幽门螺杆菌与胃上皮细胞相互作用的电镜观察

目的 研究幽门螺杆菌在体外与胃上皮细胞相互作用后的形态变化。方法 采用Ｈｐ与细胞体外共培养技术,结合透射电观察ＨＰ活菌与人胃癌细胞系ＳＧＣ－７９０１相互作用后的超微结构变化。结果 ＨＰ以多种不同的方式黏附着胃上皮细胞。高浓度的Ｈｐ（１０＾７ＣＦＵ／ｍｌ）可使体外２的胃上皮细胞发生明显病变,微绒毛丢失与胞浆内出现空泡样变等。反之,胃上皮细胞也可作用于ＨＰ,除了使其发生细胞既相互作用又相互适应,这在一     

嗜酸乳杆菌对球形幽门螺杆菌的细胞黏附和增殖作用的影响

目的研究球形幽门螺杆菌(Hp)对AGS细胞的黏附和增殖作用及热灭活状态和活菌状态嗜酸乳杆菌对其产生的影响。方法制备AGS细胞爬片,按球形Hp对照组、球形Hp与活/灭活嗜酸乳杆菌实验组加入细菌,电镜、革兰染色后光镜观察Hp对AGS细胞黏附指数,同时用荧光抗体染色法评价黏附于AGS细胞的Hp密度。运用CCK-8比色法检测球形Hp对AGS细胞的增殖影响及比较加入嗜酸乳杆菌产生的变化。结果实验组与对照组比较,球形Hp对细胞的黏附指数及黏附密度均明显下降(P0.05)。低浓度的球形Hp促进细胞增殖,高浓度的球形Hp抑制细胞增殖。两种不同状态的嗜酸乳杆菌均能降低Hp对AGS细胞增殖的影响。结论球形Hp对AGS细胞具有黏附能力并随浓度不同对其产生不同增殖影响,嗜酸乳杆菌能降低其作用。     

幽门螺杆菌对胃上皮细胞系SGC-7901细胞生长的影响

研究证明 ,幽门螺杆菌 (Ｈｅｌｉｃｏｂａｃｔｅｒｐｙｌｏｒｉ,Ｈｐ)是引起慢性Ｂ型胃炎的病因 ,是消化性溃疡的重要致病因子 ,且与胃癌发生有关[1] 。关于Ｈｐ引起胃黏膜病变的确切机制 ,尚未完全明了。为了认识活体条件下Ｈｐ的发病机制 ,本研究用体外细胞培养技术 ,观察了Ｈｐ活菌对胃上皮细胞系生长与增殖的影响。材料与方法一、Ｈｐ活菌悬液的制备Ｈｐ国际标准菌株ＮＣＴＣ116 37接种于含 5 %羊血的厌氧菌分离培养基平板 ,置于 37℃微需氧环境中 (5 %Ｏ2 、10 %ＣＯ2 、5 %Ｈ2 、80 %Ｎ2 )培养 ,3ｄ后收集细菌 ,用ＰＢＳ制成细菌悬液 …     

幽门螺杆菌脂多糖通过调节MDM2表达对胃上皮细胞恶性转化的影响

背景:幽门螺杆菌脂多糖(Hp-LPS)在诱导胃癌发生中起重要作用。目的:探讨Hp-LPS对MDM2基因表达的调节作用,及其对胃上皮细胞恶性转化的影响。方法:分别以Hp-LPS (1μg/mL)和大肠埃希菌(E. coli)-LPS(1μg/mL)刺激人胃上皮细胞株GES-1和胃癌细胞株HGC-27、MKN45 24 h,以蛋白质印迹法检测MDM2表达。构建MDM2启动子区荧光素酶报告基因质粒并转染HGC-27细胞,以Hp-LPS刺激细胞24 h后,检测报告基因相对荧光素酶活性。构建MDM2 RNA干扰质粒并分别转染三株细胞,行流式细胞术细胞凋亡检测和Transwell细胞侵袭实验。建立裸鼠MKN45、HGC-27细胞胃癌异种移植瘤模型并予尾静脉注射siRNA-hMDMA2-3,观察移植瘤生长情况。结果:Hp-LPS可增加MDM2基因启动子转录活性,上调胃上皮细胞和胃癌细胞MDM2蛋白表达。转染siRNAhM DMA2-3的GES-1、HGC-27、MKN45细胞,细胞凋亡较转染阴性对照siRNA者显著增加(P 0. 05),侵袭能力显著减弱(P 0. 05)。经siRNA-hMDMA2-3干预的异种移植瘤模型裸鼠,肿瘤生长抑制率显著高于以阴性对照siRNA干预者(P 0. 05)。结论:Hp-LPS可能通过诱导MDM2表达引起胃上皮细胞恶性转化,而抑制MDM2表达可促进胃上皮细胞和胃癌细胞凋亡并抑制其侵袭能力,发挥抑制胃上皮细胞恶性转化和胃癌生长的作用。     

幽门螺杆菌和非甾体抗炎药对胃上皮细胞增殖的影响

目的　从体外研究的角度探讨幽门螺杆菌 (Hp)和非甾体抗炎药 (NSAID)对胃上皮细胞增殖的影响及其相互作用。方法　胃癌细胞株AGS与Hp和 (或 )吲哚美辛、阿司匹林体外共培养 ,通过MTT比色法和WesternBlot法检测增殖细胞核抗原 (PCNA) ,观察Hp和NSAID对胃上皮细胞增殖的影响。结果　MTT比色法显示CagA阳性的标准Hp菌株NCTC116 37能明显促进胃上皮细胞增殖 ,吸光度 (A)值明显升高 (P <0 .0 5 ) ,而CagA阴性的标准Hp菌株NCTC12 90 8则未发现有促增殖作用 ,且Hp对上皮细胞生长的效应取决于Hp作用的密度 ,在低密度 (3.2× 10 4～ 4 .0× 10 6CFU/ml)时NCTC116 37促进胃上皮细胞生长 ,在高密度 (>2× 10 7CFU/ml)时则抑制其生长 (P <0 .0 5 )。吲哚美辛和阿司匹林均能抑制胃上皮细胞生长 (P <0 .0 5 ) ,并呈浓度依赖性。当Hp和NSAID共同作用于AGS细胞时 ,Hp的促生长作用被逆转 ,呈现出抑制细胞生长的效应 ,A值明显降低 (P <0 .0 5 )。PCNA的WesternBlot检测结果发现 ,Hp菌株NCTC116 37可明显促进细胞PCNA的表达 ,而吲哚美辛和阿司匹林则抑制其表达 ,当两者共同作用于AGS细胞时 ,PCNA的表达明显减弱。结论　Hp对胃上皮细胞的增殖效应与Hp的密度及菌株差异有关 ,CagA阳性的Hp易促进胃上皮细胞生长 ,NSAID可抑制其     

胃恶性肿瘤与幽门螺杆菌L型感染的关系

应用组织切片革兰染色、免疫组化染色和电镜等技术，对136例胃恶性肿瘤进行幽门螺杆菌L型检测。结果，革兰染色幽门螺杆菌L型检出阳性率为77．2％，免疫组化染色L型检出阳性率为71．3％，两者具有一致性（P＞0．05）。此外，革兰染色在91例瘤细胞的胞浆内同时查见L型菌，透射电镜在腺癌和淋巴瘤细胞质内也见到L型菌。提示，幽门螺杆菌L型感染与胃恶性肿瘤关系密切，很可能是其致癌因子之一。     

幽门螺杆菌抑制胃粘蛋白的合成

胃粘蛋白是一种大分子的糖蛋白,它是胃粘液层的主要成分,能保护胃粘膜表面免受各种化学、酶、机械、微生物等的损伤。研究表明,幽门螺杆菌(Hp)感染者中,胃粘膜表面粘蛋白含量降低,而一旦清除Hp,则可使粘蛋白含量恢复。此研究旨在观察Hp对体外培养的胃粘膜上皮细胞合成粘蛋白能力的影响。 方法:运用~3H-葡萄糖胺渗入法和高效液相     

Cag A阳性幽门螺杆菌对胃癌细胞lncRNA DLEU2表达及上皮间质转化的影响

目的 探讨细胞毒素相关蛋白A(Cag A)阳性幽门螺杆菌（Hp）对胃癌细胞长链非编码RNA淋巴细胞白血病缺失基因2(DLEU2)表达及上皮间质转化的影响。方法 体外培养胃癌细胞系HGC-27、AGS、MGC-803、MKN-28以及正常胃黏膜上皮细胞系GES1，采用RT-qPCR法检测DLEU2表达，并选择DLEU2表达最高的胃癌细胞进行后续实验。将Hp NCTC标准菌株26695或11637分别与正常胃黏膜上皮细胞和胃癌细胞共培养0、3、9、12 h，采用RT-qPCR法检测DLEU2表达。采用Western blotting法检测与NCTC 26695或11637共培养3、9、12 h正常胃黏膜上皮细胞和胃癌细胞Cag A表达以及共培养3、9、12 h神经钙黏着蛋白（N-cadherin）、波形蛋白（Vimentin）表达。同时，分析与NCTC 26695或NCTC 11637共培养的胃癌细胞DLEU2、Cag A表达与N-cadherin、Vimentin表达的关系。结果 HGC-27、AGS、MGC-803、MKN-28细胞DLEU2相对表达量均高于GES1细胞（P均<0...     

不同幽门螺杆菌培养滤液对胃上皮细胞端粒酶活性的影响

目的　分析不同幽门螺杆菌 (H .pylori)培养滤液对胃上皮细胞端粒酶活性的影响。 方法　分别以来自胃癌患者和来自慢性浅表性胃炎患者的cagA+ vacAs1a/m2和来自慢性浅表性胃炎患者的cagA+ vacAs1b/m2、cagA-vacAs1a/m2的H .pylori培养滤液与胃上皮细胞共孵育 ,然后撤除刺激物继续培养 ,观察不同时间的DNA合成速率和端粒酶活性。 结果　cagA+ vacAs1a/m2和cagA+ vacAs1b/m2Hp培养滤液与胃上皮细胞共孵育 6h ,诱导了胃上皮细胞端粒酶活性表达上调 ,DNA合成速率增加 ,cagA+ vacAs1a/m2的作用明显强于cagA+ vacAs1b/m2 ,撤除刺激物继续培养 2 4h后 ,其改变得以逆转 ;cagA-vacAs1a/m2H .pylori培养滤液与胃上皮细胞共孵育 ,其端粒酶活性和DNA合成速率无明显改变。 结论　不同H .pylori培养滤液对胃上皮细胞端粒酶活性的影响各异 ,cagA+ vacAs1a/m2Hp的毒性产物显著地诱导了胃上皮细胞端粒酶活性表达上调     

温脾平疡汤对幽门螺杆菌体外抑菌作用的试验研究

目的探讨温脾平疡汤对幽门螺杆菌的抑菌效果。方法取菌悬液1 ml分别加入到含不同浓度中药制剂液的试管中,同时设不含药对照管。将含药管和不含药对照管同时置于36℃微需氧环境下培养24 h。从各管中取材料,经适当稀释后涂布于布氏肉汤血琼脂培养基平板上,于36℃微需氧环境下培养72 h,分别计算平板上存活菌落数。结果不同浓度的温脾平疡汤对幽门螺杆菌均有明显抑制作用,浓度在50～60 mg/ml。存在一临界浓度,这一浓度可提高杀菌效果,使幽门螺杆菌的存活菌数由107下降到106CFU/ml。说明浓度升高杀菌作用增强。结论温脾平疡汤对幽门螺杆菌体外有明显抑菌作用。     

Prevention of Helicobacter pylori infection by lactobacilli in a gnotobiotic murine model.

BACKGROUND: Helicobacter pylori is a bacterium which causes gastric inflammatory diseases. Oral inoculation of H pylori usually results in only a temporary colonisation without a successful infection in the stomach of conventional mice in which lactobacilli are the predominant indigenous bacteria. AIM: To determine whether lactobacilli exert an inhibitory effect on colonisation by H pylori in the stomach. METHODS: The effects of H pylori on attachment to murine and human gastric epithelial cells and the H pylori mediated release of interleukin-8 (IL-8) by these cells were examined in vitro. Lactobacillus salivarius infected gnotobiotic BALB/c mice and control germ free mice were inoculated orally with H pylori to examine whether L salivarius can inhibit colonisation by H pylori. RESULTS: L salivarius inhibited both the attachment and IL-8 release in vitro. H pylori could not colonise the stomach of L salivarius infected gnotobiotic BALB/c mice, but colonised in large numbers and subsequently caused active gastritis in germ free mice. In addition, L salivarius given after H pylori implantation could eliminate colonisation by H pylori. CONCLUSION: These findings suggest the possibility of lactobacilli being used as probiotic agents against H pylori.     

Omeprazole attenuates neutrophil-endothelial cell adhesive interaction induced by extracts of Helicobacter pylori

In this study, the influence of omeprazole on the adhesive activity of neutrophils, provided by an extract of Helicobacter pylori, was determined. Human neutrophils were collected from peripheral blood and labelled with a fluorochrome. Helicobacter pylori (NCTC 11637) was cultured and its water extract was obtained by centrifugation of the bacterial suspension. Neutrophils were incubated with the extract in a plastic plate. Percentage adherence was calculated by measuring the fluorescence of floating and adherent cells. Rat mesenteric venule was prepared on an intravital microscope and the number of neutrophils which adhered to venular endothelium was counted. Neutrophil adherence to the plastic plate was increased by the presence of H. pylori extract. Pretreatment with omeprazole significantly decreased this adherence in a dose-dependent manner (10(-6)-10(-4)mol/L). Neutrophil adherence to the mesenteric venule was also increased by H. pylori extract and significantly inhibited by omeprazole. These results indicate that the neutrophil-endothelial adhesive interaction is inhibited by omeprazole, suggesting that omeprazole prevents neutrophil recruitment to the gastric mucosa associated with H. pylori infection.     

Effects of H pylori infection on gap-junctional intercellular communication and proliferation of gastric epithelial cells in vitro

AIM： To explore the effects of H pylori infection on gap-junctional intercellular communication （GJIC） and proliferation of gastric epithelial cells in vitro.
METHODS： A human gastric epithelial cell line （SGC- 7901） cultured on coverslips was exposed overnight to intact H pylori （CagA^＋ or CagA^- strains） and sonicated extracts, respectively. GJIC between the cells was detected by fluorescence redistribution after photobleaching （FRAP） technique. Proliferation of SGC cells was determined by methylthiazolyl tetrazolium （MTT） assay.
RESULTS： When compared with control in which cells were cultured with simple medium alone, both CagA^＋ and CagA^- H pylori isolates could inhibit GJIC （CagA^＋： F = 57.98, P 〈 0.01; CagA^-： F = 29.59, P 〈 0.01） and proliferation （CagA^＋： F = 42.65, P 〈 0.01; CagA^-： F = 58.14, P 〈 0.01） of SGC-7901 cells. Compared with CagA^- strains, CagA^＋ H pylori more significantly downregulated GJIC of gastric cells （intact Hpylori： t = 13.86, P 〈 0.01; sonicated extracts： t = 11.87, P 〈 0.01） and inhibited proliferation gastric cells to a lesser extent in vitro （intact H pylori： t = 3.06, P 〈 0.05; sonicated extracts： t = 3.94, P 〈 0.01）.
CONCLUSION： Compared with CagA^- H pylori strains, CagA^＋ strains down-regulate GJIC of gastric epithelial cells more significantly and inhibit proliferation of gastric cells to a lesser extent in vitro. H pylori, especially CagA^＋ strains, may play an important role in gastric carcinogenesis.     

Periostin过表达诱导人胃癌细胞化疗耐药性的机制研究

背景：前期研究发现periostin过表达可增强人胃癌细胞株SGC-7901对顺铂和5-氟尿嘧啶（5-Fu）诱导的细胞凋亡的抵抗能力。目的：探讨periostin过表达诱导人胃癌细胞化疗耐药性的可能机制。方法：Periostin稳转组和空载体稳转组SGC-7901细胞分别以5μmol／L顺铂或10μmol／L5-Fu处理24h或不予化疗药物处理,其中periostin稳转组在化疗药物处理前可接受或不接受Akt特异性抑制剂MK-2206（1μmol／L）预处理30min。以蛋白质印迹法检测各组细胞的总Akt、磷酸化Akt（p-Akt）、p53蛋白表达,流式细胞术检测细胞凋亡。结果：periostin稳转组SGC．7901细胞Akt磷酸化水平明显高于空载体稳转组,两组间总Akt无明显差异。在经顺铂或5-Fu处理的各组SGC．7901细胞中,periostin稳转组p53蛋白表达和细胞凋亡率明显低于空载体稳转组,而MK-2206预处理可在一定程度上逆转periostin对p53表达的抑制作用及其诱导的凋亡保护效应。结论：通过激活Akt通路抑制p53表达可能是periostin过表达诱导人胃癌细胞化疗耐药性的机制之一,有望作为胃癌治疗的潜在靶点。     

Adherence and internalization of Helicobacter pylori by HEp-2 cells.

Helicobacter pylori colonizes the mucous layer of the stomach and the surface of gastric mucous cells. Although H. pylori is not generally thought of as invasive, it has been observed in the lamina propria and within vacuoles in the cytoplasm of epithelial cells. The authors report that isolates of H. pylori can enter into the cytoplasm of tissue culture epithelial cell lines such as HEp-2 cells. Intracellular uptake of H. pylori by HEp-2 cells is rapid and appears to require both the N-acetylneuraminyllactose-binding adhesin and another factor present only in living bacteria. Uptake of H. pylori was inhibited by ammonium chloride and chloroquine at concentrations that did not effect either adherence or bacterial viability. Dansylcadaverine, an inhibitor of receptor clustering and internalization, also inhibited uptake but not adherence of H. pylori. Uptake was completely inhibited when H. pylori and HEp-2 cells were incubated at 4 degrees C under conditions that did not effect bacterial adherence. Cytochalasin B, an inhibitor of phagocytosis, did not inhibit uptake. It is concluded that H. pylori is internalized either by receptor-mediated endocytosis or by a closely related pathway.     

Akt和核因子κB信号通路在胃癌细胞化学治疗耐药中的作用

目的 探讨Akt、核因子(NF)-κB信号通路在胃癌细胞化学治疗耐药中的作用,以及Akt、NF-κB信号通路的相互作用关系.方法 采用化学治疗药物阿霉素、足叶乙甙及两药联合应用Akt抑制剂(Wortmannin)或NF-κB抑制剂(MG-132)分别作用于胃癌细胞(SGC-7901).用四甲基偶氮唑盐比色法(MTT法)检测SGC-7901细胞增长率;TUNEL法和膜联蛋白V/碘化丙啶(PI)双标法检测肿瘤细胞凋亡;免疫细胞化学法检测NF-κB/P65蛋白表达;电泳迁移率实验(EMSA)法检测NF-κB-DNA结合活性的变化;Western印迹法检测磷酸化Akt或磷酸化NF-κB抑制因子(IκB)α蛋白的表达.结果 ①阿霉素和足叶乙甙均能明显抑制SGC-7901细胞的生长,并呈时间、剂量依赖性;分别联合应用Wortmannin或MG-132后能进一步抑制其生长.②化学治疗药物剂量依赖性地诱导SGC-7901细胞凋亡和NF-κB或Akt活化,联合应用MG-132或Wortmannin均能增强化学治疗药物的诱导凋亡作用和抑制NF-κB或Akt活化作用.③Wortmannin可明显抑制NF-κB的活化,而MG-132对Akt的活化无明显影响.结论 化学治疗药物诱导SGC-7901细胞凋亡的同时诱导Akt、NF-κB活化,抑制Akt、NF-κB的活化可增加化学治疗药物的疗效.     

Tributyrin inhibits human gastric cancer SGC-7901 cell growth by inducing apoptosis and DNA synthesis arrest

AIM: To evaluate the effects of tributyrin, a pro-drug of natural butyrate and a neutral short-chain fatty acid triglyceride, on the growth inhibition of human gastric cancer SGC-7901 cell. METHODS: Human gastric cancer SGC-7901 cells were exposed to tributyrin at 0.5, 1, 2, 5, 10 and 50 mmol/L(-1) for 24-72 h. MTT assay was applied to detect the cell proliferation. [(3)H]-TdR uptake was measured to determine DNA synthesis. Apoptotic morphology was observed by electron microscopy and Hoechst-33258 staining. Flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay were performed to detect tributyrin-triggered apoptosis. The expressions of PARP, Bcl-2 and Bax were examined by Western blot assay. RESULTS: Tributyrin could initiate growth inhibition of SGC-7901 cell in a dose- and time-dependent manner. [(3)H]-TdR uptake by SGC-7901 cells was reduced to 33.6 % after 48 h treatment with 2 mmol/L(-1) tributyrin, compared with the control (P<0.05). Apoptotic morphology was detected by TUNEL assay. Flow cytometry revealed that tributyrin could induce apoptosis of SGC-7901 cells in dose-dependent manner. After 48 hours incubation with tributyrin at 2 mmol/L(-1), the level of Bcl-2 protein was lowered, and the level of Bax protein was increased in SGC-7901, accompanied by PARP cleavage. CONCLUSION: Tributyrin could inhibit the growth of gastric cancer cells effectively in vitro by inhibiting DNA synthesis and inducing apoptosis, which was associated with the down-regulated Bcl-2 expression and the up-regulated Bax expression. Therefore, tributyrin might be a promising chemopreventive and chemotherapeutic agent against human gastric carcinogenesis.     

Effects of epidermal growth factor on the growth of human gastric cancer cell and the implanted tumor of nude mice

AIM：Epidermal growth factor(EGF)plays an important role in the regulation of gastrointestinal tissue growth and development,and it can stimulate epithelial proliferation,cell differentiation and growth,it has been established that the EGF can promote gastric cytoprotection and ulcer healing but the potential ability of EGF to reaulate the gastric cancer growth is unknown,This study is to investigate the influence of EGF on human gastric cancer cell and the implanted tumor growth of nude mice.METHODS:The cell growth rates of human gastric adenocarinome cell lines MKN-28,MKN-45,SGC-7901 and normal human gastric epithelial cells 3T3 were assessed when incubated with recombinant human EGF(rhEGF,0.05,0.1,0.5,1.0,10,50,100mg.L^-1)using MTT method ,The cells of MKN-28,MKN-45,SGC-7901(gastric cancer tissue 1.5mm^3)were implanted in the BALB/cA nude mice for 10 days,The EGF was given intraperitoneally(15,30,60μg.kg^-1)for 3 weeks.The body weights of the tumor bearing animals and their tumor mass were measured afterwards to assess the mitogenic effect of rhEGF in the nude mice.RESULTS:Within the concentration range of 0.05-100mg.L^-1 rhEGF could increase the cell growth of normal 3T3 cells (cell growth rate 100%vs 102.8%,P&lt;0.05)but partially restrain the gastric cancer cell growth,The latter effect was related to cell differentiation ,In 15-60μg/kg rhEGF groups,the mean implanted tumor mass of MKN-28 cell were 1.75g,1.91g,2.08g/NS group 1.97g(P&gt;0.05),the mean tumor mass of SGC-7901 cell were 1.53g,1.07g,1.20g/NS group 1.07g(P&gt;0.05),and for MKN-45 cell,the tumor mass were respectively 1.92g,1.29g,1.77g/NS group 1.82g(P&gt;0.05) So rhEGF had no obvious effect on implanted MKN-28,SGC-7901 and MKN-45 tumor growth.CONCLUSION:EGF has no stimulating effect on the human gastric cancer cell growth neither in vitro nor in vivo.