1.酵母双杂交诱饵质粒的构建及鉴定提取幽门螺杆菌标准毒株NCTC11637基因组DNA,PCR扩增VacA片段,并将其克隆入pGBKT7载体构建pGBKT7-VacA诱饵质粒。通过酶切和测序鉴定诱饵质粒构建成功后,将诱饵质粒转化宿主酵母菌AH109后,提取酵母总蛋白,Western-Blot分析重组蛋白的活性。同时分别检测了诱饵质粒的毒性、渗漏和自激活活性。结果显示构建的诱饵质粒对酵母菌无毒性、无渗漏也未表现自激活活性。2.酵母双杂交猎物质粒的构建及鉴定RT-PCR扩增ANT各异构体——ANT1、ANT2、ANT3基因,并分别将它们克隆入pGADT7载体构建pGADT7-ANT1、pGADT7-ANT2、pGADT7-ANT3猎物质粒,进行酶切和测序鉴定。各猎物质粒分别转化宿主酵母菌Y187后,提取酵母总蛋白,Western-Blot分析各重组蛋白的活性。一系列活性和毒性检测结果表明全部质粒均具有天然活性、对宿主未表现毒性,并且无渗漏和自激活活性。3.酵母双杂交试验将转化了诱饵质粒pGBKT7-VacA的酵母菌AH109分别与转化了猎物质粒pGADT7-ANT1、pGADT7-ANT2、pGADT7-ANT3的酵母菌Y187两两配对交配培养,富集交配后的合子铺不同严谨程度营养缺陷培养基,X-α-Gal检测报告基因的激活,结果未检测到报告基因的转录,表明VacA与ANT在酵母双杂交系统内未发生相互作用。4.VacA免疫共沉淀载体的构建及鉴定以pGBKT-VacA质粒DNA为模板,PCR扩增得到VacA片段并将其克隆入pCMV-Myc载体中构建pCMV-Myc-VacA表达质粒。将构建好的质粒采用脂质体法瞬时转染AGS细胞,Western Blot检测重组质粒在AGS细胞内的表达,结果表明重组蛋白得到正确表达。5.ANT各异构体免疫共沉淀载体的构建及鉴定分别以pGADT7-ANT1、pGADT7-ANT2、pGADT7-ANT3质粒DNA为模板,PCR扩增ANT1、ANT2、ANT3基因片段,并将其分别克隆入pCMV-HA载体中构建pCMV-HA-ANT1、pCMV-HA-ANT2、pCMV-HA-ANT3融合表达质粒。将构建好的各质粒采用脂质体法分别瞬时转染AGS细胞,Western Blot检测各重组质粒在AGS细胞内的表达,结果表明各重组蛋白均得到正确表达。6 .免疫共沉淀试验将pCMV-Myc-VacA载体分别与pCMV-HA-ANT1、pCMV-HA-ANT2、pCMV-HA-ANT3载体配比瞬时共转染AGS细胞,提取细胞总蛋白进行免疫共沉淀试验,分别用VacA抗体沉淀ANT(ANT1、ANT2、ANT3)和ANT抗体沉淀VacA,验证二者在细胞内的蛋白相互作用。结果表明VacA与ANT之间不能发生免疫共沉淀反应。更多还原

【Abstract】 Helicobacter pylori (Hp) is most common pathogenic bacteria, which infection presently affects approximately one-half of the world’s population and seven hundred million people in our country, and the health people are very susceptible to it.Hp leads to chronic gastritis, the most frequent chronic inflammation worldwide.It is also aetiologically associated with gastric and duodenal ulcer, mucosa associated lymphoid tissue (MALT) gastric lymphoma and gastric cancer (GC). Since then, data from several epidemiological, interventional and experimental studies have been gathered, confirming the causal link between Hp and GC. Ecological studies mostly confirm the geographical association between the prevalence of Hp and prevalence of GC.It has been evident for over 20 years that Hp is involved in the development of gastric cancer; in 1994, the WHO concluded that Hp is a definite or class I carcinogen in humans.Prospective studies reveal that the risk for development of gastric cancer is much greater in Hp-infected populations than in uninfected populations. The development of GC is a multistep process that is multi-factorial. The most important risk factor is the infection with Hp. The pathogenesis of GC includes a sequence of events that begins with Hp-induced chronic superficial gastritis, progressing towards atrophic gastritis, intestinal metaplasia, dysplasia and eventually GC. The“driving force”of GC is a chronic gastric inflammation, whose intensity and localization depending on the bacteria infection. The mechanisms by which chronic inflammation lead to epithelial and pre-cancerous lesions include induction of oxidative stress, perturbation of the epithelial cells proliferation/apoptosis ratio, and cytokine secretion. The end-stage of the multistep process of GC corresponds to accumulation of molecular alterations involving either the suppressor pathway (defect in tumour suppressor genes) or the mutator pathway (defect in DNA mismatch repair genes).The abnormality of gastric epithelial cells proliferation or apotosis is playing the most important roles in development to GC which caused by Hp infections.While the mechanisms of Hp associated cells apotosis are still relatively poorly defined, the elucidation may provide opportunities to develop effective strategies for GC prevention and therapy. Chronic gastritis induced by Hp is a strong risk factor for the development of distal gastric adenocarcinoma. A specific host response to Hp that may contribute to gastric carcinogenesis is epithelial cell apoptosis. VacA is one of the most important virulence factors of Hp that is extracellular and causes damage to the gastric epithelial layer as a result of vacuolation of late endosomal and lysosomal compartments of these cells. VacA-producing Hp strains are found in the vast majority of patients with GC, powerful evidences were discovered that it has important contributions to the development of GC.VacA has also been shown to be directly involved in mucosal damage in GC. This damage might be attributed to the vacuolating as well as to the apoptosis-inducing activity of VacA. Therefore, elucidation of VacA as an apoptosis-inducing factor might offer a better understanding of the pathogenesis of Hp-related diseases such as GC. The VacA is a unique proteinous cytotoxin producted by Hp that showed no striking primary sequence homology with other known bacterial toxins.The cytotoxin is an important factor in the pathogenesis of Hp, which induces vacuolating of epithelial cells and plays an important role in gastric epithelial apotosis. Hp induces cell death by apoptosis. However, the mechanism of apoptosis-inducing factor is still unknown.The virulence factor VacA is a potential candidate.Cellular vacuolation and mitochondrial cytochrome c release are independent outcomes of VacA cytotoxin activity that are each dependent on membrane channel formation. Hp VacA which causes vacuolation of gastric epithelial cells and other types of cultured cells,is known to enter mammalian cells,localizes to the mitochondrial, induced an inactivation of energy metabolism followed by mitochondrial damage,leading to impairment of the cell cycle in gastric epithelial cells,and modulates mitochondrial membrane permeability resulting in reduction of mitochondrial transmembrane potential by a mechanism dependent on toxin channel activity ultimately resulting in cytochrome c release,stimulate apoptosis via a mitochondrial- dependent pathway.Mitochondrial apoptosis pathway is endogenous cell death pathway. Mitochondria are important organelles for energy production, Ca2+ homeostasis, and cell death. In recent years, the role of the mitochondria in apoptotic has received much attention.In apoptotic, an increase of mitochondrial membrane permeability is considered to be one of the key events, although the detailed mechanism remains to be elucidated. The mitochondrial membrane permeability transition (MPT) is increase in the permeability of the mitochondrialmembrane that leads to loss ofΔψm,mitochondrial swelling, and rupture of the outer mitochondrial membrane.The MPT is thought to occur after the opening of a channel, which is termed the permeability transition pore (MPTP) and core constitution consists of the voltage-dependent anion channel (VDAC) in the outer mitochondrial membrane (OM), the adenine nucleotide translocator (ANT) in the innermembrane (IM) and cyclophilin D (Cyp D) in the mitochondrial matix. Mitochondrial membrane permeabilization can be a rate limiting step of apoptotic as well as necrotic cell death.Permeabilization of the OM and/or IM is, at least in part, mediated by the permeability transition pore complex (PTPC). The PTPC is formed in the IM/OM contact site and contains the two most abundant IM and OM proteins, ANT and VDAC,the matrix protein cyclophilin D,which can interact with ANT, as well as apoptosis-regulatory proteins from the Bax/Bcl-2 family.The MPTP is a non-specific pore, permeant to any molecule of < 1.5 kDa, that opens in the inner mitochondrial membrane under conditions of elevated matrix [Ca2+], especially when this is accompanied by oxidative stress and depleted adenine nucleotides. Opening of the MPTP causes massive swelling of mitochondria, rupture of the outer membrane and release of intermembrane components that induce apoptosis. In addition mitochondria become depolarised causing inhibition of oxidative phosphorylation and stimulation of ATP hydrolysis. Thus, in many cases, the PT appears to be the mastermind that orchestrates apoptosis. Alteration of mitochondrial membrane permeability is a central mechanism leading invariably to cell death. Indeed, extended PTPC opening is sufficient to trigger an increase in mitochondrial membrane permeability and apoptosis. Among the various PTPC components, the ANT appears to act as a bi-functional protein which, on the one hand, contributes to a crucial step of aerobic energy metabolism, the ADP/ATP translocation, and on the other hand, can be converted into a pro-apoptotic pore under the control of onco- and anti-oncoproteins from the Bax/Bcl-2 family.Additionally,VacA likes as an A-B type cytotoxin, binds to cells primarily via amino acid sequences in its B subunit—p58,and it is necessary for enzymatic activity of A subunit—p37.Meanwhile, research indicated that the residues are situated in 418 aa to 799 aa of p58 fragment madiate the development of gastric diseases through cell cycle arrest in the G1 phase and induction of apoptosis is associated with up-regulation of apoptosis related factors.To summarized,we speculated that VacA and ANT formation the complex, then changed the channel activity of mitochondrial membrane, leaded to MPT eventually. In present study, apply to Yeast Two-Hybrid and Co-immunoprecipitation to explored the hypothesis that the protein interaction between VacA and ANT isoforms,VacA and ANT formation the complex induced the mitochondria membrane transition.Expected to clarify the mechanism of VacA induce cell apotosis,for the purpose of supply new clues of molecular mechanism of Hp infections evocated GC by mitochondrial apoptosis pathway.1. Construction and identification of the“bait”plasmid of Yeast Two-Hybrid: Extracted the genome DNA from Helicobacter pylori standard strain NCTC11637, the gene encoding Vacuolating cytotoxin partial sequences was amplified by PCR and then cloned into pGBKT7 to construct the pGBKT7-VacA bait plasmid. Insert-contained plasmid was confirmed by restriction endonuclease analysis and DNA sequencing. And then the bait plasmid were transformed into the host S.cerevisiae AH109,the results of recombinant protein detection by Western Blot indicated that the protein translated correctly; meanwhile, the bait plasmid also have no cytotoxin, no leakage and no auto-activated activities. This work was important for the further verification of the protein interaction with VacA.2. Construction and identification of the“prey”plasmids of Yeast Two-Hybrid: Each open reading frame (ORF) of the ANT isoforms were amplified by RT-PCR and then cloned into the pGADT7 respectively, to construct the prey plasmids pGADT7-ANT1, pGADT7-ANT2 and pGADT7-ANT3. After proved by restrict endonuclease digesting and DNA sequencing, the prey plasmids were transformed into S.cerevisiae Y187 separately, then the expression of the prey proteins were analyzed by Western Blot severally. A series of cytotoxin and activity performances of the prey plasmids were detected, the results indicated that all of these plasmids have natural activity and have no toxincity, leakage and auto-activated activities.These achievements provided the foundation for inspect the combination partner of ANT in the Yeast Two-Hybrid System.3. Yeast Two-Hybrid: Transform AH109 with pGBKT7-VaA and transform Y187 with pGADT7-ANT1 or pGADT7-ANT2 or pGADT7-ANT3.The two types of cells were co-cultured in liquid SD/-Trp/-Leu medium.After mating,the plasmids were introduced into the same host cells,centrifuged cells and plate culture on Low-stringency,Medium-stringency, High-stringency plates sequentially.Verify the protein interaction afterβ-galactosidase assays. None of all experimental groups activated the reporter genes transcription, there were means that have no protein interactions between each other in our test system.4. Construction and Identification of the pCMV-Myc-VacA expression vector: The pGBKT7-VacA plasmid DNA as template, amplified VacA fragment by PCR, and cloned into plasmid pCMV-Myc. After identificated by restriction endonuclease digested and then transient transfected into AGS cells by using lipofectamine 2000.Detected the recombinant protein by Western Blot analysis. The results demonstrated that the recombinant protein translated correctly.This study offers the basis for further research of co-immunoprecipitation protein of VacA.5. Construction and Identification of the pCMV-HA-ANT expression vectores: Set up the Yeast Two-hybrid prey plasmids as the templates.Amplified the ANT fragments by PCR respectively.And then cloned into the HA-tag expression vector pCMV-HA. Identificated by restriction endonuclease digested and transient transfected into AGS cells by using lipofectamine 2000 separately. All the expression plasmids were proofed that they were translated exactly by Western Blot assays. 6. Co-Immunoprecipitation: The Myc-tag plasmid pCMV-Myc-VacA and the HA-tag plasmids pCMV-HA-ANT1 or pCMV-HA-ANT2, pCMV-HA-ANT3 were co-transfected into AGS cells with optimal combination. The cells were homogenized in lysis buffer, and then added the first primary antibody into the supernatant, after 4 hours incubation, added the Protein A beads in it and incubated overnight at low velocity rotary conditions. After that, the Protein A beads were pelleted, then Western Blot were performed by using another primary antibody, to verified the protein interactions at physiological circumstance.Detected the protein interactions by Co-immunoprecipitation approach. No matter how VacA immunoprec- ipitation ANT or ANT immunoprecipitation VacA, there were no phenomenons had been detected that revealed where the co-immunoprecipitation happened between each other.更多还原