DEC205在幽门螺杆菌感染胃上皮细胞中的表达

目的：探讨幽门螺杆菌及其热休克蛋白60（H．pylori—HSP60）感染与胃上皮细胞表面DEC205受体的关系。方法：分别用H．pylori、H．pylori-HSP60及E．coliLPS刺激胃上皮细胞KATOIII,利用免疫荧光染色技术观察KATOIII细胞表面DEC205蛋白的表达变化,再利用RT—PCR技术,观察细胞中DEC205mRNA对上述抗原刺激后的变化。结果：H．pylori、H．pylori—HSP60及E．coliLPS的刺激明显引起细胞表面DEC205蛋白的表达以及细胞内DEC205mRNA的产生。结论：H．pylori感染与胃上皮细胞表面的胞吞受体DEC205有着密切的关系。     

DEC205在幽门螺杆菌感染胃上皮细胞中的表达

目的:探讨幽门螺杆菌及其热休克蛋白60(H.pylori-HSP60)感染与胃上皮细胞表面DEC205受体的关系。方法:分别用H.pylori、H.pylori-HSP60及E.coli LPS刺激胃上皮细胞KATOⅢ,利用免疫荧光染色技术观察KATOⅢ细胞表面DEC205蛋白的表达变化,再利用RT-PCR技术,观察细胞中DEC205mRNA对上述抗原刺激后的变化。结果:H.pylori、H.pylori-HSP60及E.coli LPS的刺激明显引起细胞表面DEC205蛋白的表达以及细胞内DEC205 mRNA的产生。结论:H.pylori感染与胃上皮细胞表面的胞吞受体DEC205有着密切的关系。     

幽门螺杆菌感染诱导microRNA-146a表达的机制研究

目的：探讨幽门螺杆菌（H.pylori）感染引起人胃上皮细胞microRNA-146a（miR-146a）上调的分子机制。方法：分别用H.pylori重组蛋白、全菌蛋白、培养上清、感染相关炎性因子（IL-8、TNF-α、IL-1β）以及TLR配体刺激人胃上皮细胞,检测细胞miR-146a的表达;通过生物信息学软件预测和荧光素酶实验鉴定miR-146a启动子,分析诱导表达的相关信号通路。结果：除H.pylori感染相关炎性因子IL-8、TNF-α、IL-1β能够明显诱导miR-146a表达上调（P〈0.01）外,其他刺激因素均不能诱导miR-146a的显著表达;当采用RNAi技术将IL-8、TNF-α、IL-1β分别沉默,检测H.pylori诱导miR-146a表达时,各沉默组与对照组均无显著差异。软件预测显示miR-146a启动子序列中含有多个NF-κB结合位点;H.pylori能够显著增加miR-146a启动子荧光素酶报告载体的相对荧光素酶值;当启动子序列中的NF-κB结合位点发生突变,其相对荧光素酶比值显著降低（P〈0.05）。结论：H.pylori感染相关炎性因子IL-8、TNF-α、IL-1β能够诱导miR-146a表达明显上调;NF-κB信号通路在H.pylori感染诱导miR-146a的表达中发挥关键作用。     

MEK/ERK信号通路对人结膜上皮细胞增殖的影响及其机制研究

目的:探讨MEK/ERK信号通路对人结膜上皮细胞增殖的影响及其可能的机制。方法:采用不同浓度(0、12.5、25、50、100μmol/L)的MEK抑制剂PD98059处理人结膜上皮细胞(HConEpiC),通过CCK-8法检测不同浓度PD98059作用不同时间(0、12、24、48 h)对人结膜上皮细胞增殖的影响,Western blot检测不同浓度PD98059对人结膜上皮细胞ERK1/2、P-ERK1/2表达的影响。结果:相比对照组(0μmol/L),不同浓度(12.5、25、50、100μmol/L)PD98059处理后的人结膜上皮细胞增殖率明显下降,呈剂量-效应关系,且随处理时间增加(12、24、48 h)其抑制作用也显著增强,差异均有统计学意义(P0.05)。不同浓度PD98059处理人结膜上皮细胞24 h后,其ERK及p-ERK1/2表达随处理浓度增加而降低,与对照组(0μmol/L)相比差异有统计学意义(P0.05),且二者表达量与细胞增值抑制率均呈显著负相关(r=-0.995、r=-0.968,P0.05)。结论:PD98059可抑制人结膜上皮细胞增殖,这可能与其下调ERK表达和减少其活化有关。     

幽门螺杆菌感染诱导microRNA-146a表达的机制研究

目的:探讨幽门螺杆菌(H.pylori)感染引起人胃上皮细胞microRNA-146a(miR-146a)上调的分子机制。方法:分别用H.pylori重组蛋白、全菌蛋白、培养上清、感染相关炎性因子(IL-8、TNF-α、IL-1β)以及TLR配体刺激人胃上皮细胞,检测细胞miR-146a的表达;通过生物信息学软件预测和荧光素酶实验鉴定miR-146a启动子,分析诱导表达的相关信号通路。结果:除H.pylori感染相关炎性因子IL-8、TNF-α、IL-1β能够明显诱导miR-146a表达上调(P<0.01)外,其他刺激因素均不能诱导miR-146a的显著表达;当采用RNAi技术将IL-8、TNF-α、IL-1β分别沉默,检测H.pylori诱导miR-146a表达时,各沉默组与对照组均无显著差异。软件预测显示miR-146a启动子序列中含有多个NF-κB结合位点;H.pylori能够显著增加miR-146a启动子荧光素酶报告载体的相对荧光素酶值;当启动子序列中的NF-κB结合位点发生突变,其相对荧光素酶比值显著降低(P<0.05)。结论:H.pylori感染相关炎性因子IL-8、TNF-α、IL-1β能够诱导miR-146a表达明显上调;NF-κB信号通路在H.pylori感染诱导miR-146a的表达中发挥关键作用。     

幽门螺杆菌感染细胞模型的建立及感染相关microRNAs的筛选鉴定

目的：建立稳定的幽门螺杆菌（H.pylori）感染人胃上皮细胞模型;筛选并鉴定H.pylori感染相关microRNAs（miRNAs）的表达,为深入研究感染相关miRNAs的调控作用机制奠定基础。方法：将H.pylori标准株按MOI=100：1感染人胃上皮细胞,通过检测炎性细胞因子及炎症反应关键酶的表达综合评价感染模型;采用博奥公司miRNAs V3.0芯片分析细胞感染前后miRNAs表达谱变化,运用实时定量PCR技术和Northern杂交对表达显著差异的miRNAs进行分析鉴定。结果：H.pylori感染细胞24 h后,细胞分泌促炎细胞因子IL-8显著升高（P〈0.01）;启动炎症反应的关键酶COX-2的表达明显增加。芯片数据显示：H.pylori感染引起超过2倍显著差异表达的miRNAs包括：表达上调的PREDICTED-MIR191、miR-155、miR-92b、miR-30b、miR-146a、miR-16等,和表达降低的miR-181b、miR-324。实时定量PCR和Northern杂交结果显示感染相关miR-155和miR-146a在H.pylori感染细胞模型中表达均显著增加（P〈0.01）。结论：miR-155和miR-146a在感染细胞模型中的表达增加提示二者可能参与H.pylori感染的免疫调控过程。     

银杏内酯诱导原代培养神经元低氧诱导因子1α的表达及与ERK通路的关系

目的：研究银杏内酯（Gin）对氯化钴（CoCl2）诱导的化学性缺氧原代培养神经元低氧诱导因子-α（HIF—1α）表达的影响及其与细胞外信号调节激酶（ERK）信号通路之间的关系。方法：以CoCl2（125μmol／L）诱导的原代培养胚胎小鼠大脑皮层神经元为缺氧模型,观察Gin（终浓度37．5mg／L）对神经细胞形态和活力的影响,Western blot HIF—1α和磷酸化ERK（p-ERK）的表达：运用ERK特异性抑制剂PD98059观察HIF-1α表达与ERK通路之间的关系。结果：Gin能明显提高CoCl2处理的神经细胞的活力、在正常培养的皮层神经元中HIF—1α和p-ERK的表达水平较低,CoCl2处理4h后表达水平明显上调;Gin预处理24h其表达强度进一步提高PD98059能部分抑制CoCl2诱导的HIF-1α的表达,显著抑制p-ERK的表达;预加Gin能完全阻止该抑制作用：结论：Gin对CoCl2诱导的化学性缺氧损伤神经元有保护作用,该作用与HIF-1α表达上调、ERK通路的激活有关     

大黄素对HT-29细胞MAPKs信号通路活化及IL-8分泌的影响

目的：研究大黄素对IFN-和LPS刺激的人结肠癌细胞株HT-29细胞的ERK、JNK和p38MARK和IL-8表达的影响。方法：人结肠癌细胞株HT-29细胞与40ng／mL的IFN．共培养12h,再加入100ng／mLLPS刺激15min,用大黄素预处理进行干预。ELISA检测HT-29细胞内的ERK、JNK和p38MARK含量和细胞上清IL-8含量。结果：IFN-1和LPS刺激后HT-29细胞的ERK、JNK和p38MARK磷酸化水平和IL．8分泌明显升高。大黄素对p38和JNK磷酸化有明显的抑制作用,而对ERK磷酸化则没有明显抑制作用;大黄素能显著降低IFN-γ＋LPS所引起的HT-29细胞IL-8的大量产生,并且呈明显的剂量依赖关系。结论：大黄素能有效抑制IFN-γ＋LPS所引起的HT．29细胞p38和ⅢK的磷酸化,并显著降低IL-8分泌。     

幽门螺杆菌临床分离株CagA的序列比对及其对胃上皮细胞形态与功能的影响

【背景】细胞毒素相关基因A蛋白(Cytotoxin Associated Gene A Protein,CagA)是幽门螺杆菌(Helicobacter pylori)重要的效应蛋白,CagA的多态性与胃癌的发生发展密切相关。【目的】比较幽门螺杆菌临床分离株的CagA结构差异,探讨不同CagA对胃上皮细胞形态及功能的影响。【方法】对27株幽门螺杆菌的CagA序列进行比对,分析氨基酸组成差异及变异情况,用含不同CagA序列的5株H. pylori感染低恶性胃上皮细胞AGS 6 h,感染复数(Multiplicity of Infection,MOI)为30:1,显微镜观察细胞形态变化,Western Blot法检测极性调节激酶1b(Polarity-Regulating Kinase 1b,PAR1b)的表达,ELISA检测培养液中白介素8(Interleukin-8,IL-8)的浓度。【结果】临床分离株的CagA存在结构和氨基酸组成差异,西方株的EPIYA基序存在更多变异,具有完整cagA基因的菌株感染后细胞形态发生显著变化。Western Blot分析结果显示:与对照组比较,西方株NCTC 11639和H. pylori 26695感染的细胞中PAR1b的表达升高,而东亚株H. pylori GZ7感染的细胞中PAR1b表达降低,差异均有统计学意义(P0.05);突变株H. pylori GZ15及H. pylori GZ7/ΔcagA感染后PAR1b的表达无显著变化。与对照组比较,实验组中H. pylor感染的细胞培养液中IL-8浓度均增加,东亚株促进IL-8分泌的能力大于西方株。【结论】含不同CagA的幽门螺杆菌发挥不同的生物学功能。东亚株能抑制PAR1b的表达,促进IL-8分泌的能力更强,H.pylori感染引起的细胞形态变化依赖cagA基因的完整性。     

NF—κB信号通路在绿脓菌素诱导气道上皮表达IL-8的作用

探讨绿脓菌素对人气道上皮细胞株（NCI—H292细胞）表达IL-8的诱导作用及通过NF—κB信号传导通路。采用ELISA法对PCN诱导NCI-H292细胞分泌IL-8进行分析,应用westernblotting检测NF-κB的蛋白表达,观察NF—κB阻断剂对IL-8表达的影响。PCN可促进NCI-H292细胞IL-8分泌。NF—κB的阻断剂PDTC能显著抑制IL培表达（P〈0．01）。PCN可能通过NF-κB信号通路诱导呼吸道上皮细胞IL-8表达。     

Human dendritic cells respond to Helicobacter pylori, promoting NK cell and Th1-effector responses in vitro

Helicobacter pylori infection leads to chronic gastric inflammation. The current study determined the response of human APCs, NK cells, and T cells toward the bacteria in vitro. Human monocyte-derived dendritic cells (DC) were incubated with bacteria for 48 h. Intact H. pylori at a multitude of infection 5 stimulated the expression of MHC class II (4- to 7-fold), CD80, and CD86 B7 molecules (10- to 12-fold) and the CD83 costimulatory molecule (>30-fold) as well as IL-12 secretion (>50-fold) in DCs, and thereby, strongly induced their maturation and activation. CD56(+)/CD4(-) NK cells, as well as CD4(+)/CD45RA(+) naive T cells, were isolated and incubated with DCs pulsed with intact bacteria or different cellular fractions. Coculture of H. pylori-pulsed DCs with NK cells strongly potentiated the secretion of TNF-alpha and IFN-gamma. Coculture of naive T cells with H. pylori-pulsed DCs significantly enhanced TNF-alpha, IFN-gamma, and IL-2 secretion as well as T-bet mRNA levels, while GATA-3 mRNA was lowered. However, the effect appeared attenuated compared with coculture with Escherichia coli. A greater stimulation was seen with naive T cells and DCs pulsed with H. pylori membrane preparations. Intact H. pylori potently induced the maturation and activation of human monocyte-derived DC and thereby promote NK and Th1 effector responses. The strong activation of NK cells may be important for the innate immune response. Th1-polarized T cells were induced especially by incubation with membrane preparations of H. pylori, suggesting that membrane proteins may account for the specific adaptive immune response.     

Differential activation of mitogen-activated protein kinases in AGS gastric epithelial cells by cag+ and cag- Helicobacter pylori.

The aim of this study was to determine whether Helicobacter pylori activates mitogen-activated protein (MAP) kinases in gastric epithelial cells. Infection of AGS cells with an H. pylori cag+ strain rapidly (5 min) induced a dose-dependent activation of extracellular signal-regulated kinases (ERK), p38, and c-Jun N-terminal kinase (JNK) MAP kinases, as determined by Western blot analysis and in vitro kinase assay. Compared with cag+ strains, cag- clinical isolates were less potent in inducing MAP kinase, particularly JNK and p38, activation. Isogenic inactivation of the picB region of the cag pathogenicity island resulted in a similar loss of JNK and p38 MAP kinase activation. The specific MAP kinase inhibitors, PD98059 (25 microM; MAP kinase kinase (MEK-1) inhibitor) and SB203580 (10 microM; p38 inhibitor), reduced H. pylori-induced IL-8 production in AGS cells by 78 and 82%, respectively (p < 0.01 for each). Both inhibitors together completely blocked IL-8 production (p < 0.001). However, the MAP kinase inhibitors did not prevent H. pylori-induced IkappaBalpha degradation or NF-kappaB activation. Thus, H. pylori rapidly activates ERK, p38, and JNK MAP kinases in gastric epithelial cells; cag+ isolates are more potent than cag- strains in inducing MAP kinase phosphorylation and gene products of the cag pathogenicity island are required for maximal MAP kinase activation. p38 and MEK-1 activity are required for H. pylori-induced IL-8 production, but do not appear to be essential for H. pylori-induced NF-kappaB activation. Since MAP kinases regulate cell proliferation, differentiation, programmed death, stress, and inflammatory responses, activation of gastric epithelial cell MAP kinases by H. pylori cag+ strains may be instrumental in inducing gastroduodenal inflammation, ulceration, and neoplasia.     

Helicobacter pylori stimulates gastric epithelial cell MMP-1 secretion via CagA-dependent and -independent ERK activation

Because the mechanisms of Helicobacter pylori-induced gastric injury are incompletely understood, we examined the hypothesis that H. pylori induces matrix metalloproteinase-1 (MMP-1) secretion, with potential to disrupt gastric stroma. We further tested the role of CagA, an H. pylori virulence factor, in MMP-1 secretion. Co-incubation of AGS cells with Tx30a, an H. pylori strain lacking the cagA virulence gene, stimulated MMP-1 secretion, confirming cagA-independent secretion. Co-incubation with strain 147C (cagA(+)) resulted in CagA translocation into AGS cells and increased MMP-1 secretion relative to Tx30a. Transfection of cells with the recombinant 147C cagA gene also induced MMP-1 secretion, indicating that CagA can independently stimulate MMP-1 secretion. Co-incubation with strain 147A, containing a cagA gene that lacks an EPIYA tyrosine phosphorylation motif, as well as transfection with 147A cagA, yielded an MMP-1 secretion intermediate between no treatment and 147C, indicating that CagA tyrosine phosphorylation regulates cellular signaling in this model system. H. pylori induced activation of the MAP kinase ERK, with CagA-independent (early) and dependent (later) components. MEK inhibitors UO126 and PD98059 inhibited both CagA-independent and -dependent MMP-1 secretion, whereas p38 inhibition enhanced MMP-1 secretion and ERK activation, suggesting p38 negative regulation of MMP-1 and ERK. These data indicate H. pylori effects on host epithelial MMP-1 expression via ERK, with p38 playing a potential regulatory role.     

Helicobacter pylori encoding the pathogenicity island activates matrix metalloproteinase 1 in gastric epithelial cells via JNK and ERK

Helicobacter pylori colonizes the human gastric epithelium and induces an inflammatory response that is a trigger for gastric carcinogenesis. Matrix metalloproteinases (MMPs) have recently been shown to be up-regulated in gastric epithelial cells infected with H. pylori and might contribute to the pathogenesis of peptic ulcer. The aim of this study was to extend the knowledge about the effect of H. pylori infection on MMP-1 expression by gastric epithelial cells, the kinetics of induction, the pathogenetic properties of the bacterium, and the intracellular signaling pathways required for MMP-1 up-regulation. Expression of MMP-1 was induced more than 10-fold by co-culture of AGS+cells with H. pylori strains carrying the pathogenicity island (PAI). H. pylori strains with mutations in the PAI and a defective type IV secretion system had no effect on MMP-1. Double immunofluorescence revealed strong MMP-1 staining in epithelial cells of gastric biopsies at sites of bacterial attachment. In vitro, MMP-1 is up-regulated by interleukin-1beta and tumor necrosis factor-alpha, but these regulatory mechanisms are not operating in H. pylori infection as shown by inhibitory antibodies. Specific inhibitors of JNK kinase and ERK1/2 kinase were found to suppress the H. pylori-induced MMP-1 expression and activity. AGS cells treated with antisense MMP-1 showed a significantly reduced potential to degrade reconstituted basement membrane. Our results suggest that in gastric epithelial cells, H. pylori up-regulates MMP-1 in a type IV secretion system-dependent manner via JNK and ERK1/2. Induction of MMP-1 is further implicated in complex processes induced by H. pylori, resulting in tissue degradation and remodeling of the gastric mucosa.     

Helicobacter pylori environmental interactions: effect of acidic conditions on H. pylori-induced gastric mucosal interleukin-8 production

To explore the interactions between the host, environment and bacterium responsible for the different manifestations of Helicobacter pylori infection, we examined the effect of acidic conditions on H. pylori-induced interleukin (IL)-8 expression. AGS gastric epithelial cells were exposed to acidic pH and infected with H. pylori[wild-type strain, its isogenic cag pathogenicity island (PAI) mutant or its oipA mutant]. Exposure of AGS cells to acidic pH alone did not enhance IL-8 production. However, following exposure to acidic conditions, H. pylori infection resulted in marked enhancement of IL-8 production which was independent of the presence of the cag PAI and OipA, indicating that H. pylori and acidic conditions act synergistically to induce gastric mucosal IL-8 production. In neutral pH environments H. pylori-induced IL-8 induction involved the NF-kappaB pathways, the extracellular signal-regulated kinase (ERK)-->c-Fos/c-Jun-->activating protein (AP-1) pathways, JNK-->c-Jun-->AP-1 pathways and the p38 pathways. At acidic pH H. pylori-induced augmentation of IL-8 production involved markedly upregulated the NF-kappaB pathways and the ERK-->c-Fos-->AP-1 pathways. In contrast, activation of the JNK-->c-Jun-->AP-1 pathways and p38 pathways were pH independent. These results might explain the clinical studies in which patients with duodenal ulcers had higher levels of IL-8 in the antral gastric mucosa than patients with simple H. pylori gastritis.