幽门螺杆菌感染与胃癌相关基因的筛选及预后分析

目的通过分析幽门螺杆菌感染胃黏膜组织和胃癌细胞系后的差异基因变化,并在癌症基因组图谱(The Cancer Genome Atlas,TCGA)数据库和肿瘤基因芯片(Oncomine)数据库进行验证,探究幽门螺杆菌导致胃癌发生、发展的分子机制。方法分析基因表达汇编(Gene Expression Omnibus,GEO)数据库幽门螺杆菌感染相关芯片集GSE5081与GSE70394,绘制维恩图查找幽门螺杆菌感染后共同上调的差异基因。对共同上调的差异基因进行功能富集分析。通过TCGA和Oncomine数据库验证差异基因在胃癌中的表达。利用Kaplan-Meier Plotter数据库和GEPIA数据库分析差异基因表达高低与胃癌患者预后是否存在相关性。结果通过差异基因筛选和维恩分析,两个芯片集共有21个共同上调差异基因。GO分析发现共同上调差异基因主要富集在对细菌来源分子的反应、趋化因子CXCR受体结合、中性粒细胞趋化作用等相关的基因功能上;KEGG通路主要富集在癌症通路、TNF信号通路、趋化因子信号通路等。STRING以及PPI数据库分析发现21个基因中PRDM1、IL10、NRP1、BIRC3、GNG13、CXCL1、CXCL2、CXCL3、CXCL8基因存在有网络关系,属于关键枢纽基因。通过TCGA和Oncomine数据库筛选及验证,发现在胃癌组织中NRP1、CXCL1、CXCL8基因明显上调,结果差异有统计学意义(TCGA数据库中,三者P值均小于0.05,Oncomine数据库中,NRP1:t=4.607,P0.01;CXCL1:t=5.854,P0.01;CXCL8:t=5.316,P0.01)。在Kaplan-Meier Plotter数据库(210615-at芯片:P0.01;210510-s-at芯片:P0.01;212298-at芯片:P0.01)以及GEPIA数据库(P0.01)两个数据库中,NRP1的高表达均与胃癌的预后负相关。结论不同的数据库均显示NRP1、CXCL1、CXCL8三个基因与幽门螺杆菌感染相关,同时在胃癌中高表达,并且NRP1的高表达与胃癌的不良预后相关,这些结果为进一步探究幽门螺杆菌导致胃癌发生、发展的分子机制提供了重要基础。     

温补脾肾法对脾肾阳虚型UC大鼠结肠组织趋化因子信号通路的影响

目的寻找脾肾阳虚型UC的特异性靶点。方法 96只Wistar大鼠随机分为模型组、高剂量组、中剂量组、低剂量组、SASP组,治疗组给予相应药物灌胃治疗。分别选取空白组大鼠结肠组织与模型组大鼠病变部位结肠组织进行高通量测序。RT-qPCR法检测筛选的趋化因子的基因表达。结果与模型组大鼠相比较,根据q-value≦0.05,fold-change≧1.5筛选出空白组大鼠差异表达的基因。通过GO功能分类分析显示,差异基因功能主要富集在生物过程(biological process,BP)、细胞成分(cellular component,CC)、分子功能(molecular function,MF)三个层面。通过差异基因KEGG富集分析发现趋化因子信号通路中CXCL1、CXCL2、CXCR2、CXCL6、CCL7、CCL12基因表达显著上调;并通过RT-qPCR法验证,以上因子的基因表达变化与测序结果一致,经温补脾肾方药治疗后,以上因子表达明显下调。结论脾肾阳虚型溃疡性结肠炎趋化因子信号通路中CXCL1、CXCL2、CXCR2、CXCL6、CCL7、CCL12基因表达显著上调,可作为UC黏膜炎症活动的客观指标。具有温补脾肾作用的理中汤合四神丸复方中药颗粒可以有效下调以上因子的表达,减缓炎症反应,促进受损伤的结肠黏膜的修复。     

胃癌组织中肿瘤相关成纤维细胞与正常成纤维细胞之间转录组学研究及验证

胃癌组织中肿瘤相关成纤维细胞(carcinoma associated fibroblasts, CAFs)是胃癌微环境的重要成分,主要来源于正常成纤维细胞(normal fibroblasts, NFs)的活化,对胃癌的发生发展有重要作用,但是两者之间的基因表达差异并不完全清楚。本研究选取从人胃癌组织中分离获得的CAFs及NFs 各3组,进行转录组学研究,筛选出3组细胞中交集且差异倍数较大的基因12个,用Omicsbean在线工具对差异基因进行Gene Ontology (GO)功能及KEGG通路富集,构建蛋白质相互作用调控网络;最后用RT-qPCR验证CAFs和NFs中差异基因的表达。结果显示,筛选出的12个差异表达基因主要参与NF-κB信号、炎症、细胞黏附、细胞表面受体和细胞因子等功能,上述功能均与肿瘤的发生发展密切相关。RT-qPCR检测发现,与NFs相比,CAFs中BCL2A1、NKX3-2、CXCL12、TNFAIP3、FOS、CDH4及CLDN1表达上调;ATF3、CYFIP2、CCL11、KLF2及GDF15基因表达下调,差异均具有统计学意义(P＜0.05)。结果提示,胃癌CAFs与NFs中存在肿瘤相关的差异表达基因,这些差异基因可能在胃癌微环境中发挥重要作用。     

龋病感染牙髓相关基因的筛选与生物信息学分析

目的:综合应用生物信息学技术,从分子水平对龋坏牙髓与正常牙髓的差异基因进行筛选分析,初步探讨其作用机制。方法:从GEO基因表达数据库中下载龋坏牙髓相关芯片数据集,采用MORPHEUS在线筛选差异表达基因,结合DAIVID、STRING等在线分析工具对差异表达基因进行GO功能富集分析及KEGG通路分析,后用Cytoscape软件构建蛋白质相互作用网络。结果:共筛选出375个差异表达基因,其中表达上调253个、下调122个,主要涉及免疫应答、炎症反应、细胞因子应答和生物矿化组织发育等生物过程,以及抗原加工提呈和NF-κB信号等生物通路。通过蛋白质互作网络构建分析发现,MMP9、IL-8、PTPRC、CXCR4等10个基因处于核心节点位置。结论:借助生物信息学方法能得到可靠的相关差异基因信息,能够有效指导进一步的研究。得到的差异基因可以作为龋病诊断的指示因子和机制研究的候选基因。     

幽门螺杆菌感染促进胃炎儿童胃粘膜细胞的增殖

本研究旨在探讨幽门螺杆菌感染对小儿慢性胃炎患者细胞增殖的影响,使用内镜检查消化不良患者的上消化道症状,使用改良的Giemsa染色检测胃粘膜活组织中幽门螺杆菌,用苏木精/曙红和改良的吉姆萨染色活组织,并通过光学显微镜研究染色后胃粘膜样品组织病理学变化,RT-PCR检测各组胃粘膜细胞中调控细胞凋亡的Bcl-2、Bcl-xl、Bax和PCNA的mRNA表达,提取胃粘膜细胞蛋白质,利用蛋白质免疫印迹分析蛋白质浓度。组织化学染色结果表明,与对照相比,患有胃炎和幽门杆菌感染后的胃炎患者胃粘膜细胞明显增加,且幽门螺杆菌感染后细胞增殖更显著(p<0.05);幽门螺杆菌感染后Bcl-2和Bcl-xl,PCNA在患者体内表达显著上调(p<0.05),而细胞促凋亡因子Bax基因在胃炎患者感染幽门螺杆菌后被显著下调(p<0.05),蛋白免疫印迹分析Bcl-2,Bcl-xl,Bax和PCNA蛋白表达趋势与基因表达一致,说明结果可靠。幽门螺杆菌感染会显著提高慢性胃炎儿童患者胃粘膜细胞的增殖。     

肝血窦内皮细胞介导的他莫昔芬对非酒精性脂肪性肝炎治疗作用的转录组学分析

摘要 目的：探究他莫昔芬对饮食诱导的非酒精性脂肪性肝炎小鼠肝血窦内皮细胞的代谢、炎症及纤维化等通路基因表达的影响。方法：采用8周龄的雄性C57BL/6小鼠,给予MCD饲料喂养6周后,每天腹腔注射一次他莫昔芬（100 mg/kg）,持续5天。分离并收集肝血窦内皮细胞,加入1 mL TRIzol试剂吹打至沉淀消失,放入-80℃冰箱保存。样本后续送至基迪奥生物公司进行转录组测序并在Omicsmart平台进行生物信息学分析。部分生物信息学分析数据来自已经发表的文献并通过Omicshare分析平台分析。结果：转录组测序发现,差异基因KEGG和GO分析发现差异基因在免疫和炎症通路富集。通过分析肝脏内皮特异性代谢基因表达,我们发现他莫昔芬治疗逆转了NASH过程中部分代谢基因的下调,以及NASH过程中CCL2、CXCL2、CXCL5和VCAM-1等促炎基因和Col1a1、Col1a2、Col3a1、Tgfb2、和Timp1等促纤维化基因的表达上调。同时,GSEA分析也显示他莫昔芬抑制了炎症和纤维化通路的表达。结论：他莫昔芬可能通过逆转非酒精性脂肪性肝炎对小鼠肝血窦内皮细胞代谢基因的改变以及炎症及纤维化相关基因的上调来治疗非酒精性脂肪性肝炎。     

京海黄鸡柔嫩艾美耳球虫感染后盲肠转录组分析

为了探究京海黄鸡感染柔嫩艾美尔球虫(E. tenella)后盲肠组织差异表达基因,以及球虫感染分子应答过程和免疫应答机制,试验采用RNA-seq技术对E. tenella感染和非感染组第7天的盲肠组织进行转录组测序,筛选差异表达基因,并进行差异基因的功能、通路富集分析。结果表明,在感染和非感染组中有显著差异的表达基因2 830个(P0.05),其中1 419个基因上调,1 411个基因下调。随机选取10个差异基因进行qRT-PCR验证,结果显示差异基因的表达倍数与RNA-seq检测结果显著相关(r=0.988,P0.000),决定系数达0.975。GO分析表明,有2 356个差异基因获得GO功能注释,显著富集的前30个GO terms主要涉及细胞交流、信号转导、血管生成、氧化还原酶活性等。KEGG分析发现差异基因显著富集的信号通路有黏着斑、细胞外基质-受体相互作用、过氧化物酶体增殖物激活受体等。这些通路中的差异基因有ANGPTL4、ACSL5、VEGFC、CD44和MAKP10等,提示这些基因在宿主柔嫩艾美耳球虫感染过程中发挥重要作用。     

SYBR Green实时荧光定量PCR快速检测口腔幽门螺杆菌

目的建立一种快速、灵敏、特异的鉴定幽门螺杆菌实时荧光定量PCR方法。方法利用SYBR Green实时荧光定量PCR反应体系对口腔幽门螺杆菌进行检测。鉴定结果与临床常规鉴定方法相比较,评价其敏感度、特异度及重复性。结果通过48例样品的检测,结果显示实时荧光定量PCR法检测标本的鉴定结果与常规PCR鉴定方法的结果对比,特异度为100%,敏感度为100%;最小能检测到102个拷贝数的重组质粒;批内重复试验和批间重复试验结果均与常规鉴定方法结果相符。结论实时荧光定量PCR法鉴定口腔幽门螺杆菌,特异度和敏感度高,重复性好,且快速、简便。该方法有望成为检测口腔幽门螺杆菌感染的一种快速有效的方法。     

幽门螺杆菌抗生素耐药机制研究进展

幽门螺杆菌(Helicobacter pylori,H.pylori)感染可引起消化性溃疡、胃粘膜相关淋巴组织淋巴瘤和胃癌。随着抗生素耐药性的问题越来越严重,耐药机制的研究也不断深入。分子检测方法,尤其是核酸检测技术,可高效、快速、准确地检测幽门螺杆菌抗生素耐药基因及突变,对幽门螺杆菌感染的临床治疗发挥重要的指导作用,同时也可对幽门螺杆菌抗生素耐药性进行大规模及时有效监控。本文讨论了关于幽门螺杆菌抗生素耐药机制并着重总结了相关耐药基因及突变。     

鸡miR-9不同组织表达差异及其功能预测分析

miRNA在动物生长发育过程中有重要作用. 本文采用定制茎环反转录引物,利用实时荧光定量PCR技术构建miR-9在鸡2个阶段11个组织中的表达谱,同时用TargetScan5.1与PicTar两种计算方法对其进行靶基因预测,交集的基因集合分别进行GO（gene ontology）富集分析和生物通路富集分析. 结果表明,采用实时定量PCR检测的miR-9在鸡下丘脑中的表达量和高通量测序结果一致;采用实时定量PCR在对不同组织定量结果表明,miR-9在0日龄鸡的肾脏、下丘脑、腿肌和大脑中高丰度表达,在成年鸡表达量较高为大脑、腿肌、心脏、小脑和下丘脑.在同一组织的0日龄和成年鸡中表达呈现时序性,除肝脏的表达量差异不显著,其他10个组织miR-9表达量差异显著（P<0.05）.预测到交集靶基因有160个.涉及到多个KEGG通路和GO富集中.GO分类结果显示,这些基因分布于63个群中,其中基因超过45个基因群集有26个群,与代谢有关的群有11个. 其它与发育、调控等过程有关. 在KEGG通路分析中,显著的通路有细胞骨架调控、细胞增殖和分化有关的通路（P<0.01）等5个通路. 表明miR 9基因的表达有组织和时序特异性,靶基因参与细胞代谢、生长发育和调控.这些结果为进一步验证miR 9基因在在脑中调控生长发育过程中的作用奠定了基础.     

Inflammatory gene profiles in gastric mucosa during Helicobacter pylori infection in humans

Helicobacter pylori infection is associated with an inflammatory response in the gastric mucosa, ultimately leading to cellular hyperproliferation and malignant transformation. Hitherto, only expression of a single gene, or a limited number of genes, has been investigated in infected patients. cDNA arrays were therefore used to establish the global pattern of gene expression in gastric tissue of healthy subjects and of H. pylori-infected patients. Two main gene expression profiles were identified based on cluster analysis. The data obtained suggest a strong involvement of selected Toll-like receptors, adhesion molecules, chemokines, and ILs in the mucosal response. This pattern is clearly different from that observed using gastric epithelial cell lines infected in vitro with H. pylori. The presence of a "Helicobacter-infection signature," i.e., a set of genes that are up-regulated in biopsies from H. pylori-infected patients, could be derived from this analysis. The genotype of the bacteria (presence of genes encoding cytotoxin-associated Ag, vacuolating cytotoxin, and blood group Ag-binding adhesin) was analyzed by PCR and shown to be associated with differential expression of a subset of genes, but not the general gene expression pattern. The expression data of the array hybridization was confirmed by quantitative real-time PCR assays. Future studies may help identify gene expression patterns predictive of complications of the infection.     

Towards in situ tissue repair: human mesenchymal stem cells express chemokine receptors CXCR1, CXCR2 and CCR2, and migrate upon stimulation with CXCL8 but not CCL2

The recruitment of bone marrow CD34- mesenchymal stem- and progenitor cells (MSC) and their subsequent differentiation into distinct tissues is the precondition for in situ tissue engineering. The objective of this study was to determine the entire chemokine receptor expression profile of human MSC and to investigate their chemotactic response to the selected chemokines CCL2, CXCL8 and CXCL12. Human MSC were isolated from iliac crest bone marrow aspirates and showed a homogeneous population presenting a typical MSC-related cell surface antigen profile (CD14-, CD34-, CD44+, CD45-, CD166+, SH-2+). The expression profile of all 18 chemokine receptors was determined by real-time PCR and immunohistochemistry. Both methods consistently demonstrated that MSC express CC, CXC, C and CX(3)C receptors. Gene expression and immunohistochemical analysis documented that MSC express chemokine receptors CCR2, CCR8, CXCR1, CXCR2 and CXCR3. A dose-dependent chemotactic activity of CXCR4 and CXCR1/CXCR2 ligands CXCL12 and CXCL8 (interleukin-8) was demonstrated using a 96-well chemotaxis assay. In contrast, the CCR2 ligand CCL2 (monocyte chemoattractant protein-1, MCP-1) did not recruited human MSC. In conclusion, we report that the chemokine receptor expression profile of human MSC is much broader than known before. Furthermore, for the first time, we demonstrate that human MSC migrate upon stimulation with CXCL8 but not CCL2. In combination with already known data on MSC recruitment and differentiation these are promising results towards in situ regenerative medicine approaches based on guiding of MSC to sites of degenerated tissues.     

Gene expression analysis of host innate immune responses during Lethal H5N1 infection in ferrets

How viral and host factors contribute to the severe pathogenicity of the H5N1 subtype of avian influenza virus infection in humans is poorly understood. We identified three clusters of differentially expressed innate immune response genes in lungs from H5N1 (A/Vietnam/1203/04) influenza virus-infected ferrets by oligonucleotide microarray analysis. Interferon response genes were more strongly expressed in H5N1-infected ferret lungs than in lungs from ferrets infected with the less pathogenic H3N2 subtype. In particular, robust CXCL10 gene expression in H5N1-infected ferrets led us to test the pathogenic role of signaling via CXCL10's cognate receptor, CXCR3, during H5N1 influenza virus infection. Treatment of H5N1-infected ferrets with the drug AMG487, a CXCR3 antagonist, resulted in a reduction of symptom severity and delayed mortality compared to vehicle treatment. We contend that unregulated host interferon responses are at least partially responsible for the severity of H5N1 infection and provide evidence that attenuating the CXCR3 signaling pathway improves the clinical course of H5N1 infection in ferrets.     

Chemokines determine local lymphoneogenesis and a reduction of circulating CXCR4+ T and CCR7 B and T lymphocytes in thyroid autoimmune diseases

Chemokines and their corresponding receptors are crucial for the recruitment of lymphocytes into the lymphoid organs and for its organization acting in a multistep process. Tissues affected by autoimmune disease often contain ectopic lymphoid follicles which, in the case of autoimmune thyroid disorders, are highly active and specific for thyroid Ags although its pathogenic role remains unclear. To understand the genesis of these lymphoid follicles, the expression of relevant cytokines and chemokines was assessed by real time PCR, immunohistochemistry and by in vitro assays in autoimmune and nonautoimmune thyroid glands. Lymphotoxin alpha, lymphotoxin beta, C-C chemokine ligand (CCL) 21, CXC chemokine ligand (CXCL) 12, CXCL13, and CCL22 were increased in thyroids from autoimmune patients, whereas CXCL12, CXCL13, and CCL22 levels were significantly higher in autoimmune glands with ectopic secondary lymphoid follicles than in those without follicles. Interestingly, thyroid epithelium produced CXCL12 in response to proinflammatory cytokines providing a possible clue for the understanding of how tissue stress may lead to ectopic follicle formation. The finding of a correlation between chemokines and thyroid autoantibodies further suggests that intrathyroidal germinal centers play a significant role in the autoimmune response. Unexpectedly, the percentage of circulating CXCR4(+) T cells and CCR7(+) B and T cells (but not of CXCR5) was significantly reduced in PBMCs of patients with autoimmune thyroid disease when they were compared with their intrathyroidal lymphocytes. This systemic effect of active intrathyroidal lymphoid tissue emerges as a possible new marker of thyroid autoimmune disease activity.     

Migration potential and gene expression profile of human mesenchymal stem cells induced by CCL25

Recruitment of mesenchymal stem cells (MSC) to tissue damages is a promising approach for in situ tissue regeneration. The physiological mechanisms and regulatory processes of MSC trafficking to injured tissue remain poorly understood. However, the pivotal role of chemokines in MSC recruitment has already been shown.The aim of this study was to determine the migratory potential and the gene expression profile of MSC stimulated with the CC chemokine CCL25 (TECK). Bone marrow derived human MSC were exposed to different doses of CCL25 in a standardized chemotaxis assay. Microarray gene expression profiling and pathway analysis were performed for CCL25 stimulated MSC.Maximum migration of MSC towards CCL25 was observed at 10³ nM. Microarray analysis revealed an induction of molecules directly involved in chemotaxis and homing of bone marrow cells (CXCL1-3, CXCL8, PDE4B), cytoskeletal and membrane reorganisation (CXCL8, PLD1, IGFBP1), cellular polarity (PLD1), and cell movement (CXCL1-3, CXCL6, CXCL8, PTGS2, PDE4B, TGM2). Respective chemokine secretion was confirmed by protein membrane-array analysis. The activation of CXCR2 ligands (CXCL1-3, CXCL5-6, CXCL8) and a LIF-receptor/gp130 ligand (LIF) indicated an involvement of the respective signaling pathways during initiation of chemotaxis and migration.These results suggest CCL25 as a new potential candidate for further in situ regeneration approaches.