Analysis of Helicobacter pylori binding site on HEp-2 cells and three cell lines from human gastric carcinoma

Helicobacter pylori (H. pylori) is a pathogen responsible for chronic gastritis and peptic ulcer diseases. It colonises the gastric mucus layer and adheres to the gastric epithelial cell surface. As this adherence is the first step of infection, it is important to study the adherence mechanism. The aim of this study was to analyse the specific binding assay of H. pylori to HEp-2 cells and three gastric phenotype cell lines, AGS, MKN-45 and AZ-521. H. pylori NCTC 11637 grown on agar plates was harvested and used in experiments. H. pylori was inoculated to pre-cultured cell monolayers. Adhered bacteria were labelled with an anti-H. pylori antibody and an FITC-conjugated secondary antibody and quantified by using a fluorescent plate reader. Microbial adherence to HEp-2 cells increased with incubation time and incubated concentration of H. pylori. No further increase was obtained with four or more hours of incubation or with a concentration of 4 x 10(7) bacteria/well or more. Scatchard analysis revealed a linear plot and the Bmax value was 88.3. Similar adherence patterns were obtained when AGS, AZ-521 and MKN-45 cells were used for adherence assays, but they had a lower binding affinity than HEp-2 cells and AZ-521. MKN-45 cells had less receptors than HEp-2 and AGS cells. In conclusion, H. pylori adhered to the cell surface could be quantified by this assay method. H. pylori adhesion to cell surfaces has a single population of binding site and one type of binding site on HEp-2, AGS, AZ-521 and MKN-45 cells.     

幽门螺杆菌诱导的差异蛋白基因在胃癌组织中的表达

目的研究幽门螺杆菌(Hp)诱导的差异蛋白基因在胃癌中的表达及其与胃癌生物学行为的关系。方法实时荧光定量RT-PCR检测Hp诱导的乳酸脱氢酶(LDH)、二氢硫辛酰胺脱氢酶(DLD)、mRNA前体处理因子19同系物(PRP/PSO4)、ATP合酶和Ran-特异性GTP酶活化蛋白(RanGAP)等差异蛋白基因在70例胃癌组织、淋巴结组织及癌旁组织中的表达,分析其表达与临床病理参数的关系。结果 LDH、DLD、PRP/PSO4和ATP合酶基因在胃癌组织中的表达量分别是癌旁组织的3.32,3.36,4.37及2.73倍(P均<0.05),在淋巴结组织中的表达量分别是癌旁组织的2.91,2.72,3.16及2.46倍(P均<0.05);LDH、DLD基因表达与肿瘤大小及生存时间、pTNM分期及伴有淋巴结转移有关(P均<0.05),PRP/PSO4及ATP合酶仅与淋巴结转移有关(P<0.05)。结论 LDH、DLD、PRP/PSO4和ATP合酶基因在胃癌及淋巴结组织中高表达,其LDH和DLD的高表达与胃癌患者预后不良相关。     

幽门螺旋杆菌感染和FasL蛋白表达在胃癌发生中的变化

目的 探讨幽门螺旋杆菌(H.pylori)感染和Fas配体(FasL)蛋白表达在胃癌(GC)发生发展中的变化.方法 选取2010年1月至2011年9月在上海浦东新区南汇中心医院诊断的胃部病变169例患者,其中慢性浅表性胃炎(CSG)25例、慢性萎缩性胃炎(CAG)28例、肠化生(IM)37例、异型增生(Dy)38例和胃癌(GC)41例.分别检测胃黏膜组织中H.pylori感染和FasL的蛋白表达,分析H.pylori感染和FasL的蛋白在GC发生发展中的相关性.结果 H.pylori感染率和FasL蛋白阳性表达率随着病情的加重而逐渐增高,Dy组及GC组H.pylori感染率比CSG组明显升高(65.8％和75.6％ vs 16.0％)(P＜0.05);IM组、Dy组及GC组FasL蛋白阳性表达率比CSG组明显升高(64.9％、78.9％和92.7％ vs 28.0％)(P＜0.05) ;GC组FasL蛋白阳性表达率比CAG组及IM组明显升高(92.7％vs 60.7％;92.7％ vs 64.9％)(P＜0.05).除CSG外,H.pylori感染与CAG、IM、Dy和GC胃黏膜组织中的FasL蛋白表达具有一致性,差异有统计学意义(P＜0.01).结论 FasL蛋白表达和H.pylori感染对评估胃黏膜癌前病变发展趋势有重要的临床价值,有助于早期发现和诊断GC.     

正常上皮细胞特异性-1基因在胃癌细胞株中的表达及甲基化调控

目的:研究正常上皮细胞特异性-1(NES1)基因表达及其CpG岛甲基化状况与胃癌的关系。方法:分别培养胃癌细胞MKN-28(高分化)、SGC-7901、AGS(均中分化)、MKN-45(低分化)、HGC-27(未分化)和原代正常胃上皮细胞。提取细胞RNA,经RT-PCR检测NES1在各细胞株中的表达。以不同浓度DNA去甲基化抑制剂5-aza-dc处理胃癌细胞,RT-PCR检测其NES1mRNA表达。提取细胞基因组DNA,甲基化修饰后MSP分析其CpG岛甲基化状态。结果:各肿瘤细胞株中NES1mRNA表达较胃正常上皮细胞有不同程度降低。甲基化检测显示NES1表达减少的胃癌细胞株均存在不同程度的NES1基因外显子3CpG岛甲基化。NES1表达降低的胃癌细胞株经5-aza-d处理后NES1表达上调。结论:NES1胃癌细胞株中低表达,表达下调与该基因外显子3周围CpG岛甲基化有关。5-aza-dc可使NES1表达降低的胃癌细胞株重新表达NES1mRNA。提示NES1基因CpG岛甲基化可能是NES1在胃癌细胞系中表达缺失的分子机制。     

幽门螺杆菌感染对胃黏膜三叶因子2表达的影响

目的探讨幽门螺杆菌感染(Hp)对慢性胃炎胃黏膜三叶因子2(TFF2)表达的影响。方法采用免疫组化及半定量的分析方法检测Hp阳性组与Hp阴性组,以及Hp根除前后胃窦黏膜TFF2表达量的变化。结果TFF2阳性物质表达于胃黏膜上皮近基底部的胃腺细胞的细胞质内,呈棕黄色颗粒;与Hp阴性组比较,Hp阳性组的TFF2染色评分显著减少(P<0.05);根除Hp后的胃黏膜较根除前TFF2的表达量显著增加(P<0.05)。结论Hp感染导致胃黏膜TFF2表达减少,提示TFF2在Hp相关性胃炎的发病机制中起一定作用。     

COX-2蛋白在胃癌组织、胃癌SGC7901、MGC823细胞株中的表达及意义

目的　探讨环氧合酶 2 (COX 2 )在胃癌组织及胃癌培养细胞中表达及分布情况及其与胃癌生物学活性的关系。方法　应用免疫组织化学、免疫细胞化学、Westernblot方法检测COX 2在胃癌组织、胃癌细胞SGC790 1中的表达 ,并探讨其于与胃癌临床病理的关系。结果　胃癌组织中COX 2蛋白的表达率 95 2 % (4 0 4 2 ) ,15例为低表达 ,2 5例为高表达。与正常粘膜细胞比较COX 2在胃癌组织中的表达明显增强 (P <0 0 5 )。COX 2在高、中分化型腺癌的高表达率为 81 8%为 (18 2 2 ) ,在低分化腺癌中阳中高表达率为 4 0 % (8 2 0 ) ,二者比较有显著性差异 (P <0 0 5 )。COX 2的表达与不同性别、年龄、病期及有无淋巴转移的患者间无显著差异。免疫细胞化学及Westernblot证实在胃癌组织及胃癌细胞SGC790 1中有COX 2过表达。结论　COX 2在胃癌组织中、胃癌细胞系SGC790 1中过表达 ,但并不表达在胃癌细胞系MGC80 3中。COX 2蛋白的表达与胃癌细胞的分化有关 ,与年龄、性别、肿瘤大小、肿瘤分期及淋巴转移无关     

transgelin基因在幽门螺杆菌感染胃癌细胞及人胃癌组织中的表达

目的研究幽门螺杆菌(Helicobacter pylori,HP)感染胃癌细胞及人胃癌、淋巴结组织中转凝蛋白基因(transgelin)的表达水平,探讨HP感染、transgelin基因与胃癌发生、发展的相关机制。方法用HP感染胃癌细胞系AGS和SGC-7901,同时收集胃癌患者手术切除的胃癌组织、切缘组织和淋巴结组织,提取及纯化总RNA。实时荧光定量PCR检测transgelin mRNA的表达水平,分析其与临床病理参数的关系。结果 transgelin mRNA在HP感染的AGS、SGC-7901细胞中的表达量增加,分别为对照细胞的11.39倍(t=30.025,P=0.000)、3.41倍(t=5.677,P=0.005);transgelin mRNA在胃癌组织、淋巴结组织中的表达增加,分别是切缘组织的6.48倍(t=2.290,P=0.026)、2.64倍(t=2.043,P=0.046);Ⅳ期胃癌组织transgelin mRNA表达量增加,是Ⅰ~Ⅱ期的2.32倍(t=2.111,P=0.049);淋巴结转移组是无淋巴结转移组的2.93倍(t=2.123,P=0.043)。结论HP可上调transgelin基因的表达,transgelin基因可能参与胃癌的发生、发展,且与p TNM分期及淋巴结转移相关。     

Efficacy of sulforaphane in eradicating Helicobacter pylori in human gastric xenografts implanted in nude mice

Sulforaphane, an isothiocyanate abundant in the form of its glucosinolate precursor in broccoli sprouts, has shown in vitro activity against Helicobacter pylori. We evaluated the effect of sulforaphane in vivo against this bacterium by using human gastric xenografts in nude mice. H. pylori was completely eradicated in 8 of the 11 sulforaphane-treated grafts. This result suggests that sulforaphane might be beneficial in the treatment of H. pylori-infected individuals.     

幽门螺杆菌对胃癌患者微小核糖核酸-146、人类表皮生长因子受体2表达影响研究

目的 探讨幽门螺杆菌对胃癌患者微小核糖核酸-146(miR-146)、人类表皮生长因子受体2(HER2)表达的影响.方法 选取自2018年1月至2018年12月收治的54例胃癌患者为研究对象,根据幽门螺杆菌感染情况分为阴性组(n=16)与阳性组(n=38).收集胃癌患者胃黏膜标本,实时定量聚合酶链式反应检测标本中miR...     

胃黏膜癌前病变中bcl-2蛋白表达与幽门螺杆菌感染的关系

目的 观察幽门螺杆菌(HP)感染后胃黏膜癌前病变中bcl—2蛋白表达的特点，探讨服在胃癌发生发展过程中的作用。方法 应用免疫组织化学SP法及Warthin-Starry嗜银染色(WS染色)，对56例慢性胃炎、46例慢性胃炎伴肠化生、34例异型增生进行bcl—2蛋白表达及服检测的研究。结果 胃黏膜肠化生组及异型增生组的服感染率及bcl—2蛋白阳性表达率均明显高于慢性胃炎组(P＜0．05)；86例HP阳性者bcl-2蛋白表达39例，阳性率45．3％，50例HP阴性者bcl—2蛋白表达12例，阳性率24％，两者比较，差异显著(P＜0．05)。结论 在慢性胃炎、肠上皮化生、异型增生的进展过程中，bcl—2蛋白的表达水平在增高。HP感染在胃癌发生、发展过程中起一定作用。bcl—2蛋白的表达可能是服致癌的作用机制之一。     

胃粘膜肥大细胞及C-反应蛋白与Hp致病关系的探讨

目的　探讨胃粘膜肥大细胞 (MC)及C 反应蛋白 (CRP)与幽门螺杆菌 (Hp)致病的关系。方法　采用改良甲苯胺蓝染色法检测 1 2 0例患者胃粘膜中MC计数及脱颗粒 ,免疫比浊法测定血清CRP水平。结果　①Hp阳性患者胃粘膜MC计数及脱颗粒比显著高于Hp阴性患者 (P <0 .0 1 ) ;②不同Hp感染性胃病患者之间胃粘膜MC计数及脱颗粒比也有差异 ,消化性溃疡 (PU)和慢性浅表性胃炎 (CSG)患者胃粘膜MC计数及脱颗粒比显著高于慢性萎缩性胃炎(CAG)患者 (P <0 .0 1 ) ;③Hp阳性患者与HP阴性患者的CRP水平无明显差异。结论　胃粘膜肥大细胞及其脱颗粒参与了Hp致病而导致胃粘膜受损 ,CRP与Hp感染致病无关     

幽门螺旋杆菌感染与胃黏膜癌前病变中bcl-2蛋白表达的关系

目的研究幽门螺杆菌(Hp)感染的胃黏膜癌前病变bcl-2蛋白表达的变化,探讨HP在胃癌发生发展过程中的作用。方法应用石蜡切片及免疫组化方法,对58例慢性胃炎、42例慢性胃炎伴肠化生、36例异型增生进行比bcl-2蛋白表达及HP检测研究。结果 胃黏膜化生组及异型增生组的Hp感染率及bcl-2蛋白阳性表达率均明显高于慢性胃炎组p<0.05;84例Hp阳性者bcl-2蛋白表达34例,阳性率40.5％,52例HP阴性者bcl-2蛋白表达11例,阳性率21％,两者比较,差异显著p<0.05。结论 肠上皮化生、异型增生的胃黏膜HP的感染及bcl-2蛋白的表达水平同步增高,说明Hp感染在胃癌发生、发展过程中起一定作用。     

胃癌细胞系中甲基化与叶酸干预对多种基因的调控研究

目的：胃癌的发生从分子发病水平上说是一个涉及多种癌基因、抑癌基因、凋亡相关基因等的异常和积累的多步骤的过程。DNA甲基化在基因表达中起调控作用已成为共识[1]。各种肿瘤的发生过程中有癌基因的低甲基化和抑癌基因的高甲基化，因为甲基化程度与基因表达呈负相关，所以引起抑癌基因的表达减弱或缺失及癌基因的表达增强。5-氮脱氧胞苷（5-aza-2’-deoxycytidine, 5-aza-dC）通过抑制甲基化酶使基因去甲基化，通过5-aza-dC干预能够观察甲基化对基因表达的影响。胃癌发生中也有抑癌基因的高甲基化及癌基因的低甲基化现象，但是迄今为止未见同时研究同一细胞系的多种基因的甲基化对基因表达和细胞周期的影响。本研究对此作了有益的探讨，以期为以后的胃肠道肿瘤的治疗提供分子水平的依据。本文主要探讨不同分化的胃癌细胞系在不同浓度的5-氮脱氧胞苷（5-aza-2’-deoxycytidine, 5-aza-dC）和甲基化食物供体叶酸的化学干预下癌基因、抑癌基因和与凋亡有关的基因与甲基化调控的关系。方法： 培养高分化、中分化和未分化的胃癌细胞MKN－45、MKN－28、HGC－27，分别以不同浓度的5-aza-dC干预细胞，以同一浓度的叶酸干预细胞，部分细胞48小时后再以不同浓度的5-aza-dC干预。提取细胞的RNA，用RT-PCR的方法检测p16INK4A、p21WAF1、p73、c-myc、c-Ha-ras等多种基因的表达情况。结果：在不同分化的三种胃癌细胞中基因表达受甲基化调控的程度也不同。抑癌基因中p16INK4A在干预前MKN－45和HGC－27细胞表达，而在用5-aza-dC干预后表达增强，且不同的胃癌细胞株表达增强与5-aza-dC干预的时间与浓度不同有关，p21WAF1、p73没有明显变化；癌基因中的c-myc、c-Ha-ras变化不明显；所有胃癌细胞系中叶酸干预及叶酸联合5-aza-dC干预前后所以基因的mRNA表达没有明显变化。结论：不同分化人胃癌细胞中，甲基化修饰对癌基因、抑癌基因、与凋亡有关的基因的调控差异明显。其中的机制研究是否与个体差异有关。但是在肿瘤细胞中的叶酸干预后对甲基化的调控不敏感，可能是因为肿瘤细胞对叶酸的甲基化供体已经不起作用，与癌前病变补充叶酸不同。     

A novel ligand in lymphocyte-mediated cytotoxicity: expression of the beta subunit of H+ transporting ATP synthase on the surface of tumor cell lines

Extracellular adenosine triphosphate (eATP) has been suggested to play a role in lymphocyte-induced tumor destruction. We now provide evidence that a protein responsible for ATP synthesis in mitochondria may also play a physiologic role in major histocompatibility complex- independent, lymphocyte-mediated cytotoxicity. A 51.5-kD protein (p51.5) bearing structural and immunologic characteristics of the beta subunit of H+ transporting ATP synthase (E.C. 3.6.1.34, beta-H+ATPase, published molecular mass of 51.6 kD) was detected on the plasma membrane of three different human tumor cell lines studied. NH2- terminal amino acid sequence analysis of purified p51.5 from K562 tumor cells revealed 100% homology of 16 residues identified in the first 21 positions to the known sequence of human mitochondrial beta-H+ ATPase. Antibody directed against a 21-mer peptide in the ATP binding region of beta-H+ ATPase (anti-beta) reacted with only one band on Western blots of whole tumor extracts and tumor membrane extracts suggesting that the antiserum reacts with a single species of protein. Anti-beta reacted with the cell membranes of tumor cells as determined by fluorescence- activated flow cytometry and immunoprecipitated a 51.5-kD protein from surface-labeled neoplastic cells (but not human erythrocytes and lymphocytes). Purified p51.5 bound to human lymphocytes and inhibited natural killer (NK) cell-mediated cytotoxicity. Furthermore, anti-beta treatment of the K562 and A549 tumor cell lines inhibited NK (by > 95%) and interleukin 2-activated killer (LAK) cell (by 75%) cytotoxicity, respectively. Soluble p51.5 upon binding to lymphocytes retained its reactivity to anti-beta suggesting that the ATP binding domain and the lymphocyte-receptor binding domain reside in distinct regions of the ligand. These results suggest that beta-H+ ATPase or a nearly identical molecule is an important ligand in the effector phase (rather than the recognition phase) of a cytolytic pathway used by naive NK and LAK cells.     

幽门螺杆菌VacA蛋白体外诱导胃上皮AGS细胞内HMGB1的表达

摘要：目的：探讨幽门螺杆菌（Hp）感染的胃上皮AGS细胞内高迁移率族蛋白B1(high mobility group box 1, HMGB1)的表达。 方法：Hp11638（CagA⁺,VacA⁺）和Hp11638突变株（Hp11638M,CagA⁺,VacA^-）的提取液与AGS细胞共同温育后,收集细胞及培养上清液。裂解AGS细胞,western blot分析AGS细胞内HMGB1的表达,ELISA法检测培养上清液中HMGB1的水平。 结果:Hp11638提取液刺激AGS细胞后HMGB1表达量为(123.33±25.2) μg/mL,明显高于Hp11638M提取液刺激后的(46.67±7.23) μg/mL（q=8.49,P<0.01）。Hp11638和Hp11638M提取液刺激的AGS细胞培养上清液中HMGB1的水平分别为（115.59±16.62）和（48.32±6.30） ng/mL,差异有统计学意义（q=12.25,P<0.01）。 结论:在胃炎发生、发展过程中,VacA蛋白是刺激细胞中HMGB1高表达的主要因子。     

胶质瘤中RNA编辑酶ADAR2,ADAR3 mRNA表达及意义

目的观察不同恶性程度的胶质瘤细胞中RNA编辑酶ADAR2（RED1），ADAR3（RED2）mRNA水平的表达。方法对原代培养的正常人脑胶质细胞和胶质瘤细胞SHG-44，U-251，BT-325，应用ADAR2，ADAR3全长序列合成引物及合成的特异引物，用逆转录PCR及图像分析法检测ADAR2，ADAR3 mRNA水平的表达。ADAR基因表达水平用基因／β-肌动蛋白（β-aetin）灰度比值表示。结果ADAR2在正常人脑胶质细胞中表达极弱，在低恶性度的胶质瘤SHG-44细胞中呈弱表达，在高恶性度的胶质瘤U-251，BT-325细胞中明显表达。ADAR3在正常人脑胶质细胞及苯乙酸处理前后的胶质瘤细胞中，均未见表达。结论ADAR2 mRNA水平高表达，可能与高恶性度胶质瘤的发生有关。     

SOCS-3 mRNA在胃癌组织及胃癌细胞系中的表达

目的探讨人胃癌组织及胃癌细胞系中细胞因子信号转导抑制因子3（suppressors of cytokine signaling-3，SOCS-3）的表达。方法应用RT-PCR法测定10例人正常胃组织、10例人胃癌组织及人胃癌细胞系SWWC中SOCS-3的表达。结果与正常胃组织相比较，本研究中所测定的人胃癌组织（P〈0．05）及人胃癌细胞系SWWC（P〈0．001）SOCS-3 mRNA的表达呈降低趋势。结论在人胃癌的发生、发展过程中SOCS-3基因发生显著变化。     

Urease test of the gastric content deposits for diagnosis of Helicobacter pylori infection in the gastric mucosa

A rapid urease test was applied to the examination of the deposit of gastric juice for diagnosing H. pylori in the gastric mucosa. Two hundred and twenty patients blindly randomized were examined in a case control study. The standard rapid urease test kit Jatrox-H.p.-Test (Rohm Pharma, Germany) was used to determine urease activity in the deposit of gastric juice and duodenal [n = 110 (Group 1)] and gastroduodenal [n = 110 (Group 2)] mucosae. Giemsa staining was employed as a comparison method to examine H. pylori infection in the gastric and duodenal mucosae. The availability of regions of duodenal metaplasia was confirmed by periodic acid-Schiff and alcian blue (Serva) staining tests (pH 1.0 and 2.5, respectively). The results of evaluation of the efficiency of the rapid urease test of gastric juice deposit and gastric and duodenal mucosae in Groups 1 and 2 were as follows: sensitivity (SE) (0.97, 0.99, 0,96), specificity (SP) (0.97, 0.97, 0.99), prevalence (0.64, 0.67, 0.24), test accuracy (TA) (0.96, 0.98, 0.98), negative (0.95, 0.97, 0.99) and positive (0.98, 0.99, 0.96) predictive values; positive (38.8, 33.0, 96.0) and negative (0.03, 0.01, 0.04) likelihood ratios. It is expedient to employ the rapid urease test for the diagnosis of H. pylori infection in the stomach (Se 96-99%, Sp 97%, TA 97-98%). When the test of gastric juice deposit and gastric biopsy is positive, the probability of gastric H. pylori availability is 98-99%. When the test is negative (the probability of H. pylori absence is 95-97%), duodenal biopsy is made. When the test of duodenal biopsy is positive, the probability of H. pylori availability is 96%. When it is negative, the probability of H. pylori absence is 99%. An algorithm of use of the rapid urease test to diagnose H. pylori in different intestinal parts (stomach, duodenum) has been developed.