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。     

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（Helicobacter pylori,Hp）小鼠感染模型。方法：建立Hp经口感染SPF级小鼠的动物模型,取小鼠胃粘膜组织,利用PCR技术、尿素酶实验、细菌培养等方法检测接种小鼠,对结果进行判定。结果：Hp可感染C57BL／6小鼠并在小鼠胃部定植。     

幽门螺杆菌(SS1)感染及其胃炎的小鼠模型

目的 :建立感染幽门螺杆菌 (Helicobacterpylori,H pylori)SS1株BALB/c小鼠感染模型 ,研究H pylori胃内定植及胃黏膜病理变化。 方法 :BALB/c小鼠胃内分别接种体外培养的H pyloriSS1株 (实验组 )或PBS(对照组 ) ,组织学方法评价H pylori定植及胃黏膜病理变化。结果 :所有对照组小鼠胃组织未见H pylori定植 ,胃组织也未见明显的炎症反应 ;而所有实验组小鼠在感染H pylori 12周后 ,胃黏膜表面的黏液层及胃小凹顶端可见大量H pylori,胃体及胃窦交界处、胃体及胃底交界处最多 ;胃组织可见到不同程度的炎性反应 ,感染H pylori 2 4周后 ,胃组织炎性反应加重。结论 :用胃内接种方法建立了小鼠H pylori感染及其相关性胃炎的模型。     

卡介苗ERP基因缺失突变株在小鼠体内毒力的研究

为初步研究卡介苗ERP基因缺失突变株对小鼠的毒性作用,将卡介苗ERP基因缺失突变株和BCG分别皮下接种BALB/c小鼠后,取小鼠脾脏和肺脏匀浆,将匀浆液稀释后接种于罗氏培养基,培养3周后计菌落数(CFU);同时观察肺部病理改变,并比较和分析免疫16周时各组小鼠肺、肝、脾的脏器系数.结果表明,卡介苗ERP基因缺失突变株较BCG对BALB/c小鼠毒性低.     

H.pylori抗体鸡蛋制品对H.pylori感染BALB/c小鼠的实验研究

目的通过幽门螺杆菌(Helicobacter pylori,H.pylori)标准株的接种,建立BALB/c小鼠感染H.pylori胃炎动物模型,评价H.pylori抗体鸡蛋制品对小鼠感染性胃炎的预防效果。方法将灭活的H.pylori国际标准菌株(NCTC 11637)作为抗原,对产蛋鸡进行免疫。免疫后收集鸡蛋,对达到效价的鸡蛋,无菌采集卵黄。BALB/c小鼠60只,适应性喂养1周后,随机分为5组,Ⅰ组为胃炎模型组,Ⅱ组为生理盐水组,Ⅲ、IV、V组分别为低剂量、中剂量和高剂量卵黄抗体组,每组12只。Ⅰ组予H.pylori菌液灌胃造模,Ⅱ组先予生理盐水灌胃后再予H.pylori菌液灌胃对照,Ⅲ、IV、V组分别用不同剂量抗H.pylori卵黄抗体灌胃后再予H.pylori菌液灌胃造模。小鼠均于距最后一次灌胃后8周全部处死,用微需氧细菌培养检测H.pylori定植;HE染色观察小鼠胃黏膜病理组织学改变。结果在接种H.pylori后第8周,Ⅰ组和Ⅱ组小鼠胃内均有大量H.pylori定植,感染率为91.7%,Ⅲ组的感染率是58.3%,IV和V组的感染率均小于30%。结论 H.pylori抗体鸡蛋制品可以抑制BALB/c小鼠感染H.pylori,抗体的保护作用与抗体的剂量呈正相关。     

重组血型抗原结合黏附素的免疫原性初步探讨

目的 构建幽门螺杆菌(Helicobacter pylori, Hp)血型抗原结合黏附素(blood group antigen-binding adhesin,babA)基因,表达纯化重组BabA蛋白;初步探索口服免疫BabA配伍双突变大肠埃希菌(E.coli)热不稳定毒素(double mutant heat-labile toxin, dmLT)佐剂在BALB/c小鼠中的免疫保护作用。方法 从Hp SS1株基因组DNA中扩增babA编码基因,构建原核表达质粒pET28a-babA,转化入E.coli BL21(DE3)感受态细胞,IPTG诱导表达,His标签纯化。将纯化的BabA蛋白配伍dmLT免疫佐剂口服免疫BALB/c小鼠,生理盐水对照;末次免疫后2周小鼠眼眶静脉采血分离血清,分析特异性体液免疫应答;然后进行Hp胃部活菌感染,感染27周采集脾淋巴细胞进行细胞免疫分析并对小鼠胃部细菌定植量进行检测。结果 重组质粒pET28a-babA基因序列正确;重组蛋白BabA以包涵体形式表达,相对分子质量56 000,纯度为95.9%;重组BabA蛋白配伍dmLT佐剂口服免疫小鼠可诱导产生...     

利用无传染性耻垢分枝杆菌建立小鼠结核病模型的探索

目的:利用耻垢分枝杆菌(M.smegmatismc2155)建立C57BL/6小鼠结核病模型。方法:每天以高剂量(5×107CFU)耻垢分枝杆菌给C57BL/6小鼠腹腔注射,连续感染4周,检测耻垢分枝杆菌对小鼠的致病性。分别于2周和4周处死小鼠,无菌条件下解剖小鼠取肺、脾脏组织匀浆,进行组织内细菌活力检测;通过嗜酸性染色进行分枝杆菌的鉴定;同时进行病理切片的制备,观察肺和脾脏组织的病理变化;最后进行菌体DNA的提取和基因检测,根据上述指标确定小鼠结核病模型的建立是否成功。结果:腹腔感染小鼠2周后,模型组小鼠只有脾脏组织匀浆液出现抗酸染色阳性菌落,肺部组织未见阳性菌落。腹腔感染小鼠4周后,模型组小鼠肺、脾脏组织匀浆液中均可见大量抗酸染色阳性的菌落;组织病理学观察结果显示:小鼠肺组织主要表现为以中性粒细胞为主的炎性病变;基因检测结果表明:模型组小鼠肺组织匀浆液中可检测到耻垢分枝杆菌特异性3-磷酸甘油醛脱氢酶(gap)基因,而脾脏组织未扩增出耻垢分枝杆菌特异性基因。结论:通过腹腔注射无致病性耻垢分枝杆菌方法,成功建立C57BL/6小鼠结核病发生模型,为结核分枝杆菌与宿主相互作用研究提供安全的疾病模型。     

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.     

幽门螺旋杆菌感染与胃癌中Shh和C-myc表达的关系

为了探讨胃癌中幽门螺旋杆菌(Hp)感染和Sonic Hedgehog(Shh)、C-myc表达,它们之间的相关性以及胃癌发生的可能机制,采用免疫组化法检测89例胃癌组织及20例正常胃上皮组织中Shh及C-myc的表达。并采用快速尿素酶试验,组织病理学检测两种方法检查Hp。实验结果显示,胃癌组织Shh的表达要明显高于正常上皮组织,二者之间有显著差异(P<0.05);胃癌组织C-myc的表达水平也高于正常胃上皮组织,二者之间有显著差异(P<0.05);Hp阳性的C-myc阳性表达率明显高于Hp阴性,二者之间有显著差异(P<0.05);Shh表达阳性率在Hp阳性和阴性胃癌中无显著差异(P>0.05)。结果提示,胃癌的发生与癌基因Shh及C-myc的过度表达有关,Hp感染的致癌机制中可能有癌基因C-myc参与。     

幽门螺杆菌基因分型与胃病关系研究进展

幽门螺旋杆菌(Helicobacter pylori,Hp)已被国际癌症组织确认为胃部疾病最主要的致病因子。近年来对于Hp菌株的基因分型、流行病学和致病性等方面的研究逐步深入,越来越多的研究成果证实对Hp基因分型进行细化可为精准医疗提供依据。Hp可依照Cag A羧基末端磷酸化位点、Vac A信号区(s)中间区(m)过渡区(i)缺失区(d)和粘附因子(OMPs)进行基因分型。Hp基因型差异可导致不同毒性作用,从而引起不同临床结果和疗效预后。因此对Hp基因分型可为胃病的防治提供重要依据。本文就Hp基因分型方法、基因分型及其与胃部疾病研究进展进行如下综述。     

小鼠溃疡性结肠炎模型的建立及病理组织学比较

目的建立乙酸,右旋葡聚糖硫酸钠（dextran sodium sulfate,DSS）,幽门螺杆菌（Helicobacter pyliri）小鼠溃疡性结肠炎（ulcerative colitis）动物模型,通过病理学对比观察,选择最佳的小鼠溃疡性结肠炎动物模型。方法将40只清洁级BALB/c小鼠随机分为4组,实验组中Ⅰ组采用乙酸刺激法诱发溃疡性结肠炎,Ⅱ组采用饮用3.5%DSS溶液诱发结肠炎,Ⅲ组采用幽门螺杆菌感染小鼠诱发溃疡性结肠炎,对照组饮用蒸馏水。观察小鼠每日的体重,大便性状和隐血情况,以及结肠大体形态和组织病理学改变。结果乙酸,右旋葡聚糖硫酸钠均可引起小鼠疡性结肠炎。结论 DSS诱发的小鼠溃疡性结肠炎是一种较理想的UC动物模型,可作为研究UC发病机制和药物治疗较理想的工具。     

Helicobacter pylori-associated gastric pro- and antioxidant formation in Mongolian gerbils

Helicobacter pylori colonized gastric mucosa is manifest in a significant neutrophil infiltration with an extensive level of oxyradical formation. Mongolian gerbil is one of the excellent models for H. pylori-infection. The present study was designed to investigate pro- and antioxidant formation in the stomach of H. pylori-positive gerbils. Fourteen male Mongolian gerbils (MGS/Sea) were orally inoculated with H. pylori (ATCC43504) (Hp group) and 15 gerbils were inoculated with the culture media (Control). H. pylori infection was confirmed by the serum anti-H. pylori IgG test. Each gerbil was evaluated 6 or 12 weeks after the inoculation. Neutrophil infiltration was assessed by the tissue MPO activity. Mucosal oxidative stress was evaluated by thiobarbituric acid-reactive substances (TBARS), total glutathione contents, glutathione peroxidase (GSHPx) activity and Cu-, Zn-superoxide dismutase (SOD) activity. In Hp group, the H. pylori was persistently infected until 12 weeks. The level of MPO activity was significantly higher in Hp group at 6 and 12 weeks. Although the levels of TBARS and total glutathione were within the same range as controls at 6 weeks, they were significantly increased at 12 weeks. However, GSHPx activity was significantly increased at 6 weeks, but became the same range with the controls at 12 weeks. SOD activity showed no significant increase in Hp group at 6 and 12 weeks. In conclusion, H. pylori inoculation induced gastric mucosal neutrophil activation and pro-oxidant formation and also increased total glutathione contents, one of the mucosal antioxidants in gerbils.     

BABL/c小鼠感染大肠杆菌O127模型的建立及TGF-β1因子的荧光定量检测

目的建立BABL/c小鼠感染大肠杆菌O127的模型及TGF-β1的荧光定量PCR方法检测方法。方法通过灌胃的方式建立肠炎性小鼠模型,并利用嵌合荧光定量的方法对其结肠TGF-β1定量分析实验。结果成功建立了肠炎性小鼠模型,荧光定量PCR试验结果表明实验组TGF-β1表达量明显高于对照组。结论利用经口灌胃的方法可以成功的建立肠炎性小鼠动物模型,TGF-β1定量方法可以作为模型建立的评价标准。     

H9N2亚型猪流感病毒感染BALB/c小鼠动物模型的建立

目的建立H9N2亚型猪流感病毒感染BALB/c小鼠动物模型,为研究病毒致病机制提供模型动物。方法通过滴鼻的方法将H9N2亚型猪流感病毒感染BALB/c小鼠,观察小鼠的症状和组织病理变化。结果 BALB/c小鼠的临床症状明显,病理变化典型。结论 H9N2亚型猪流感病毒感染BALB/c小鼠的疾病模型成功建立。     

Profiling and identification of eubacteria in the stomach of Mongolian gerbils with and without Helicobacter pylori infection

Background. Mongolian gerbils are frequently used to study Helicobacter pylori‐induced gastritis and its consequences. The presence of an indigenous bacterial flora with suppressive effect on H. pylori may cause difficulties with establishing this experimental model. Aim. The aim of the present study was to determine bacterial profiles in the stomach of Mongolian gerbils with and without (controls) H. pylori infection. Methods. Gastric tissue from H. pylori ATCC 43504 and CCUG 17874 infected and control animals were subjected to microbial culturing and histology. In addition, gastric mucosal samples from H. pylori ATCC 43504 infected and control animals were analyzed for bacterial profiling by temporal temperature gradient gel electrophoresis (TTGE), cloning and pyrosequencing of 16S rDNA variable V3 region derived PCR amplicons. Results. Oral administration of H. pylori ATCC 43504, but not CCUG 17874, induced colonization and gastric inflammation in the stomach of Mongolian gerbils. Temporal temperature gradient gel electrophoresis (TTGE) and partial 16S rDNA pyrosequencing revealed the presence of DNA representing a mixed bacterial flora in the stomach of both H. pylori ATCC 43504 infected and control animals. In both cases, lactobacilli appeared to be dominant. Conclusion. These findings suggest that indigenous bacteria, particularly lactobacilli, may have an impact on the colonization and growth of H. pylori strains in the stomach of Mongolian gerbils.     

Cutaneous leishmaniasis in anti-IgM-treated mice: enhanced resistance due to functional depletion of a B cell-dependent T cell involved in the suppressor pathway

The contribution of B cells and antibodies to either the resistance or susceptibility to cutaneous leishmaniasis has been investigated in mouse strains rendered B cell-deficient by treatment with anti-mouse IgM antisera from birth (mu-suppressed). These studies confirm that immunity to cutaneous disease in a normally resistant mouse strain (C3H/HeJ) is independent of antibody, but that B cells and/or antibodies are required for the evolution of suppressed DTH and the consequent disease susceptibility of BALB/c mice. Anti-IgM-treated BALB/c mice, which lacked detectable anti-leishmanial antibodies during the course of infection, displayed a sustained DTH response to leishmanial antigen and were able to control their cutaneous lesions. The enhanced resistance of mu-suppressed mice could be completely abrogated by transfer of suppressor T cells from infected control animals into mu-suppressed mice before their infection. Thus the suppressor T cells, which are generated during leishmanial infection in BALB/c mice, can effect suppression in the absence of antibody. Evidence that B cells or antibodies are required for the generation of suppressor T cells was demonstrated by using BALB/c mice in which suppressor T cells fail to be generated during infection as a result of prior sublethal irradiation. Splenic T cells from normal mice could overcome the resistance conferred by sublethal irradiation, whereas splenic T cells from mu-suppressed mice could not. Thus the enhanced resistance of mu-suppressed BALB/c mice appears to be a consequence of their lack of functional expression of a B cell-dependent T cell critical to the suppressor pathway.     

Growth Inhibitory,Bactericidal, and Morphostructural Effects of Dehydrocostus Lactone from Magnolia sieboldii Leaves on Antibiotic-Susceptible and -Resistant Strains of Helicobacter pylori

Helicobacter pylori is associated with various diseases of the upper gastrointestinal tract, such as gastric inflammation and duodenal and gastric ulcers. The aim of the study was to assess anti-H. pylori effects of the sesquiterpene lactone dehydrocostus lactone (DCL) from Magnolia sieboldii leaves, compared to commercial pure DCL, two previously known sesquiterpene lactones (costunolide and parthenolide), (–)-epigallocatechin gallate, and four antibiotics. The antibacterial activity of natural DCL toward antibiotic-susceptible H. pylori ATCC 700392 and H. pylori ATCC 700824 strains (MIC, 4.9 and 4.4 mg/L) was similar to that of commercial DCL and was more effective than costunolide, parthenolide, and EGCG. The activity of DCL was slightly lower than that of metronidazole (MIC, 1.10 and 1.07 mg/L). The antibacterial activity of DCL was virtually identical toward susceptible and resistant strains, even though resistance to amoxicillin (MIC, 11.1 mg/L for PED 503G strain), clarithromycin (49.8 mg/L for PED 3582GA strain), metronidazole (21.6 mg/L for H. pylori ATCC 43504 strain; 71.1 mg/L for 221 strain), or tetracycline (14.2 mg/L for B strain) was observed. This finding indicates that DCL and the antibiotics do not share a common mode of action. The bactericidal activity of DCL toward H. pylori ATCC 43504 was not affected by pH values examined (4.0–7.0). DCL caused considerable conversion to coccoid form (94 versus 49% at 8 and 4 mg/L of DCL for 48 h). The Western blot analysis revealed that urease subunits (UreA and UreB) of H. pylori ATCC 43504 were not affected by 10 mM of DCL, whereas UreA monomer band completely disappeared at 0.1 mM of (–)-epigallocatechin gallate. Global efforts to reduce the level of antibiotics justify further studies on M. sieboldii leaf-derived materials containing DCL as potential antibacterial products or a lead molecule for the prevention or eradication of drug-resistant H. pylori.     

Reduced expression of STAT4 and IFN-gamma in macrophages from BALB/c mice

BALB/c mice have been shown to easily induce Th2 type responses in several infection models. In this study, to examine the mechanisms of Th2 dominant responses in BALB/c mice, we assessed several macrophage functions using C3H/HeN, C57BL/6, and BALB/c mouse strains. Peritoneal macrophages from three strains of mice equally produced IL-12 by stimulation with LPS plus IFN-gamma. However, IFN-gamma production in response to IL-12 or IL-12 plus IL-18 was much lower in macrophages from BALB/c mice than other strains. IFN-gamma produced by activated macrophages induced IL-12R mRNA expression in T cells and macrophages themselves depending on their amount of IFN-gamma; namely, macrophages from BALB/c mice induced lower expression of IL-12R. Intracellular levels of STAT4 were much lower in macrophages from BALB/c mice. However, other STATs, such as STAT1 or STAT6, were expressed similarly in the three mouse strains. STAT4 and IFN-gamma production by other cell types such as T cells and B cells were equal in C3H/HeN and BALB/c mice. These results indicate that macrophages from Th2-dominant BALB/c mice have different functional characters compared with other mouse strains; that is, STAT4 expression and IFN-gamma production are reduced, which is one of the causes to shift to Th2-type responses.