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Peptic ulcer is a common and frequently encountered disease. H2 receptor blocker and several other drugs have been used in treating peptic ulcer (PU) since many years ago, but there still remain a rather large number of intractable ulcer and recurrent ulcer. Recently the discovery of Helicobacter pylori (HP), as well as the relationship between HP and PU relapse was confirmed, but there still lacks of perfect therapeutic program for anti-HP infection(1). From June 1993 to August 1996, we used a pure TCM preparation Moluo Yangping granule (摩罗疡平冲剂, MYG) in treating 64 PU patients, and satisfactory results have been obtained. The report is as follows.METHODSPatient Selection According to the diagnostic standard of PU worked out by Ministry of Health in “Guiding Principles for Clinical Study of New Chinese Drugs”, 126 PU patients with typical symptoms and signs were enrolled, who were confirmed to have PU before treatment with biopsy sample performed urease quick diagnostic method under gastroscopy, and pathological special stain were microscopically examined to verify the HP infection. Basal acid output (BAO), maximal acid output (MAO), peak acid output (PAO) and the amount of parietal cells were measured in all the patients.
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胃肠病学研究的一次突破--2005年诺贝尔生理学或医学奖介绍
2005年诺贝尔生理学或医学奖共同授予了两位澳大利亚科学家John Robin Warren和BarryJames Marshll,表彰他们的研究:"幽门螺杆菌以及它在胃炎和消化性溃疡病中的作用"(The bacterium Helicobacter pylori and its role in gastritis and peptic ulcer disease)[1].
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胃黏膜活检幽门螺杆菌染色方法比较
幽门螺杆菌(Helicobacter pylori,HP)是一种革兰阴性杆菌,人群中HP的感染率较高,其感染与慢性胃炎、消化性溃疡和胃癌等相关,清除HP有利于胃黏膜炎症程度的减轻[1-2].检测HP感染是诊断、药物疗效分析等方面的重要手段,检测方法较多,每种方法各有优点及不足,由于条件和技术的要求,限制了某些方法的应用[3-4].
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与克拉霉素耐药相关的幽门螺杆菌基因23S rRNA序列分析
幽门螺杆菌是寄生于人体胃部的革兰阴性杆菌,是胃溃疡、十二指肠球部溃疡和胃癌的主要致病因素之一,与胃黏膜相关淋巴组织淋巴瘤关系密切.随着大环内酯类抗生素广泛应用于治疗幽门螺杆菌感染,幽门螺杆菌的耐药现象日益严重.现已证实,幽门螺杆菌对克拉霉素耐药主要与其23S rRNA基因的点突变有关.为此,本研究通过对江苏苏中沿海地区分离培养的幽门螺杆菌菌株的23S rRNA部分基因片段进行序列分析,希望了解本地区幽门螺杆菌耐药菌株的基因突变和耐药的关系.
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幽门螺杆菌18000u外膜蛋白编码基因的克隆及表达
目的:构建含幽门螺杆菌(Hp)18000 u外膜蛋白编码基因的重组载体并在E.coli BL2l中表达.为Hp的疫苗开发、快速诊断试剂盒的研究奠定基础.方法:用PCR从Hp染色体中,扩增18000 u外膜蛋白编码基因片段.将目的基因与pET32a(+)同时经BamH I,HindⅢ双酶切、纯化、连接,转化含有目的基因的重组载体.以含目的基因片段的重组载体转化大肠杆菌BL21(DE30)并表达.表达产物经纯化后,用ELISA法检测其抗原性.结果:经酶切、测序分析表明,插入的基因片段为Hpl8000 u的外膜蛋白编码基因,与Tomb等报道相比较,有2%编码基因发生变异,1.7%氨基酸残基改变.经SDS-PAGE分析发现,融合基因表达的蛋白为38000 u,其中pET32a(+)表达的蛋白约为20000u,可溶性表达产物占全菌总蛋白的21.3%.重组蛋白经Ni-NTA琼脂糖树脂纯化后,其纯度达95%以上.ELISA法检测显示,该重组蛋白可被Hp阳性患者的血清所识别,具有良好的抗原性.结论:成功地克隆并表达Hp18000 u的外膜蛋白编码基因,为Hp蛋白质疫苗的研制和快速诊断试剂盒的研究奠定了基础.
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Hp对慢性萎缩性胃炎内皮素及一氧化氮水平影响的实验与临床研究
目的:试图通过实验与临床研究观察Hp根除前后慢性萎缩性胃炎(CAG)NO和ET水平的改变,从这一角度出发探讨Hp相关CAG的发病机制.方法:(1)实验研究:采用SS1 Hp菌株,3%水杨酸钠、5%乙醇,5 mmol/L去氧胆酸钠灌胃,加以饥饱失常,制作Hp相关CAG大鼠模型.模型大鼠随机分成3组:(a)Hp根除组:根除Hp三联(德诺、阿莫西林、甲硝唑)治疗2 wk.(b)模型自然恢复组:采用蒸馏水灌胃.(c)正常对照组:正常大鼠.治疗结束后3 mo处死全部大鼠,进行Hp、病理组织学检查及血清一氧化氮(NO)、内皮素(ET)含量测定.(2)临床研究:选择Hp阳性CAG患者42例,Hp阴性CAG患者25例,Hp阳性慢性浅表胃炎(CSG)21例,分别进行Hp、病理组织学检查及血中NO和ET测定.结果:实验CAG模型中,Hp根除组血清NO、ET含量[(3 672±1 845)ng/ml,(181.14±22.5)×10-3ng/ml]较模型自然恢复[(5 887±1 896)ng/ml,(211.67±34.36)×10-3ng/ml]显著减低,P<0.01,但仍未达正常水平[(2461±949)ng/ml,(135.42±27.46)×10-3ng/ml].临床研究Hp阳性CAG患者,血清NO(5 834±1 896)ng/ml、ET(91.18±34.19)×10-3ng/ml明显高于Hp阴性CAG[(2 773±1 896)ng/ml,(68.37±1424)×10-3ng/ml],P<0.01,也高于Hp阳性CSG患者[(3 420±1 024)ng/ml,(68.90±19.47)×10-3ng/ml],P<0.01.结论:Hp相关CAG血中NO和ET水平显著升高,二者呈正相关,根除Hp后,NO和ET水平显著降低.
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幽门螺杆菌融合蛋白HspA-UreB的表达和免疫学活性
目的:构建表达幽门螺杆菌(H pylori)融合蛋白HspA-UreB的重组表达质粒,并研究其免疫学活性.方法:定向克隆方法将郑州分离Hp菌株MEL-HP27的hspA和ureB基因融合连接入原核表达载体pET30(a),构建重组表达质粒pET-HU27.该质粒转化大肠杆菌BL21后经IPTG诱导,SDS-PAGE分析融合蛋白HspA-UreB表达情况.Ni2+柱亲和层析纯化融合蛋白,与小鼠免疫血清进行Western blot分析,检测融合蛋白的免疫反应性.结果:特异PCR法与质粒酶切鉴定证实重组表达质粒pETHU27构建成功.SDS-PAGE分析显示在82.1 KDa处出现特异蛋白带,占菌体总蛋白的21%,亲和层析法获得纯度为91%的纯化融合蛋白,经免疫小鼠制备的血清可以识别该融合蛋白.结论:成功构建表达H pylori HspA-UreB融合蛋白的重组表达质粒,表达的融合蛋白具有良好的免疫反应性.
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幽门螺杆菌感染者胃黏膜中内质网分子伴侣Grp94的表达
目的:探讨幽门螺杆菌感染对胃黏膜组织中内质网分子伴侣Grp94mRNA及蛋白水平表达的影响及意义.方法:应用半定量RT-PCR方法及Western blot方法检测感染与未感染幽门螺杆菌的胃黏膜组织中Grp94mRNA及蛋白的表达情况.结果:未感染组28例,Grp94mRNA表达量为0.424±0.055,感染组32例,Grp94mRNA表达量为0.882±0.082.两组相比感染组Grp94mRNA的表达量明显高于未感染组(P<0.01);未感染组Grp94蛋白表达量为0.427±0.036,感染组Grp94蛋白表达量为0.671±0.072,两组相比感染组Grp94蛋白表达量明显高于未感染组(P<0.01).结论:幽门螺杆菌感染可使胃黏膜增加Grp94mRNA的表达及蛋白的合成,这可能有利于胃黏膜的自身保护作用.
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胃食管反流病与幽门螺杆菌相关性胃炎及胃肠激素的关系
目的:通过对胃食管反流病(GERD)患者幽门螺杆菌(H pylori)感染、Hpylori相关性胃炎的程度及血清胃泌素(Gas)、胃动素(Mot)水平的检测,探讨Hpylori与GERD之间的关系.方法:GERD患者53例按内镜表现分为非糜烂性反流病(NERD)和反流性食管炎(RE)2组,结合病检评估胃窦、胃体黏膜炎症及食管黏膜损伤,采用放免法检测空腹血Gas,Mot水平.H pylori检测采用血清抗体法,组织银染及尿素酶依赖试验(14C-尿素呼气试验或快速尿素酶试验),三项试验中二项阳性定为Hpylori感染.20名正常健康人作对照.32例NERD患者18例Hpylori阳性,14例阴性;2l例RE患者12例Hpylori阳性,9例阴性.RE组Ⅰ级食管炎11例,Ⅱ级7例,Ⅲ级3例.53例GERD中,Hpylori(+)30例,Hpylori(-)23例.结果:RE组Mot水平显著低于对照组(360±126 vs 440±110 μg/L,aP<0.05).NERD组Mot水平与对照组相比差异无显著性(P>0.05).NERD和RE二组患者Gas水平与对照组相比均无显著性差异(P>0.05),Hpylori(+)GERD患者Gas水平显著高于对照组(35.8±11.6 vs 28.5±10.6μg/L,bP<0.05).Hpylori(+)GERD患者与Hpylori(-)组相比,胃窦胃炎及胃体胃炎均有高度显著性差异(P<0.005),但2组食管炎程度并无显著性差异(P>0.05),胃炎的程度与Hpylori感染有关但与食管炎的程度无关.结论:Mot与RE发病有关.Hpylori(+)GERD患者有较高Gas水平及明显胃窦及胃体炎症,但Hpylori性胃炎与食管病变分级无关.
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幽门螺杆菌vacA毒性片段与hpaA融合基因的原核表达
目的:构建幽门螺杆菌vacA毒性片段(v)与hpaA融合基因的原核表达载体,并诱导表达,为制备具有治疗与预防作用的疫苗奠定基础.方法:通过设计带有编码IL-3N端12个氨基酸引物,用PCR技术从pQE30-V质粒扩增出有接头的v基因,克隆至质粒pTrc99A-HpaA中与hpaA融合.将融合基因插入原核表达载体pQE30,再将pQE30-V-HpaA转化大肠杆菌DH5α,经IPTG诱导表达,SDS-PAGE分析表达结果,Western blotting鉴定其抗原性.结果:融合蛋白的相对分子量约为65 000,能与幽门螺杆菌感染的人阳性血清发生抗原抗体反应.结论:重组表达质粒pQE30-V-HpaA表达成功,为进一步研究其免疫学活性及制备疫苗提供了材料.
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人幽门螺杆菌热休克蛋白A编码基因的克隆、表达及抗原性研究
目的:构建含人幽门螺杆菌(Hpylori)热休克蛋白A编码基因的重组载体、进行核甘酸序列分析,并在E. coliBL21中表达,研究其抗原性,为疫苗的开发奠定基础.方法:利用分子克隆技术从Hpylori DNA染色体中,扩增热休克蛋白A编码基因片段;将目的基因与载体pET32a(+)同时经kpnⅠ、BamH Ⅰ双酶切、纯化、连接后,转化含有目的基因的重组载体;以含目的基因片段的重组载体转化大肠杆菌BL21(DE30)并表达;表达产物经纯化后,用Western blot法检测其抗原性.结果:经酶切、测序分析表明,插入的基因片段为Hpylori热休克蛋白A编码基因,与GenBank报道的相比较,有1.6%的碱基(bp)发生变异,1.6%的氨基酸残基改变.经SDS-PAGE分析发现,融合基因表达的蛋白Mr为33×103,其中pET32a(+)表达的蛋白Mr约为20×103,可溶性表达产物占全菌总蛋白的18.96%.重组蛋白经Ni+-NTA琼脂糖树脂纯化后,其纯度达95%以上.用Westernblot方法检测显示,该重组蛋白可被Hpylori阳性患者的血清所识别,具有良好的抗原性.结论:成功地克隆并表达了H pylori热休克蛋白A码基因,为Hpylori蛋白质疫苗的研制和快速诊断试剂盒的研究奠定了良好的基础.
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C57BL/6小鼠幽门螺杆菌感染动物模型的建立
目的:建立幽门螺杆菌(Hpylori)小鼠感染模型.方法:以C57BL/6小鼠为实验动物,经口感染接种Hpylori悉尼株(SS1),置层流柜中饲养.用HE染色、石碳酸-碱性品红染色、免疫组织化学染色、尿素酶实验、细菌培养等方法检测接种小鼠,并用PCR、基因测序方法对胃组织细菌、胃组织分离培养细菌DNA进行分析.结果:小鼠胃组织分离培养的细菌与接种细菌形态及特性相同.胃窦隐窝可观察到细菌定植,胃黏膜下层可见炎症细胞浸润.胃组织DNA中成功扩增出H pylori 16SrRNA及cagA基因的目的片段,基因测序结果显示与接种菌株序列完全一致.结论:H pylori SS1经口接种C57BL/6小鼠2 mo后可成功在胃内定植并导致成慢性胃炎.
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幽门螺杆菌细胞毒素相关抗原A的表达纯化及其临床研究
目的:cagA+Hp的感染与胃癌的相关性在亚洲有不同报道,本研究应用重组幽门螺杆菌毒素相关抗原A(cagA)建立检测血清中抗CagA抗体的方法,以探讨本地区cagA+Hp的感染与胃癌的相关性.方法:构建表达幽门螺杆菌Cagg抗原的表达载体,工程菌经IPIG诱导,表达的CagA包含体经Ni柱纯化,变性、复性、透析并经活性鉴定后,抗原被点在硝酸纤维素膜上或用以包被ELISA板,建立检测正常人及胃癌患者血清抗Cag3抗体的DIGFA(斑点金免疫渗滤试验,简称滴金法)和ELISA法.结果:重组克隆pltSETcagA的工程菌可表达M36000的目的蛋白,表达形式为包含体;CagA包含体经纯化后SDS-PAGE显示纯度达98%以上,活性经ELISA证实;正常人64例及胃癌患者血清50例中,cagA+Hp的感染率分别为29.7%和62%,胃癌患者cagA+Hp的感染率明显高于正常人(P<0.01).结论:我们建立的检测血清抗CagA抗体的DIGFA和ELISA均具有良好的可重复性;胃癌患者cagA+Hp的感染比例明显高于正常人,CagA阳性幽门螺杆菌的感染与胃癌有关,CagA可作为制备防治caen+Hp感染的疫苗的后备抗原.CagA可作为幽门螺杆菌疫苗制备的候选抗原,检测患者血清幽门螺杆菌CagA抗体,有可能为胃癌的发生提供预警作用.
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砂土鼠胃黏膜幽门螺杆菌长期感染的病理改变
目的:用Hp标准菌株感染砂土鼠以建立Hp感染的长期动物模型,探讨Hp的可能致病机制.方法:取SPF级砂土鼠90只,随级分为实验组60只,对照组30只,实验组接种Hp菌液0.5 ml/只,连续3 d,每天1次,对照组则不做相应处理,接种Hp1,2,3,6mo分别处死实验组和对照组各3只,余下鼠继续喂养30mo后全部处死实验组34只(剔除实验中研究结束前死亡的14只砂土鼠)和对照组8只.取动物胃黏膜组织分别进行肉眼观察、HE染色、AB/PAS染色、Hp、CD3、BrdU免疫组化染色,评价Hp感染后胃黏膜病理组织学改变.结果:Hp接种后所有实验组动物胃黏膜Hp检查阳性,对照组动物未有Hp定植.病理组织学检查Hp感染1mo开始炎症反应明显,随着感染时间延长,炎症越来越重,感染30mo后胃黏膜出现大小不等的隆起性病变,出现典型的肠上皮化生及少数不典型增生改变,未见显著萎缩性胃炎及胃癌发生,对照组无炎症反应.结论:本研究表明Hp长期感染砂土鼠引起胃黏膜严重炎症反应、肠上皮化生及不典型增生,该模型的建立对于Hp致病机制的研究具有重要的应用价值.
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AIM To study the relationship among typing of Traditional Chinese Medicine (TCM) and Helicobacterpylori infection, expression of oncogene and tumor suppresser genes in gastric cancer and precancerouslessions.METHODS According to TCM typing, 120 patients with chronic superficial gastritis, intestinal metaplasia,atypical hyperplasia and gastric cancer were divided into 4 groups: 21 patients with coexistence of cold andheat syndrome (group R), 22 patients with in coordination between the liver and the spleen (group U), 29patients with deficiency of the spleen-yin (group I) and 48 patients with insufficiency of the spleen-yang(group H). Protein expression of c-myc, p21 and p53 were detected immunohistochemically, and Hp wereconfirmed by modified Giemsa method.RESULTS The Hp infection of the group H was significantly higher (72.9%) than that of group R(38.1%, P<0.01) and group U (40.9%, P<0.01). Expression of c-myc, p21 and p53 were significantlyrelated to Hp infection and severity of gastric mucosa lesions (group H>group I>group U>group R).CONCLUSION Hp infection, expression of oncogene and tumor suppresser genes were related to TCMtyping. These parameters were helpful in identification of symptoms and signs and TCM differentiationdiagnosis.
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AIM To investigate the diagnostic value of CagA IgG in serum.METHODS Seventy three patients with peptic ulcer infected with HP were eradicated by antibioticstherapy. At pretreatment, wk9 and wk20 after treatment, the detection of Hp in gastric muscosa bybacteriologic method were performed, and CagA and whole-cell antigen of HP igG in serum by ELISAmethod were also performed at the same time.RESULTS The IgG titres of Hp CagA and whole-cell antigen changes in accordance with the efficacy ofHp eradicated. The former with an earlier appearance and a greater number of cases decreased to normallevel in comparison with the latter.CONCLUSION CagA IgG is a better index for observing the effectiveness of the eradication of Hp.
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Chronic gastritis ( CG ) and peptic ulcer ( PU ) are frequently-occurring diseases. It is now well recognized that Helicobacter pylori (Hp) is a major factor that leads to CG and PU[1-8] In order to study the relationship among T lymphocyte subsets, NO, Hexosamine and Hp infection in patients with chronic gastric diseases, the levelsof blood T lymphocyte subsets, plasma NO and hexosamine in gastric mucosa were measured respectively in 30 patients with CG and 32 patients of PU + CG.
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Helicobacter pylori (H. pylori ) is a major causativebacterium of chronic gastritis, peptic ulcer and mucosaassociated lymphoid tissue lymphoma in humans, and associated with an increased risk of gastric cancer[1 -8]. An important virulant factor of H. pylori is the vacuolating cytotoxin ( VacA ) encoded by vacA that induces cytoplasmic vacuolation in target cells both in vitro and in vivo[9-11]. VacA is produced as a 140 kDa precursor which contains an N-terminal signal peptide and an approximately 33 kDa C-terminal outer membrance exporter. The precursor is cleaved at both N-terminal and C-terminal and secreted into the extracellular milieu as a 95 kDa mature protein. The mature protein futher undergoes specific cleavage to yield 37 kDa and 58 kDa subunits[12-14] Although vacA is present in all H. pylori strains, only about 50% to 60% of strains can induce vacuolation of epithelial cells as assessed by the HeLa cell assay. vacA shows considerable genetic variation in H. pylori isolated from all over the world and contains at least two variable regions. The s region exists as sl or s2 allelic types. Among type sl strains, subtypes sla and slb have been identified. The m region occurs as ml or m2 allelic types. Specific vacA genotype of H. pylori strains are associated with the production of the cytotoxin in vitro, epithelial damage in vivo, and clinical consequences[15-27]. The other virulant factor is the cytotoxin-associated protein (CagA) encoded by the cytotoxin-associated gene (cagA). The cagA gene is present in about 60% to 70% of strains and all of these strains express the cagA. The presence of cagA is also associated with the production of the cytotoxin in vitro, and clinical outcome[24-30]. The aim of this study was (i) to identify vacA genotypes and cagA status of H. pylori isolated from Chinese patients; (ii) to evaluation the relatioship beween vacA genotypes, cagA status and related gastroenterological disorders.