幽门螺杆菌内毒素对小鼠成纤维细胞和人胃粘膜组织细胞的侵入和损害

应用免疫胶金技术证实幽门螺杆菌内毒素（ＨＰ—ＬＰＳ）作用于小鼠成纤维细胞后５ｍｉｎ即能侵入胞浆,６０ｍｉｎ后可在细胞核中检出ＨＰ—ＬＰＳ结合的胶金颗粒。免疫组织化学法检测结果表明,６５．４％的胃粘膜活检标本组织中有ＨＰ—ＬＰＳ存在。受ＨＰ—ＬＰＳ作用的小鼠成纤维细胞和ＨＰ—ＬＰＳ检测阳性的胃炎患者活检标本细胞均出现相似的超微结构病变,如粗面内质网扩张、膜旁核糖体颗粒脱落、线粒体嵴或外膜消失等。实验结果表明幽门螺杆菌内毒素是该菌致慢性胃炎的主要致病物质之一。     

幽门螺杆菌与冠心病关系的病例对照研究

通过病例对照研究方式探讨幽门螺杆菌与冠心病发生之关系。以性别、年龄、职业相同为条件,按病例,对照为1:1,以微剂量法~(14)C—尿素呼气试验调查62对配对个体幽门螺杆菌感染状况。结果显示冠心病病人幽门螺杆菌感染率为64.5％(40/62),对照组感染率为34.0％(21/62)。相对危险度为2.73,95％可信区间为1.22～11.50。结果提示幽门螺杆菌感染与冠心病发生有一定关系。     

幽门螺杆菌及IL-17高表达与原发性IgA肾病的关系

目的 探讨幽门螺杆菌感染及细胞免疫与IgA肾病的关系.方法 收集40例原发性IgA肾病(IgAN组)和32例非IgA相关原发性肾小球肾炎(n-IgAN组)患者,并比较两组患者血清幽门螺杆菌抗体(Hp-IgG)、IL-17、IgA、补体C3、尿蛋白定量和肾小球新月体形成情况.结果 与n-IgAN组比较,IgAN组Hp感染率增高(45.0％比31.3％),血清IL-17水平[(208.69±27.10)ng/L比(171.48±21.44) ng/L,P＜0.05]和新月体检出率(51.90％比5.30％,P＜0.05)升高,IgA/C3比值增大[(2.71±1.37)比(2.05±0.95)].所有幽门螺杆菌感染的患者中,合并IgA肾病者多于合并非IgA肾病者(64.29％比35.71％).幽门螺杆菌感染与否与血清IL-17呈正相关(r=0.581,P＜0.01),IL-17水平与新月体检出率呈正相关(r=0.484,P＜0.01).结论 IgA肾病患者普遍存在幽门螺杆菌感染和IL-17高表达.幽门螺杆菌可能通过影响IL-17相关的细胞免疫状态而参与IgA肾病的发生.     

幽门螺杆菌感染与小儿复发性腹痛的相关性研究

目的研究幽门螺杆菌感染与小儿复发性腹痛的相关性。方法采用~(13-)C尿素呼气试验(~(13-)C-UBT)检测60例健康儿童和187例复发性腹痛(RAP)儿童的幽门螺杆菌感染率,观察幽门螺杆菌根除后RAP患儿的腹痛缓解率,并对RAP儿童在性别、年龄、病程、腹痛方式等方面进行比较.结果 RAP儿童和对照组的幽门螺杆菌感染的阳性率分别为31.4％、15％,两组比较有显著差异(p<0.05),幽门螺杆菌阳性的RAP儿童进行抗幽门螺杆菌治疗后,幽门螺杆菌已根除和未根除的腹痛缓解率分别为78.5％、37.5％,两组比较有显著差异(p<0.05)。在3～5岁、6～9岁、9～12岁3个年龄组的幽门螺杆菌感染率分别为42％、32％、32％,三组比较无显著差异(p>0.05).幽门螺杆菌感染与RAP儿童的性别、平均年龄、病程及腹痛方式之间进行比较,亦无显著差异(p>0.05)。结论幽门螺杆菌感染与复发性腹痛有密切的相关性,可能为复发性腹痛的主要致病因素,根除幽门螺杆菌治疗可缓解复发性腹痛儿童的腹痛症状,幽门螺杆菌感染与复发性腹痛儿童的性别、病程及腹痛方式无关.     

根治幽门螺杆菌对老年反流性食管炎患者预后的影响

目的探讨老年反流性食管炎根治幽门螺杆菌对预后的影响。方法反流性食管炎患者85例,分为观察组41例:常规治疗并接受幽门螺杆菌根治治疗;对照组44例:常规治疗。结果治疗4和8 w后,观察组治疗有效率低于对照组(χ2=4.730,P=0.030;χ2=4.595,P=0.032)。治疗4和8 w后,观察组黏膜Ⅰ级愈合率均低于对照组(P<0.05)。治疗后4和8 w,观察组胃食管反流病量表(GerdQ)评分高于对照组(P<0.05)。治疗后2年的累计复发率,观察组为36.67%(11/30),对照组为15.00%(6/40),两组差异显著(Log-rankχ2=4.691;P=0.030)。结论老年反流性食管炎治疗中根治幽门螺杆菌导致近期及远期疗效欠佳。     

幽门螺杆菌脂多糖化学组成和SDS－PAGE特性的研究

应用气液色谱法对幽门螺杆菌脂多糖中的单糖和脂肪酸进行定性和定量分析。该菌脂多糖中主要含有甘露糖、葡萄糖、半乳糖、氨基葡萄糖和鼠李糖,所含的主要脂肪酸为３─羟基─豆蔻酸、棕榈酸、月桂酸和豆蔻酸,其单糖和脂肪酸组成与大肠杆菌脂多糖相似。由于幽门螺杆菌脂多糖和大肠杆菌脂多糖在化学组成上的相似性,使两者的ＳＤＳ─ＰＡＧＥ图谱基本相同。     

幽门螺杆菌的治疗对2型糖尿病患者血糖的影响

目的探讨幽门螺杆菌治疗前后对糖尿病患者血糖的影响。方法选取2017年8月—2019年5月该院门诊和住院的405例2型糖尿病患者,其中确诊有幽门螺杆菌(Hp)感染者(观察组)198例,无幽门螺杆菌感染者(对照组)207例,分析比较两组的空腹血糖、餐后2 h血糖、糖化血红蛋白、胰岛素抵抗指数及临床特征。同时观察组成功根治幽门螺杆菌(184例)与根治前进行对比。结果 2型糖尿病感染幽门螺杆菌者的糖尿病相关指标高于无感染的2型糖尿病患者,幽门螺杆菌感染与空腹血糖、餐后2 h血糖呈正相关。而根治幽门螺杆菌后糖尿病相关指标较治疗前有所下降(P0.05)。结论幽门螺杆菌感染可导致糖尿病患者的血糖紊乱,幽门螺杆菌的根治可有效控制血糖。     

幽门螺杆菌对人上皮细胞的吸附

幽门螺杆菌对人胃粘膜的吸附是实现其感染的第一步。本研究以人上皮细胞株ＨＥｐ—２为材料对幽门螺杆菌吸附上皮细胞的性能进行了探讨，结果显示，幽门螺杆菌ＹＣ—１１Ａ株具有特异吸附于ＨＥｐ—２细胞表面的性能，其吸附能力在与ＨＥｐ—２细胞共孵育３ｈ后达到高峰，吸附率达８１％，此后吸附率虽无显著提高，但单个ＨＥｐ—２细胞吸附的幽门螺杆菌数量却随孵育时间的延长而有明显增加。在大气环境中与在含５％氧气的微氧环境中幽门螺杆菌对ＨＥｐ—２的吸附无显著差异。抗幽门螺杆菌优势抗原单克隆抗体不能有效阻断幽门螺杆菌对ＨＥｐ—２的吸附。     

幽门螺杆菌内毒素对小鼠成纤维细胞和人胃粘膜组织细胞的侵入和侵害

应用免疫胶金技术证实幽门螺杆菌内毒素作用于小鼠成纤维细胞后５ｍｉｎ即能侵入胞浆，６０ｍｉｎ后可在细胞核中检出ＨＰ－ＬＰＳ结合的胶合颗粒，免疫组织化学法检测结果表明，６５．４％，胃粘膜活检标本组织吸ＨＰ－ＬＰＳ存在，受ＨＰ－ＬＰＳ作用的小鼠成纤维细胞和ＨＰ－ＬＰＳ检测阳性的胃炎患者活检标本细胞均出现相似的超微结构病变，如粗面内质网扩张，膜旁核糖体颗粒脱落，线粒体嵴或外膜消失等。实验结果表明幽门螺杆菌     

幽门螺杆菌外膜泡在动脉粥样硬化形成中的作用

<正>幽门螺杆菌感染与冠心病的发生密切相关,幽门螺杆菌分泌的细胞毒素相关基因A(cytotoxin-associated gene A,CagA)蛋白参与动脉粥样硬化的发生。作为革兰阴性杆菌内毒素的重要载体,外膜泡具有侵袭力强、携带毒素稳定性好的特性。幽门螺杆菌释放的外膜泡携带CagA蛋白突破胃黏膜上皮细胞屏障后入血,进一步作用于血管内皮细胞,通过一系列炎性反应导致血管内皮功能紊乱,促进动脉粥样     

Fusion expression of Helicobacter pylori neutrophil-activating protein in E.coli

AIM: To produce a recombinant protein rMBP-NAP, which was fusionally expressed by Helicobacter pylori (H pylori) neutrophil-activating protein (NAP) and E. coli maltose binding protein (MBP) and to evaluate its immunoreactivity and immunogenicity. METHODS: Neutrophil-activating protein gene of H pylori (HP-napA) was subcloned from the recombinant plasmid pNEB-napA, and fused to MalE gene of expressing vector pMAL-c2x. The recombinant plasmid pMAL-c2x-napA was confirmed by restriction enzyme digestion, and then transformed into E. coli TB1. Fusion protein rMBP-NAP was induced by IPTG and identified by SDS-PAGE analysis. Soluble rMBP-NAP was purified by amylose affinity chromatography. Immunoreactivity and immunogenicity of the fusion protein were evaluated by animal experiment, Western blotting with human H pylori anti-sera. RESULTS: E.coli TB1 carrying recombinant plasmid pMAL-c2x-napA was constructed and led to a high efficiency cytosol expression of fusion protein rBMP -NAP when induced by IPTG. The molecular weight of rBMP-NAP was about 57 kD, accounting for 37.55% of the total protein in the sonicated supematant of E. coli TB1 (pMAL-c2x-napA). The purity of the fusion protein after one-step affinity chromatography was 94% and the yield was 100 mg per liter of bacterial culture. The purified fusion protein could be specifically recognized by both human anti-sera from clinical patients with H pylori infection and rabbit sera immunized by rMBP-NAP itself. CONCLUSION: Recombinant protein rMBP-NAP might be a novel antigen for vaccine development against H pylori.     

钩端螺旋体内毒素的实验研究 Ⅰ.钩端螺旋体内毒素的生物学活性

钩端螺旋体波摩那群波摩那型56608株和赛玛伦群帕托克型帕托克Ⅰ株的酚水提取物具有较大肠杆菌内毒素为弱的生物学活性。该提取物使鱟血液变形细胞溶解物凝胶化,注入家兔后能使动物出现发热、血糖升高和Shwartzman现象,但较大剂量时方能致死小鼠。小鼠内脏病理检查结果表明,各脏器均出现DIC,尤以肺组织更为明显。     

幽门螺杆菌粘附素与大肠杆菌LtB融合疫苗的口服免疫与预防试验

目的　观察幽门螺杆菌粘附素HpaA与大肠杆菌LtB融合蛋白质经口服免疫BALB/c小鼠后机体产生的免疫应答和蒙古沙鼠免疫预防作用。方法　用PCR方法扩增HpaA和LtB目的基因片段 ,将LtB与 pPIM质粒连接后 ,再与HpaA基因连接 ,转化大肠杆菌DH5α ,经酶切获得含有ltB -hpaA的pPIM -ltB/hpaA融合质粒。将重组质粒转化E coliBL2 1(DE3) ,筛选获得重组工程菌 ,经发酵、包涵体提取 ,复性 ,纯化获得LtB -HpaA。BABL/c小鼠设正常对照组、单独rHpaA对照组、CT +HpaA实验组、融合蛋白LtB -HpaA组和rLtB +rHpaA实验组 ,免疫 4周后检测血清抗原特异性IgG、IgA和胃冲洗液、肠冲洗液中sIgA。蒙古沙鼠分 3个实验组 ,正常对照 (PBS)组 ,单独HpaA免疫组和LtB -HpaA组 ,免疫 4周 ,于末次免疫后 14天灌活力良好的H pyloriSS1活菌 ,1个月处死动物 ,采集标本 ,用细菌培养和病理切片检查评价蒙古沙鼠的免疫保护作用。结果　融合蛋白LtB -HpaA组及将CT和LtB作为外粘膜佐剂的CT +HpaA组和LtB +HpaA组中血清IgA和IgG、胃冲洗液和肠冲洗液中sIgA含量明显高于单独HpaA组和对照组 ,差异非常显著 (P <0 0 0 1)。蒙古沙鼠融合蛋白rLtB-HpaA组比单纯rHpaA组效果明显 (rLtB -HpaA组保护率为 80 % ,单独rHpaA为 2 0 % )。 结论　融合蛋白质LtB     

Apoptosis induction in gastric mucous cells in vitro: lesser potency of Helicobacter pylori than Escherichia coli lipopolysaccharide, but positive interaction with ibuprofen

Non-steroidal anti-inflammatory drugs (NSAIDs) cause peptic ulcer disease, but whether they interact with Helicobacter pylori to promote damage is controversial. Moreover, the reported induction of apoptosis in gastric cells by H. pylori lipopolysaccharide (LPS) (10(-9) g/ml) contrasts with studies showing low immunological potency of this LPS. Therefore, the effects of LPS from H. pylori NCTC 11637 and Escherichia coli O111:B4 on apoptosis in a primary culture of guinea-pig gastric mucous cells were investigated in the presence and absence of the NSAID, ibuprofen. Cell loss was estimated by a crystal violet assay, and apoptosis determined from caspase activity and from condensation and fragmentation of nuclei. Exposure to E. coli LPS for 24 h caused cell loss and enhanced apoptotic activity at concentrations >or= 10(-9) g/ml, but similar effects were only obtained with H. pylori LPS at concentrations >or= 10(-6) g/ml. Although ibuprofen (250 microM) caused cell loss and apoptosis, addition of either E. coli or H. pylori LPSs further enhanced these effects. In conclusion, LPS and ibuprofen interact to enhance gastric cell loss and apoptosis. In such interactions, E. coli LPS is more potent than that of H. pylori. The low potency of H. pylori LPS may contribute to a chronic low-grade gastritis that can be enhanced by the use of NSAIDs.     

Role of sympathetic nerve activity in endotoxin induced hypotension in cats

Within 10 min of intravenous (iv) injection of E. coli endotoxin (1 mg X kg-1) in alpha-chloralose anaesthetised cats, mean blood pressure (MBP) fell significantly to 86% of the pre-endotoxin level. There followed a gradual decline in MBP, so that after 60 min MBP was depressed to 49% of the pre-endotoxin level. Preganglionic splanchnic nerve (PSN) activity decreased significantly before the rapid fall in MBP. There followed a gradual decrease in PSN activity coincident with the significant fall in MBP, so that 60 min after iv injection of E. coli endotoxin PSN activity fell to 40% of the pre-endotoxin level. Heart rate did not differ significantly from the pre-endotoxin level in the control group. However, the hypotension and the reduction of PSN activity following endotoxin were abolished by intracisternal (ic) pretreatment with phentolamine (0.5 mg X kg-1). The heart rate in this group increased significantly, by 15% at 10 min and was maintained until the end of the experiment. These results indicate that in E. coli endotoxin hypotension the blood pressure falls together with the reduction in sympathetic outflow; and suggests that stimulation of central alpha-adrenergic receptors leads to an inhibition of activity in brainstem sympathetic pathways.     

幽门螺杆菌lpp20基因与麦芽糖结合蛋白基因的融合表达与纯化

目的 研究幽门螺杆菌(Helicobacter pylori，H．pylori)lpp20基因与麦芽糖结合蛋白基因的融合表达产物的纯化方法，为幽门螺杆菌基因工程疫苗的制备建立基础。方法 采用本研究室分离的HpMEL-HP27菌株提取染色体DNA，用PCR方法从HP染色体DNA上扩增出lpp20基因片段，将目的基因插人到表达载体pMAL-c2X中，用重组质粒转化大肠杆菌(E．coil TB1)。采用IPTG进行诱导表达。用多糖树脂(amylose resin)作为填充料，制备层析柱，将可溶性菌体蛋白进行亲和层析。应用SDS-PAGE方法对纯化产物进行分析。结果 融合蛋白的分子量约为60kDa，融合蛋白的表达量约占全菌总蛋白的34％；纯化后的融合蛋白纯度达90％以上。结论 与麦芽糖结合蛋白基因融合的lpp20基因在E．coli TB1中能够高效表达；用多糖树脂作为填充料，进行亲和层析的方法，具有良好的纯化效果。     

Imbalance between plasma levels of thromboxane B2 and 6-keto-prostaglandin F1 alpha during subacute endotoxin-induced hyperdynamic sepsis or multiple organ failure syndrome in sheep.

We compared the time course of plasma and pulmonary lymph levels of thromboxane B2 (TxB2) and 6-keto-prostaglandin (PG)F1 alpha during the development of either the hyperdynamic phase of sepsis or of the multiple organ failure syndrome (MOFS) associated with sepsis in 26 chronically instrumented awake sheep with intravascular catheters and a chronic pulmonary lymph fistula. Using a continuous i.v. infusion of Escherichia coli endotoxin administered at a rate of 20 ng.kg-1.min-1 (group E20, n = 9) resulted in hyperdynamic septic shock with more than 75% of animals surviving after 72 h of continuous endotoxin administration. Infusing endotoxin at a higher dosage (40 ng.kg-1.min-1; group E40, n = 9) resulted in the development of respiratory failure and MOFS with death occurring within 55 hr of endotoxemia. Eight similarly instrumented sheep served as controls. Administration of endotoxin produced within 4 hr in both endotoxin groups a significant increase in arterial plasma concentration of TxB2, which was not significantly different between both endotoxin groups. Thereafter, plasma TxB2 concentrations progressively decreased in the E20 group to reach at 36 hr values significantly lower than those measured in control sheep not given endotoxin. In the E40 group, plasma TxB2 concentrations returned to baseline values during the development of a MOFS. The time course of TxB2 concentrations in pulmonary lymph in both endotoxin groups was similar to that measured in each group in plasma. 6-Keto-PGF1 alpha concentrations in arterial plasma and pulmonary lymph were significantly higher than in controls during the first 20 hr following the start of endotoxin infusion in both endotoxin groups and were not different between these groups. Thereafter, plasma and pulmonary lymph 6-keto-PGF1 alpha concentrations progressively returned to baseline values in the E20 group and remained at these levels up to the end of the study period (72 hr). In the E40 group, plasma 6-keto-PGF1 alpha concentrations also decreased to baseline values during the second day of endotoxemia but then significantly increased in sheep that survived more than 36 hr and developed a hypodynamic septic state. During the first 24 hr of endotoxemia, the plasma TxB2/6-keto-PGF1 alpha ratio was similar in controls and in both endotoxin groups. During the second study day, TxB2/6-keto-PGF1 alpha ratio progressively decreased in both endotoxin groups to reach and maintain values significantly lower than those measured in controls at 36 hr in the E40 group and at 52 hr in E20 group.(ABSTRACT TRUNCATED AT 400 WORDS)     

幽门螺杆菌融合蛋白HspA-UreB在大肠杆菌中的表达与鉴定

目的在大肠杆菌中表达幽门螺杆菌(简称Hp)HspA-UreB融合蛋白,并探索其免疫反应性,为Hp基因工程疫苗的研制奠定基础。方法用PCR方法扩增郑州分离Hp菌株MEL-HP27的hspA和ureB基因,分别克隆入pNEB193中。测序后,回收两种基因片段,并以hspA-ureB的顺序连接插入原核表达载体pMAL-C2x进行融合表达。采用蛋白印迹法对表达产物进行鉴定。结果特异PCR法和酶切鉴定证实融合基因hspA-ureB克隆入表达载体中;重组质粒转化大肠杆菌TB1后,经IPTG诱导3h,SDS-PAGE电泳显示在119kDa处出现一条特异蛋白带,即麦芽糖结合蛋白(MBP)与HspA-UreB的融合表达形式,约占细菌总体蛋白含量的31%;该融合蛋白与Hp免疫小鼠血清和Hp阳性病人血清的Westernblot分析结果显示,在119kDa处出现特异杂交带。结论成功地在大肠杆菌中实现了Hp融合蛋白HspA-UreB的高效表达,并证实其具有良好的免疫反应性。     

Tracing the leukocyte marker protein in lung fluids and lung-draining lymph nodes during endotoxemia in sheep

Infusion of Escherichia coli endotoxin into sheep produces a form of acute lung injury that resembles the adult respiratory distress syndrome (ARDS). A large portion of the physiologic derangements produced by E. coli endotoxin in this model is thought to be granulocyte-dependent. We measured the level of L1, a granulocyte and monocyte marker protein, in various tissues and fluids after infusion of E. coli endotoxin into sheep. In an acute study, sheep received saline or 1.25 microgram/kg E. coli endotoxin dissolved in saline, or endotoxin after hydroxyurea-induced granulocytopenia. L1 was measured by radioimmunoassay in efferent lymph from the caudal mediastinal lymph node collected between 5 and 6 h after infusion. In addition, L1 was visualized in both lung-draining and extrapulmonary lymph nodes by indirect immunofluorescence. In a chronic study, sheep were prepared with lung lymph fistulas, and L1 was measured in draining pulmonary lymph, plasma, and bronchoalveolar lavage (BAL) fluid, serially, over a 24-h period after infusion. Mean L1 level in pulmonary lymph in the acute study was 6 times higher by absolute concentration, and 19 times higher when lymph flow rates were taken into account, in the sheep that received endotoxin than in saline-infused sheep or endotoxin-infused, granulocytopenic sheep. Fluorescence was greater in the outer cortical region adjacent to subcapsular, afferent sinuses of lung draining-lymph nodes of endotoxin-treated sheep than in the comparable nodes of saline-infused sheep and endotoxin-infused granulocytopenic sheep. In endotoxin-treated sheep, extrapulmonary lymph nodes were less reactive than lung-draining nodes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Variation in the propensity to release endotoxin after cefuroxime exposure in different gram-negative bacteria: uniform and dose-dependent reduction by the addition of tobramycin

An aminoglycoside in combination with a beta-lactam antibiotic is often recommended for the treatment of severe infections. The aim of the present study was to study whether cefuroxime-induced endotoxin release could be reduced by addition of tobramycin in different Gram-negative bacteria and how endotoxin release was affected by bacterial killing rate and number of killed bacteria. Three Escherichia coli strains, 1 Klebsiella, 1 Salmonella and 1 Neisseria strain were exposed in vitro to 2, 10 and 50 x minimum inhibitory concentration of cefuroxime, tobramycin or a combination of both. The cefuroxime-induced endotoxin release in the 6 strains varied from 0.1 to 9.9 x 10(-3) EU/killed bacterium. By adding tobramycin, highly significant reductions of 96%, 93%, 97%, 86% and 85% were seen in the 3 E. coli strains and in the Klebsiella and the Salmonella strain, respectively. In the Neisseria strain, the reduction was less. Increasing doses of tobramycin or the combination led to significant endotoxin release reduction in 4/6 strains. In conclusion, addition of tobramycin reduced penicillin-binding protein-3-beta-lactam binding-induced endotoxin release in all tested Gram-negative strains, despite a large interspecies variation in the propensity to release endotoxin. Besides broadening the spectrum and increasing the killing rate, this might be of benefit in the most severe forms of sepsis.