幽门螺旋杆菌ATCC 43504感染BALB/c小鼠动物胃炎模型的建立及分析

利用幽门螺旋杆菌标准菌株建立稳定可靠的幽门螺旋杆菌(Helicobacter pylori,Hp)感染小鼠胃炎模型对Hp疫苗研制、Hp致病机制的研究及抗Hp药物的筛选具有重要意义。通过灌胃国际标准菌株幽门螺旋杆菌Hp ATCC 43504,构建了BALB/c小鼠动物胃炎模型。利用小鼠胃部Hp尿素酶活性检测、Hp的定量培养、PCR检测、组织病理学等多种方法鉴定BALB/c小鼠动物胃炎模型。通过Hp诊断试剂盒检测,造模组小鼠的胃组织能使Hp尿素酶试剂变色,呈现玫瑰红色,而健康小鼠的胃组织不能使Hp尿素酶试剂变色,依旧呈现黄色;通过小鼠胃组织匀浆液培养Hp,造模组小鼠的胃组织匀浆液均可在BHI血平板长出Hp菌落,而对照组小鼠的胃组织匀浆液不能培养出Hp菌落;利用PCR对造模组和对照组BALB/c小鼠的胃组织进行Hp检测,造模组小鼠胃组织可扩增出150 bp的DNA产物,而对照组小鼠胃组织无扩增产物;通过HE染色法观察小鼠胃部病理变化和炎症情况,与健康BALB/c小鼠比较,造模组小鼠胃粘膜和粘膜下层有大量白细胞浸润,胃部炎症明显。利用国际标准菌株Hp ATCC 43504菌株成功构建了BALB/c小鼠胃炎模型,为评价防治Hp感染药物的效果奠定了实验基础。     

幽门螺杆菌感染对DSS结肠炎小鼠 Th17/Treg细胞亚群的影响

目的 探讨幽门螺杆菌(H.pylori)感染对葡聚糖硫酸钠(DSS)诱导的小鼠溃疡性结肠炎(UC)的影响。 方法 80只雌性BALB/c小鼠随机分为空白对照组(CON组,n=20)和实验组(n=60),实验组制作DSS诱导UC模型,然后分组建立成为结肠炎组(CLi组)、H.pylori持续感染的结肠炎组(Hp组)和H.pylori根除的结肠炎组(Hp Era组),每组20小鼠。实验过程中观察小鼠一般情况和疾病活动指数(DAI);在第25天、45天时处死动物,分离外周血和结肠组织中单个核细胞,流式细胞仪检测其Th17/Treg细胞亚群变化。 结果 CLi组、Hp组、Hp Era组小鼠DAI评分显著高于CON组(均P结论 H.pylori感染使DSS结肠炎小鼠UC的Th17亚群比例降低、Foxp3+Treg细胞比例增加,这种变化似乎对DSS诱导的结肠炎具有保护作用。     

幽门螺杆菌球形体回复原形的实验研究

目的对幽门螺杆菌（Helicobacter pylori,Hp）球形体进行体内、外回复原形的比较研究,揭示其潜在的传播途径。方法在布氏肉汤的基础上设计了4种Hp再生培养基,对Hp螺旋体、原生质体及球形体进行体外培养;同时采用30只蒙古沙土鼠（Mongolian gerbil）进行体内感染定植实验,对感染小鼠胃粘膜进行Hp定量培养和组织学检测。结果Hp球形体在4种再生培养基中均未能回复生长;而在感染小鼠的体内却观察到了Hp球形体的回复定植。Hp螺旋体感染组在小鼠胃内的定植密度较高,且胃粘膜下可见大量炎症细胞浸润;而球形体感染组并未见到小鼠胃粘膜组织的明显炎症损伤,且仅有少量回复的螺旋体定植。结论Hp球形体作为一种低水平代谢休眠体,代谢活性及毒力均有所减弱,但仍具有潜在的致病性。本研究支持部分Hp球形体具有活力但体外不能培养成活这一假说,提示＂粪-口＂传播应引起更多的关注。     

幽门螺旋杆菌诱发肝硬化大鼠高氨血症的实验研究

目的：探讨幽门螺旋杆菌与肝硬化大鼠高氨血症的相关性。方法：选取32 只SD大鼠,将其随机分为正常组(n=8)、Hp 感染组 (n=8)、肝硬化组(n=8)、肝硬化合并Hp 感染组(n=8)。肝硬化组及肝硬化合并Hp 感染组给予50%四氯化碳橄榄油腹腔注射;正常 组与Hp 感染组给予同量的橄榄油腹腔注射。从建立模型的第9 周开始,Hp 感染组与肝硬化合并Hp 感染组每周感染2 次Hp,共 8 次。记录各组大鼠的一般状态,检测大鼠血氨水平及胃粘膜Hp数量。结果：与正常组比较,肝硬化组及肝硬化合并Hp感染组体 重较低(P＜0.05);与肝硬化组比较,肝硬化合并Hp 感染组体重较低(P＜0.05)。与正常组比较,其余各组胃粘膜Hp 数量较高(P＜ 0.05),与肝硬化组与Hp 感染组比较,肝硬化合并Hp 感染组胃粘膜Hp 数量高(P＜0.05)。与正常组比较,肝硬化组及肝硬化合并 Hp 感染组血氨水平高(P＜0.05),与肝硬化组比较,肝硬化合并Hp 感染组血氨水平高(P＜0.05)。结论：幽门螺旋杆菌能诱发肝硬 化大鼠高氨血症     

抗VacA+CagA+幽门螺杆菌IgY的抗感染作用研究

为研究抗VacA CagA 幽门螺杆菌(Hp)IgY的抗感染作用,以VacA CagA Hp为抗原免疫蛋鸡,聚乙二醇法和水稀释法从鸡卵黄中提取抗-VacA CagA Hp-IgY,酶联免疫吸附实验(ELISA)测定IgY抗体效价。建立胃腔感染VacA CagAHp的昆明系小鼠模型,观察抗-VacA CagA Hp-IgY对小鼠胃腔感染VacA CagA Hp的防治效果。ELISA法测定IgY效价均为1∶20,480;抗-VacA CagA Hp-IgY防治小鼠胃腔感染VacA CagAHp效果较理想,IgY高、中剂量组效果优于阳性对照组(P<0.05);低剂量组效果等同于阳性对照组(P>0.05)。抗-VacA CagA Hp-IgY较好的体内抗感染作用,提示该IgY有望成为较理想的治疗VacA CagA Hp感染的生物制剂。     

幽门螺杆菌致人胃病的小鼠模型研究

目的建立幽门螺杆菌致人胃病的小鼠模型.方法用Ⅰ型幽门螺杆菌(Helicobacter pylori,Hp)对三种SPF级小鼠(BALB/c、NIH、 KM)采用循环滴喂攻击等方法处理,从菌体定值、抗体水平、病理变化三个方面分14、39、69、105 d进行测试.结果 Hp可持续定植于各供试小鼠胃粘膜上,并刺激Hp抗体(IgG)维持一较高水平,Hp在小鼠上主要引起以胃粘膜变性坏死为主,伴有淋巴细胞浸润的炎症反应,这为Hp致人胃病的症状相似.结论为Hp的致病机理,治疗药物和疫苗研究提供了良好的小动物模型.     

幽门螺杆菌表面抗原免疫保护作用的体外与活体研究

目的：调查幽门螺杆菌（Hp)几种表面蛋白体外对T细胞增殖的影响和在小鼠体内的免疫保护作用。方法：评价Hp全菌抗原、尿原酶（Urease)、黏附素（hpaA)、外膜蛋白25（Hop25)和38（Hop38)对人外周血T细胞及小鼠CD4^ T细胞增殖的影响;与佐剂合用,评价上述重组蛋白对小鼠Hp感染的免疫预防作用。结果：Urease和Hop25可刺激人及鼠T细胞增殖,hapA只能刺激Hp^ PBL增殖,而Hop38则有毒性作用;Hop25和Hop38均可产生60％的完全保护,hpaA可产生100％的部分保护即降低细菌定植密度,而Urease只能产生40％的部分保护。结论：外膜蛋白可能是一组高效的Hp疫苗免疫原;其长期免疫效果及对T细胞功能的活体调节作用尚需进一步评价。国际上尚未见相关报道。     

健康体检人群幽门螺杆菌感染的影响因素分析及其与胃蛋白酶原和颈动脉粥样硬化的关系研究

摘要 目的：分析健康体检人群幽门螺杆菌（Hp）感染的影响因素,并进一步探讨健康体检人群Hp感染与胃蛋白酶原（PG）和颈动脉粥样硬化（CAS）的关系。方法：选取2021年3月～2022年3月在我院体检的146例接受Hp筛查的健康体检者,根据碳13尿素呼气试验结果分为Hp阳性组（n=62）和Hp阴性组（n=84）。采用单因素和多因素Logistic回归分析健康体检人群Hp感染的影响因素,比较两组血清PGⅠ、PGⅡ、PGⅠ/PGⅡ水平和CAS比例。Spearman相关系数分析Hp阳性组DOB值与血清PGⅠ、PGⅡ、PGⅠ/PGⅡ和颈动脉内中膜厚度（CIMT）的相关性。结果：单因素分析显示,与Hp阴性组比较,Hp阳性组年龄更大,家族Hp感染史、吸烟、饮酒、饮食口味辛辣、经常摄入腌制食品、共用餐具比例更高,经常喝茶、经常摄入水果/蔬菜比例更低（均P＜0.05）。多因素Logistic回归分析显示,年龄增加、家族Hp感染史、吸烟、饮食口味辛辣、经常摄入腌制食品、共用餐具为健康体检人群Hp感染的独立危险因素,经常喝茶、经常摄入水果/蔬菜为独立保护因素（均P＜0.05）。Hp阳性组血清PGⅠ、PGⅡ水平和CAS比例高于Hp阴性组,PGⅠ/PGⅡ低于Hp阴性组（均P＜0.05）。Spearman相关系数显示,Hp阳性组DOB值与血清PGⅠ、PGⅡ和CIMT呈正相关,与PGⅠ/PGⅡ呈负相关。结论：年龄增加、家族Hp感染史、吸烟、饮食口味辛辣、经常摄入腌制食品、共用餐具是健康体检人群Hp感染的危险因素,而经常喝茶、经常摄入水果/蔬菜为健康体检人群Hp感染的保护因素,Hp感染与血清PG水平变化和CAS密切相关。     

幽门螺旋杆菌重组尿素酶B亚基的纯化及其免疫学性质的研究

目的:幽门螺旋杆菌(Hp)尿素酶是Hp重要的定制因子和致病因子,Hp尿素酶活性位点位于Hp尿素酶B亚基(UreB),研发基于UreB的Hp疫苗是一种很有前景的防治Hp感染的策略。方法:主要利用基因克隆技术从幽门螺旋杆菌标准菌株SS1(Hp SS1)获得Hp尿素酶B亚基基因,并构建含有重组Hp尿素酶B亚基(rUreB)基因的重组表达载体pET-rUreB及其重组菌株;重组菌株经蛋白表达和优化后,利用Ni-NTP镍离子亲和层析和DEAE Sepharose FF阴离子交换层析纯化重组尿素酶B亚基(rUreB),并进一步通过腹腔注射免疫BALB/c小鼠,研究rUreB的免疫学性质。结果:通过基因克隆技术成功获得了Hp尿素酶B亚基基因,并成功构建了重组表达载体pET-rUreB及其重组菌株BL21(DE3)/pET-rUreB,经蛋白表达优化及纯化,可获得高纯度(96.5%)的重组蛋白rUreB。重组蛋白rUreB辅以弗氏佐剂腹腔注射免疫BALB/c小鼠,经间接ELISA鉴定小鼠能够产生针对天然Hp尿素酶和UreB的高滴度特异性抗体,且能够显著性抑制Hp尿素酶的活性。结论:重组Hp尿素酶B亚基能够在大肠杆菌表达系统中获得较高水平的表达,具有较高的免疫学特异性,其抗体能够有效抑制Hp尿素酶活性。为研究基于尿素酶的防治Hp感染的Hp疫苗奠定了一定的实验基础。     

Th1细胞因子(IFN-γ)在幽门螺杆菌致病中的作用

目的：评价Thl细胞因子IFN-γ在幽门螺杆菌(Hp)感染时对胃上皮细胞的作用。方法：胃上皮细胞经IFN-γ处理后,流式细胞术测定表面MHC-Ⅱ类分子的表达和Hp的黏附,ELISA法测定细胞因子对Hp致胃上皮细胞凋亡的影响。结果：IFN-γ可诱导胃上皮细胞表达MHCR类分子,进而增加Hp的黏附。IFN-γ本身即可诱导胃上皮细胞凋亡,并可促进Hp诱导的胃上皮凋亡。结论：Thl细胞因子IFN-γ参与并加剧了Hp感染所致的胃黏膜炎症。     

Type II restriction endonucleases from Helicobacter pylori include an enzyme with a novel recognition sequence

Type II restriction endonuclease activities of Helicobacter pylori strain Roberts and of the type strain H. pylori NCTC 11637 were detected and analysed by conventional techniques. The endonucleases were partially purified, their optima for activity and their recognition and cleavage sites were determined. H. pylori (Roberts) contained at least two enzymes: HpyBI was an isoschizomer of RsaI (GT/AC) and HpyBII was of a novel specificity (GTN/NAC). H. pylori NCTC 11637 was found to contain an isoschizomer of EcoRV (HpyCI: GAT/ATC) and at least one other enzyme which was too unstable to characterise.     

Alanine racemase from Helicobacter pylori NCTC 11637: purification, characterization and gene cloning

The Helicobacter pylori NCTC 11637 alanine racemase gene, alr1, was cloned based on a putative alanine racemase gene, alr, of H. pylori 26695. The protein, Alr1, was purified to homogeneity from Escherichia coli MB2795 cells harboring the alr1 gene. The protein exclusively catalyzes the conversion of l-alanine to the d-isomer with K(m) and V(max) values of 100 mM and 909 mumol min(-1) mg(-1), respectively. The values are 16-fold higher than those for the reaction in the reverse direction. The molecular weight of Alr1 is 42,000 by SDS-PAGE, and 68,000 by gel-filtration analysis. The optimal pH and temperature are pH 8.3 and 37 degrees C, respectively, in good accordance with the characteristics shown by the alanine racemase purified from H. pylori NCTC 11637 cells. Pyridoxal 5'-phosphate was suggested to be the cofactor. The physiological function of Alr1 is discussed regarding energy production in the microbial cells.     

异源表达的幽门螺杆菌cag4蛋白有溶菌糖基转移酶活性

摘要【目的】构建融合基因原核表达载体pET-28a- cag4,并表达重组融合蛋白cag4,分析重组融合蛋白的酶活性,为新型抗生素（或是抗菌药物）的研发提供作用靶位。【方法】本研究利用PCR技术从幽门螺杆菌NCTC11637中克隆了cag4基因;经T-A克隆,酶切鉴定,构建了原核表达载体pET-28a- cag4;经测序鉴定正确后,转化进入大肠埃希菌 BL21(DE3)进行异源表达。利用IPTG体外诱导后,经SDS-PAGE和Western Bolt鉴定目的蛋白表达后,采用Ni2＋-NTA柱在变性条件下纯化目的蛋白,并对重组蛋白进行透析复性处理。将SDS煮沸法获得的溶壁微球菌肽聚糖掺入SDS-PAGE作为底物,进行酶谱分析。【结果】在大肠埃希菌 BL21(DE3)中获得高效表达的重组蛋白; 经SDS-PAGE和Western Bolt鉴定表达后,采用Ni2＋-NTA柱在变性条件下纯化,并进行透析复性处理。将SDS煮沸法获得的溶壁微球菌肽聚糖掺入SDS-PAGE作为底物,进行酶谱分析,表明目的蛋白具有明显的肽聚糖水解活性; 通过监测浊度下降速率,比较其在不同pH条件下活性的变化,即?A/（min?mg protein）,结果表明,幽门螺杆菌cag4蛋白具有溶菌糖基转移酶活性。【结论】幽门螺杆菌cag4蛋白具有溶菌糖基转移酶活性。     

Protection against Helicobacter pylori infection by intestinal immunisation with a 50/52-kDa subunit protein

A mouse model of Helicobacter pylori infection was used to evaluate the vaccine antigen potential of the citrate synthase homologue protein purified from the H. pylori NCTC 11637 strain. Mice were immunised with the protein by intra-Peyer's patch immunisation. This route gives maximal intestinal immunisation and was used to screen oral vaccine candidate antigens without the added complication of simultaneously testing oral delivery systems. Two weeks post-immunisation mice were infected with Sydney strain H. pylori and 4 weeks after infection the mice were killed and the level of H. pylori infection in the stomach determined. Pre-immunisation with the 50/52-kDa protein led to a 84-91% reduction in H. pylori infection compared to unimmunised controls.     

Helicobacter pylori bacterial ghost containing recombinant Omp18 as a putative vaccine

Helicobacter pylori bacterial ghosts, HPBG, were generated by PhiX174 mechanism and loaded with recombinant Omp18, which were then applied in therapeutic immunization of Hp-infected C57BL/6 mice. Recombinant Omp18 loaded HPBG plus cholera toxin stimulated serum anti-Hp and Omp18-specific antibodies which resulted in significant reduction of gastric Hp colonization (P < 0.05).     

幽门螺杆菌Ⅳ型分泌系统HP0527-C基因的克隆表达及其生物学功能的初步研究

为克隆表达幽门螺杆菌（Helicobacter pylori,H.pylori）NCTC 11637 hp0527-c 基因,探讨其生物学功能。设计引物扩增,T-A克隆,测序正确后构建表达载体pET-28a- hp0527-c,转化大肠杆菌BL21,经IPTG诱导表达后以Ni2＋-NTA柱获得纯度为98％重组蛋白。经Western blot鉴定正确后,免疫新西兰大白兔获得了较高滴度的多克隆抗体;将重组蛋白作用于BGC-823细胞,RT-PCR检测细胞IL-8mRNA表达增高;同时用MTT法来检测重组蛋白对细胞增殖的影响,结果呈现一定量的时间和剂量依赖性。已成功克隆了HP0527-C的重组蛋白,初步探讨了其与细胞之间的相互作用,为进一步阐明H.pylori致病机制奠定了基础。     

球形与螺旋形幽门螺杆菌基因表达差异的研究

目的：从基因转录水平了解球形幽门螺杆菌(Helicobacter pylori,Hp)基因表达的变化。方法：幽门螺杆菌标准株NCTC ll637和2株临床分离株,经抗生素—灭滴灵诱导球变。提取等菌量螺旋形和球形Hp的RNA,RT—PCR扩增Hp25种基因,这些基因包括毒力基因(kat、aphA、rdxA、frxA、cysS、ureB glmM、cagA、vacA、fldA、iceAl、hpaA、fliD、napA、oipA、alpAB、babB、hopZ);看家基因(scoB、atpD、g1nA、recA)和功能不明的外膜蛋白基因(hopW、hopX)。扩增产物经分子定量成像系统进行扫描定量。结果和结论：等菌量的球形HpRNA量比螺旋形Hp减少。NCTC ll637、F44和F49菌株的螺旋菌分别有25、20、19个基因RT—PCR阳性,对应的球形菌相应的基因RT—PCR也为阳性。定量分析结果表明：被检测的基因在球形菌中的表达均比螺旋菌降低。提示球形菌致病性的降低与基因的低表达有关。3株球形菌不同基因表达的变化并不完全一致,表达量变化较小且各菌间较一致的是g1mM、oipA、g1nA;表达量变化较大且菌问较一致的基因是：napA、sodB、recA、fliD。     

两株乳酸菌在小鼠体内对幽门螺杆菌抑制作用的初探

成功地建立了小鼠幽门螺杆菌感染模型,并对两株在体外对幽门螺杆菌有显著抑制作用的乳酸菌,进行了体内预防与治疗作用的初步验证。通过尿素酶反应、细菌培养、病理组织切片检验三种指标测试,证明J16发酵乳能够有效预防幽门螺杆菌感染;小鼠胃中乳酸菌数量和幽门螺杆菌数量相反。     

Leptin receptor signaling is required for vaccine-induced protection against Helicobacter pylori

Background:  A vaccine against Helicobacter pylori would be a desirable alternative to antibiotic therapy. Vaccination has been shown to be effective in animal models but the mechanism of protection is poorly understood. Previous studies investigating the gene expression in stomachs of vaccinated mice showed changes in adipokine expression correlated to a protective response. In this study, we investigate a well-characterized adipokine-leptin, and reveal an important role for leptin receptor signaling in vaccine-induced protection.
Materials and Methods:  Leptin receptor signaling-deficient (C57BL/Ks Lepr^db), wild-type C57BL/Ks m littermates and C57BL/6 mice were vaccinated, and then challenged with H. pylori . Levels of bacterial colonization, antibody levels, and gastric infiltrates were compared. The local gene expression pattern in the stomach of leptin receptor signaling-deficient and wild-type mice was also compared using microarrays.
Results:  Interestingly, while vaccinated wild-type lean C57BL/6 and C57BL/Ks m mice were able to significantly reduce colonization compared to controls, vaccinated obese C57BL/Ks Lepr^db were not. All mice responded to vaccination, i.e. developed infiltrates predominantly of T lymphocytes in the gastric mucosa, and made H. pylori -specific antibodies. A comparison of expression profiles in protected C57BL/6 and nonprotected C57BL/Ks Lepr^db mice revealed a subset of inflammation-related genes that were more strongly expressed in nonprotected mice.
Conclusions:  Our data suggest that functional leptin receptor signaling is required for mediating an effective protective response against H. pylori .     

Helicobacter pylori infection in wild-type and cytokine-deficient C57BL/6 and BALB/c mouse mutants

Helicobacter pylori causes gastroduodenal ulcer disease in humans. T lymphocytes and their cytokines are thought to play a substantial role in the control of H. pylori infection. To determine the importance of T helper (Th) cytokines and background genes we investigated the natural course of H. pylori infection in BALB/c and C57BL/6 wild-type or mutant mice deficient for either interleukin (IL)-4 or interferon (IFN)-gamma. H. pylori SPM 326 persisted for at least six months in C57BL/6 but was cleared by BALB/c wild-type mice nine weeks postinfection. H. pylori was recovered more frequently from IFN-gamma(-/-) BALB/c and IFN-gamma( -/-) C57BL/6 mice than from the respective wild-type animals. In contrast, IL-4 deficiency had no detectable effect on H. pylori recovery rates from either strain of mice. Our data suggest a protective role of IFN-gamma by mediating inflammation in murine H. pylori infection. In addition, our data emphasize that background genes which differ between BALB/c and C57BL/6 mice regulate the clearance of H. pylori.