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。     

ERK介导幽门螺杆菌HSP60感染胃上皮细胞的IL-8分泌

目的：探讨幽门螺杆菌热休克蛋白60（H．pylori—HSP60）感染胃上皮细胞后ERK与白介素-8（IL-8）分泌的关系。方法：利用ELISA技术,对活菌（IntactH．pylori）、死菌（Heat—killedH．pylori）及H．pylori—HSP60刺激胃上皮细胞KATOIII的IL_8蛋白分泌水平进行分析,观察IL-8随以上抗原浓度梯度的变化及ERK抑制剂PD98059对其分泌量的影响;利用Westernblot技术,观察KATOⅢ胞中磷酸化ERK随IntactH．pylori、Heat—killedH．pylori及H．pylori—HSP60刺激时间的变化状况。结果：IL-8的分泌随着IntactH．pylori、Heat—killedH．pylori及H．pylori—HSP60刺激浓度的升高而增高;H．pylori刺激KATOⅢ胞1h后ERK开始表达,其中IntactH．pylori在9h时表达达到高峰,Heat·killedH．pylori在24h时达到高峰,而H．pylori-HSP60刺激KATOⅢ胞6h后ERK开始表达,9h时达到高峰;PD98059抑制了H．pylori—HSP60诱导的IL-8的分泌。结论：ERK介导了H．pylori—HSP60感染的胃上皮细胞的IL-8的分泌。     

幽门螺杆菌疫苗

可观的证据认为H．pylori重建了胃免疫系统。H.pylori对胃上皮细胞的结合给了NF-kB转录因子激活和RANTES、GROa、MIP-1a、MCP-IⅠ?、IL-1β、IL-6、IL-8、IL-12和TNFa产生的信号。最近的证据显示，控制宿主炎症应答程度的IL-1基因多态性与胃病相关。H.pylori感染上调了CD11b／CD18整合素及其受体、     

幽门螺杆菌急性感染对胃上皮细胞凋亡的影响

目的 探讨幽门螺杆菌(Helicobacter pylori)急性感染对GES 1细胞凋亡的影响,揭示H. pylori引起GES 1细胞凋亡变化的分子机制。 方法 将H. pylori临床分离株SBK与胃上皮细胞GES 1按不同比例(感染复数MOI分别为50∶1和100∶1)共培养24 h,建立H. pylori急性感染模型。采用流式细胞仪分析GES 1的凋亡,通过Western Blot检测凋亡相关蛋白Bcl xL、Bcl 2、Bax、Caspase 3和NF κB p65的表达。经H. pylori感染的GES 1细胞为处理组细胞,未经H. pylori感染的GES 1细胞即为对照细胞。使用SPSS 21.0软件对结果进行统计学分析。 结果 GES 1细胞经H. pylori处理24 h后,与对照细胞相比,MOI为50∶1(t=11.040,P结论 H. pylori急性感染通过改变线粒体途径中凋亡相关蛋白Bax、Bcl 2及Bcl xL的表达促进GES 1细胞凋亡,且GES 1细胞的凋亡程度与H. pylori的感染复数有关。     

幽门螺杆菌感染新型原代人胃上皮细胞模型的建立

目的 培养三维(3D)人胃黏膜上皮类器官,转为二维(2D)原代胃黏膜细胞培养,并建成人2D原代胃上皮细胞的幽门螺杆菌感染模型。 方法 (1)从正常人胃上皮组织中分离胃腺,在含有多种生长调节和凋亡抑制等混合因子的培养基中,依附于基质胶而培养成3D类器官;(2)利用免疫荧光技术鉴定胃上皮类器官的相关分子标记;(3)研究正常原代胃上皮细胞被幽门螺杆菌感染后的形态学变化,利用免疫印迹技术鉴定幽门螺杆菌感染相关蛋白的表达水平。 结果 成功培养出可长期传代的人胃上皮3D类器官,具有典型的人胃黏膜上皮分子标记。而且3D类器官转为2D平面培养的原代胃上皮细胞,可作为幽门螺杆菌的体外原代细胞感染模型。 结论 3D胃上皮类器官,可作为2D原代胃上皮细胞的持久来源,为研究幽门螺杆菌感染人体胃上皮的分子机制带来个体化的新模型。     

幽门螺杆菌对胃上皮细胞间隙连接超微结构的影响

通过实验和临床观察幽门螺杆菌(Helicobacter pylori)对胃上皮细胞间隙连接超微结构的影响,从细胞间隙连接角度探讨H. pylori致癌机制．将不同H. pylori菌株与BGC-823细胞共培养24 h或 48 h,用原位固定与原位包埋法透射电镜观察细胞间隙连接超微结构变化．对70例胃癌患者,用快速尿素酶试验、碱性品红染色和¹⁴C尿素呼气实验检测H. pylori,PCR法检测H. pylori CagA基因,及透射电镜观察胃上皮细胞间隙连接超微结构变化．结果显示,未加H. pylori组BGC-823细胞可见较多细胞连接及连接复合体,加H. pylori各组细胞的连接数、单位周长连接数与单位周长连接长度均小于未加H. pylori组,而细胞间隙最小宽度大于未加H. pylori组(P < 0.001或P < 0.005),且CagA⁺ 的NCTC J99组、临床株GC 01组和NCTC 11639组细胞连接数、单位周长连接数均小于CagA^- 的NCTC 12908组(P < 0.001或P < 0.05),NCTC J99组与临床株GC 01组细胞单位周长连接长度短于NCTC 12908组(P < 0.001)．胃癌患者H. pylori感染组细胞连接数、单位周长连接数与单位周长连接长度均小于无H. pylori感染组,细胞间隙最小宽度大于无H. pylori感染组(P < 0.001),且CagA⁺ H. pylori感染者细胞连接数、单位周长连接数与单位周长连接长度均小于CagA^- H. pylori感染者,细胞间隙最小宽度大于CagA^- H. pylori感染者．上述结果表明,胃上皮细胞间隙连接改变与H. pylori感染,特别是CagA⁺ H. pylori感染有关．     

金双歧联合四联疗法对幽门螺杆菌感染的治疗

目的观察金双歧联合四联疗法根除幽门螺杆菌(H.pylori)的临床疗效。方法选取福建省立金山医院南院2015年8月1日至2017年8月1日300例H.pylori感染患者,随机分为A组(50例,采用雷贝拉唑、枸橼酸铋钾、克拉霉素和阿莫西林治疗)、B组(50例,在A组基础上联用金双歧治疗)、C组(50例,采用雷贝拉唑、枸橼酸铋钾、阿莫西林和呋喃唑酮治疗)、D组(50例,在C组基础上联用金双歧治疗)、E组(50例,采用雷贝拉唑、枸橼酸铋钾、呋喃唑酮和四环素治疗)、F组(50例,在E组基础上联用金双歧治疗)。观察各组患者的临床疗效及不良反应。结果 A、B、C、D、E和F组患者的H.pylori根除率按PP分析分别为81.4%、95.7%、82.2%、95.8%、97.9%和97.9%,而各组患者H.pylori根除率按ITT分析分别为70.0%、88.0%、74.0%、92.0%、92.0%和94.0%,A组和B组,C组和D组H.pylori根除率的PP分析和ITT分析比较差异有统计学意义(P0.05);E组和F组根除率的PP分析和ITT分析比较差异无统计学意义(P0.05)。A、B、C、D、E和F组的总不良反应率分别为30.2%、10.9%、31.1%、10.4%、53.2%和22.9%,其中A组和B组,C组和D组,E组和F组总不良反应率差异均有统计学意义(P0.05)。结论金双歧联合四联疗法可提高H.pylori根除率,减少患者不良反应,可推荐用于H.pylori感染患者的根除治疗。     

幽门螺杆菌对胃上皮细胞间隙连接超微结构的影响(英文)

通过实验和临床观察幽门螺杆菌（Helicobacter pylori）对胃上皮细胞间隙连接超微结构的影响,从细胞间隙连接角度探讨H.pylori致癌机制.将不同H.pylori菌株与BGC-823细胞共培养24h或48h,用原位固定与原位包埋法透射电镜观察细胞间隙连接超微结构变化.对70例胃癌患者,用快速尿素酶试验、碱性品红染色和14C尿素呼气实验检测H.pylori,PCR法检测H.pyloriCagA基因,及透射电镜观察胃上皮细胞间隙连接超微结构变化.结果显示,未加H.pylori组BGC-823细胞可见较多细胞连接及连接复合体,加H.pylori各组细胞的连接数、单位周长连接数与单位周长连接长度均小于未加H.pylori组,而细胞间隙最小宽度大于未加H.pylori组（P〈0.001或P〈0.005）,且CagA＋的NCTCJ99组、临床株GC01组和NCTC11639组细胞连接数、单位周长连接数均小于CagA-的NCTC12908组（P〈0.001或P〈0.05）,NCTCJ99组与临床株GC01组细胞单位周长连接长度短于NCTC12908组（P〈0.001）.胃癌患者H.pylori感染组细胞连接数、单位周长连接数与单位周长连接长度均小于无H.pylori感染组,细胞间隙最小宽度大于无H.pylori感染组（P〈0.001）,且CagA＋H.pylori感染者细胞连接数、单位周长连接数与单位周长连接长度均小于CagA-H.pylori感染者,细胞间隙最小宽度大于CagA-H.pylori感染者.上述结果表明,胃上皮细胞间隙连接改变与H.pylori感染,特别是CagA＋H.pylori感染有关.     

融合表达DEC205单链抗体和NS3蛋白的HCV DNA疫苗的免疫原性研究

为研发新型HCV DNA疫苗并探讨优化其免疫原性的策略,我们分析靶向树突状细胞(Dendritic cells,DC)的分子对HCV DNA疫苗免疫原性的影响。我们基于抗小鼠DC细胞表面分子DEC205/CD205的单克隆抗体DEC205的单链分子,构建可单独表达DEC205单链抗体或者与HCV非结构蛋白NS3融合表达的DNA表达质粒,并构建单独表达HCV非结构蛋白NS3的DNA表达质粒;经瞬时转染法鉴定HCV NS3及其与DEC205单链抗体融合蛋白的表达;随后采用注射结合电转的方式免疫Balb/C小鼠并研究各疫苗的体液(NS3特异性IgG抗体)与细胞免疫(IFN-γELISPOT)效果。结果表明:DEC205单链抗体基因与HCV NS3编码基因的融合可显著增强NS3特异的免疫应答;采用皮内注射加卡钳电极电转的方式可以产生最强的NS3特异性抗体和T细胞免疫反应。因此,通过DEC205单链抗体与HCV DNA疫苗靶抗原融合可明显增强免疫应答效果。该策略为HCV及其他类似病原的新型DNA疫苗研制提供重要依据。     

幽门螺杆菌细胞空泡毒素与胃上皮细胞的表皮生长因子信号转导

细胞空泡毒素是幽门螺杆菌的一种重要的外分泌毒素,但其确定的致病机制尚不清楚。近年来研究发现,细胞空泡毒素不仅直接导致细胞的空泡毒性,还可能通过干扰胃黏膜上皮细胞内及细胞间的信息传递,尤其是与表皮生长因子有关的信号转导过程,影响上皮细胞的生长、增殖及组织的修复,是幽门螺杆菌致病机制的重要环节之一。     

Modulation of Activation-Induced Cytidine Deaminase by Curcumin in Helicobacter pylori-Infected Gastric Epithelial Cells

Background: Anomalous expression of activation‐induced cytidine deaminase (AID) in Helicobacter pylori‐infected gastric epithelial cells has been postulated as one of the key mechanisms in the development of gastric cancer. AID is overexpressed in the cells through nuclear factor (NF)‐κB activation by H. pylori and hence, inhibition of NF‐κB pathway can downregulate the expression of AID. Curcumin, a spice‐derived polyphenol, is known for its anti‐inflammatory activity via NF‐κB inhibition. Therefore, it was hypothesized that curcumin might suppress AID overexpression via NF‐κB inhibitory activity in H. pylori‐infected gastric epithelial cells. Materials and Methods: MKN‐28 or MKN‐45 cells and H. pylori strain 193C isolated from gastric cancer patient were used for co‐culture experiments. Cells were pretreated with or without nonbactericidal concentrations of curcumin. Apoptosis was determined by DNA fragmentation assay. Enzyme‐linked immunosorbent assay was performed to evaluate the anti‐adhesion activity of curcumin. Real‐time polymerase chain reaction was employed to evaluate the expression of AID mRNA. Immunoblot assay was performed for the analysis of AID, NF‐κB, inhibitors of NF‐κB (IκB), and IκB kinase (IKK) complex regulation with or without curcumin. Results: The adhesion of H. pylori to gastric epithelial cells was not inhibited by curcumin pretreatment at nonbactericidal concentrations (≤10 μmol/L). Pretreatment with nonbactericidal concentration of curcumin downregulated the expression of AID induced by H. pylori. Similarly, NF‐κB activation inhibitor (SN‐50) and proteasome inhibitor (MG‐132) also downregulated the mRNA expression of AID. Moreover, curcumin (≤10 μmol/L) has suppressed H. pylori‐induced NF‐κB activation via inhibition of IKK activation and IκB degradation. Conclusion: Nonbactericidal concentrations of curcumin downregulated H. pylori‐induced AID expression in gastric epithelial cells, probably via the inhibition of NF‐κB pathway. Hence, curcumin can be considered as a potential chemopreventive candidate against H. pylori‐related gastric carcinogenesis.     

Antigen Delivery to DEC205+ Dendritic Cells Induces Immunological Memory and Protective Therapeutic Effects against HPV-Associated Tumors at Different Anatomical Sites

The generation of successful anticancer vaccines relies on the ability to induce efficient and long-lasting immune responses to tumor antigens. In this scenario, dendritic cells (DCs) are essential cellular components in the generation of antitumor immune responses. Thus, delivery of tumor antigens to specific DC populations represents a promising approach to enhance the efficiency of antitumor immunotherapies. In the present study, we employed antibody-antigen conjugates targeting a specific DC C-type lectin receptor. For that purpose, we genetically fused the anti-DEC205 monoclonal antibody to the type 16 human papillomavirus (HPV-16) E7 oncoprotein to create a therapeutic vaccine to treat HPV-associated tumors in syngeneic mouse tumor models. The therapeutic efficacy of the αDEC205-E7 mAb was investigated in three distinct anatomical tumor models (subcutaneous, lingual and intravaginal). The immunization regimen comprised two doses of the αDEC205-E7 mAb coadministered with a DC maturation stimulus (Polyinosinic:polycytidylic acid, poly (I:C)) as an adjuvant. The combined immunotherapy produced robust antitumor effects on both the subcutaneous and orthotopic tumor models, stimulating rapid tumor regression and long-term survival. These outcomes were related to the activation of tumor antigen-specific CD8⁺ T cells in both systemic compartments and lymphoid tissues. The αDEC205-E7 antibody plus poly (I:C) administration induced long-lasting immunity and controlled tumor relapses. Our results highlight that the delivery of HPV tumor antigens to DCs, particularly via the DEC205 surface receptor, is a promising therapeutic approach, providing new opportunities for the development of alternative immunotherapies for patients with HPV-associated tumors at different anatomical sites.     

VCP Phosphorylation-Dependent Interaction Partners Prevent Apoptosis in Helicobacter pylori-Infected Gastric Epithelial Cells

Previous studies have demonstrated that valosin-containing protein (VCP) is associated with H. pylori-induced gastric carcinogenesis. By identifying the interactome of VCP overexpressed in AGS cells using a subtractive proteomics approach, we aimed to characterize the cellular responses mediated by VCP and its functional roles in H. pylori-associated gastric cancer. VCP immunoprecipitations followed by proteomic analysis identified 288 putative interacting proteins, 18 VCP-binding proteins belonged to the PI3K/Akt signaling pathway. H. pylori infection increased the interaction between Akt and VCP, Akt-dependent phosphorylation of VCP, levels of ubiquitinated proteins, and aggresome formation in AGS cells. Furthermore, phosphorylated VCP co-localized with the aggresome, bound ubiquitinated proteins, and increased the degradation of cellular regulators to protect H. pylori-infected AGS cells from apoptosis. Our study demonstrates that VCP phosphorylation following H. pylori infection promotes both gastric epithelial cell survival, mediated by the PI3K/Akt pathway, and the degradation of cellular regulators. These findings provide novel insights into the mechanisms of H. pylori infection induced gastric carcinogenesis.     

体外培养的人牙胚上皮细胞的成釉分化

人表皮干细胞可以作为牙齿再生中上皮源性的种子细胞,但是其成釉分化的效率低下. 本研究分离培养了人牙胚上皮细胞,利用E13.5的小鼠牙间充质与其重组,构建重组牙胚,对其成釉分化的潜能和机制进行研究. 研究结果发现,体外培养的P1代人牙胚上皮细胞成釉率高达50%. 随着传代次数的增加,成釉率明显下降. 通过对牙上皮发育分化相关基因的表达检测和分析表明,重组牙胚成牙分化能力和成釉潜能的下降与牙上皮发育相关基因的表达状态密切相关. 特别是FGF8表达水平的下调以及PITX2不同亚型在人牙胚细胞中表达量的不均衡,可能是导致人牙胚细胞成釉潜能下降并丧失的主要原因. 本研究结果为理解牙齿再生过程中上皮源性的种子细胞的成釉机制提供了新的实验数据,对进一步提高表皮干细胞在牙齿再生过程中的成釉率有指导意义.     

高浓度葡萄糖诱导人晶状体上皮细胞发生EMT

目的:观察高浓度葡萄糖诱导人晶状体上皮细胞发生上皮-间质转分化(epithelial-to-mesenchymal transition,EMT)。方法:将人晶状体上皮细胞HLE-B3系分别培养在正常葡萄糖浓度(5.5 mmol/L)DMEM培养基和高浓度葡萄糖(35.5 mmol/L)的DMEM培养基中24小时,于培养的0 h、3 h、6 h、12 h、24 h在倒置显微镜下观察细胞形态学变化,采用免疫荧光染色检测晶状体上皮细胞中EMT相关蛋白E-cadherin及α-SMA的表达变化。结果:与正常糖浓度组相比,随着时间的延长高糖组细胞逐渐丢失上皮细胞形态,细胞变细、变长,向纤维细胞的形态转变;同时随着时间的延长,高糖组晶状体上皮细胞中E-cadherin染色的荧光强度在各时间点均低于正常糖浓度组,而α-SMA的荧光强度却明显高于正常糖浓度组,在6 h和12 h时差异显著,有统计学意义(P0.01)。结论:高浓度葡萄糖诱导人晶状体上皮细胞发生上皮-间质转分化。     

脂多糖对人支气管上皮细胞STAT1、STAT3、STAT4、STAT6表达的影响

目的观察脂多糖对人支气管上皮细胞16HBESTAT1、STAT3、STAT4、STAT6表达的影响。方法采用普通RT—PCR检测16HBE细胞STAT1、STAT3、STAT4、STAT6的mRNA表达;Western印迹检测16HBE细胞STAT1、STAT4、STAT6的蛋白表达。分别采用不同浓度的脂多糖在不同的时间点处理16HBE细胞,采用Real—timePCR的方法检测16HBE细胞STAT1、STAT3、STAT4、STAT6的mRNA表达。结果1μg／m1的LPS处理16HBE细胞1h组、0．25μg／m1的LPS处理16HBE细胞4h组、1μg／ml的LPS处理16HBE细胞4h组STAT1、STAT4的mRNA表达较正常对照组显著增高（P〈0．01）;0．25μg／ml的LPS处理16HBE细胞2h组、1μg／ml的LPS处理16HBE细胞2h组、10μg／ml的LPS处理16HBE细胞2h组STAT1、STAT4的mRNA表达较正常对照组有所增高（P〈0．05）;1μg/ml的LPS处理16HBE细胞1h组STAT6的mRNA表达较正常对照组显著增高（P〈0．01）。所有LPS处理16HBE细胞组STAT3的mRNA表达均较正常对照组减低。结论人支气管上皮细胞表达STAT1、STAT3、STAT4、STAT6的mRNA和STAT1、STAT4、STAT6的蛋白,一定剂量的脂多糖在某些时间点分别刺激了人支气管上皮细胞STAT1、sTAT4、STAT6的mRNA表达。     

EGF Modulates Expression of STAT5 in Mammary Epithelial Cells

HC11 mouse mammary epithelial cells are capable of differentiatingin vitro.By growing cells in EGF-containing medium, and upon confluence withdrawing EGF, these cells become competent at responding to lactogenic hormone treatment and expressing milk proteins. We found that during proliferation and at confluence STAT5A and STAT5B proteins were expressed at equal levels or with STAT5B being predominant. In competent cells, expression levels of STAT5A and STAT5B increased markedly with STAT5A now being the predominant form, an expression pattern resembling the expression patterns of STAT5 proteins seen during mammary gland differentiationin vivo.This suggests that EGF has a suppressive effect on STAT5 expression, in particular, STAT5A, which we conclude to be mediated through ras/raf/MEK/MAPK pathway and to a lesser extent through a PI3-kinase-mediated pathway. Furthermore, we also found that EGF regulated a nuclear phosphatase capable of dephosphorylating tyrosine-phosphorylated STAT5. Our data show that HC11 cells have retained the expression patterns of STAT5 proteins seenin vivo.This makes HC11 cells useful for studying molecular mechanisms regulating expression of STAT factors and their participation in differentiation processes of mammary gland.     

IL-22 Negatively Regulates Helicobacter pylori-Induced CCL20 Expression in Gastric Epithelial Cells

Helicobacter pylori is a Gram-negative bacterium that infects the human gastric mucosa and causes various gastric diseases. H. pylori infection induces the production of inflammatory chemokine CCL20 in gastric mucosa and leads to gastric inflammation. Given that the IL-22/IL-22R axis plays a critical role in the regulation of homeostasis and inflammation of epithelial cells at barrier surfaces, we investigated the effect of IL-22 on CCL20 expression induced by H. pylori. We demonstrated that H. pylori infection of the gastric epithelia-derived AGS cells significantly induced CCL20 expression and the induction was inhibited by IL-22. Functional analysis of the CCL20 promoter revealed that the H. pylori-induced CCL20 expression required the activation of NF-κB, and that IL-22 inhibited the induction by attenuating NF-κB activation. Knockdown of endogenous STAT3 by either short interfering RNAs or a short hairpin RNA significantly reduced the inhibitory effect of IL-22. Furthermore, STAT3 phosphorylation elicited by IL-22 was crucial for the inhibition of H. pylori-induced CCL20 expression. Consistent with the in vitro data showing that IL-22 negatively regulated H. pylori-induced CCL20 expression in gastric epithelial cells, studies on the tissue sections from patients with H. pylori infection also revealed an inverse association of IL-22 expression and CCL20 expression in vivo. Together, our findings suggest that IL-22 plays a role in the control of overproduction of the inflammatory chemokine and thus may protect the gastric mucosa from inflammation-mediated damage.