应用抑制消减杂交和cDNA芯片技术筛选流行性感冒病毒感染相关基因

病毒基因组有限的编码能力和以病毒蛋白为靶的抗病毒药物易出现耐药性,使从病毒感染宿主筛选病毒感染相关生物大分子作为抗病毒药靶和诊断标志物成为新的研究方向。为了筛选流行性感冒（流感）病毒感染相关基因,采用抑制消减杂交（suppression subtractive hybridization,SSH)技术,以流感病毒A／鲁防／93－9（H3N2）感染的MDCK细胞及正常MDCK细胞为材料,构建病毒感染特异性差减cDNA文库。从文库中随机挑取约800个克隆,PCR扩增其中插入片段,经纯化、紫外定量后,用基因芯片自动点样仪点在氨基片上,制备cDNA芯片。将流感病毒感染的MDCK细胞和正常MDCK细胞的总RNA分别用Cy3、Cy5反转录荧光标记后,与cDNA芯片杂交,用芯片扫描仪扫描获得芯片杂交信号,经阳性对照校正和归一化处理后,以如下条件作为判定基因差异表达的标准;（a)Cy3与Cy5的信号比值大于1．5（正常细胞用Cy5标记）或小于0．67（正常细胞用Cy3标记）;（b)Cy3和Cy5信号值之一必须大于1000。经cDNA芯片筛选获得了18个流感病毒感染特异性克隆,经测序和生物信息学分析发现均为流感病毒感染相关新基因EST。流感病毒感染相关基因cDNA片段的获得,为新型病毒药靶诊断标志物发现和功能研究提供了基础。     

利用基因芯片技术筛选HIV-1F亚型基因限制性显示探针

为筛选限制性显示技术制备的HIV 1F亚型基因探针 ,应用基因芯片打印仪将其有序地打印在玻片上制备基因芯片 .在随机引物延伸的过程中进行HIV样品的荧光标记 ,然后与芯片进行杂交 .杂交后清洗玻片并干燥 ,对芯片进行扫描 ,分析各探针的杂交信号 .从中筛选了 14个基因片段作为芯片下一步研究的探针 .实验证明 ,限制性显示技术是一种制备基因芯片探针的实用方法     

一种标记cDNA芯片探针的新方法

探讨mRNA长片段反转录PCR技术(RT-LDPCR)在cDNA芯片微量探针标记和信号放大中的应用.首先提取BEP2D细胞的总RNA,然后用两种不同的方法进行标记,一种为RT-LDPCR,用荧光素Cy3-dCTP进行标记;另一种为传统的RNA反转录,用荧光素Cy5-dCTP进行标记.将两种方法标记好的探针等量混合后与含有440个点(44个基因)的cDNA芯片同时杂交,发现二者具有很高的一致性(0.5＜Cy3/Cy5＞2.0).由于RNA反转录法为cDNA芯片探针标记的传统方法,从而验证了RT-LDPCR用于cDNA芯片探针标记的可行性.RT-LDPCR具有对样品总RNA的需要量少和可对样品中信号进行放大的优点,特别适合于对材料来源受到限制的RNA进行标记.     

五倍子小鼠肝脏毒性的基因表达谱分析

昆明种小鼠（雄性,体重20±2g）随机分为正常对照组和五倍子给药组。给药组每日灌胃五倍子提取物（0．2mL／10g体重,相当于8g五倍子生药／1kg体重）一次,连续给药30天。对照组灌胃等量生理盐水。之后分别提取两组小鼠的肝组织总mRNA,经反转录分别用Cy3,Cy5荧光标记．制备用于芯片杂交的cDNA探针。两种探针等量混合后与小鼠全基因组寡核苷酸芯片杂交．杂交信号经芯片扫描仪获取并用GenePix Pro4．0软件分析。结果表明,五倍子给药组中有461条基因差异表达,其中上调基因267条,下调基因194条,功能已知基因373条,功能未知基因88条。通过对这些差异表达基因的生物学功能及生物通路分析,它们主要涉及代谢、DNA结合与转录、蛋白质合成与修饰、细胞骨架及黏附因子、细胞周期与分化、离子通道与受体、信号转导、免疫、细胞凋亡等。上述研究结果对分析五倍子肝损伤机制可能具有十分重要的作用。     

Affymetrix基因表达谱芯片在新疆维吾尔族与汉族胰腺癌组织样本间差异表达基因筛选中的运用

目的:运用基因表达谱芯片筛选并分析新疆维吾尔族与汉族胰腺癌组织样本间的差异表达基因。方法:收集我院2014年1月至2016年6月间行手术切除的维吾尔族与汉族胰腺导管细胞癌组织并提取总RNA,选取经Nanodrop 2000与Agilent 2100仪器质检合格的样本总RNA采用Affymetrix基因表达谱芯片筛选出差异表达基因并绘制统计图,运用基因本体(GO)分析及信号通路(Pathway)分析对这些差异表达基因的生物信息进行汇总分析。结果:通过基因表达谱芯片分析,新疆维吾尔族与汉族胰腺癌组织样本间共检测到1063个基因存在差异表达,在维吾尔族胰腺癌标本中显著上调表达的基因共281个,差异表达倍数最高的为IGLV1-44基因(差异倍数:9.99)下调表达的基因共782个,差异表达倍数最高的为CPB1基因(差异倍数:33.76);在Gene Ontology数据库中共检索到815个上述差异表达基因具有明确的GO分类,差异表达倍数最高的为CPB1基因(差异倍数:33.76);Pathway分析中共检测到30条信号通路包含有上述差异表达基因,共涉及196个基因,其中以FAK信号通路差异表达基因富集程度最高,差异表达倍数最高的基因为COL11A1基因(差异倍数:5.02)。结论:基因表达谱芯片分析结果显示,在新疆维吾尔族与汉族胰腺癌组织样本间存在大量的差异表达基因,这些基因与胰腺癌的增殖分化、侵袭转移及多药耐药等特性密切相关,且参与了多条生物体内重要信号转导通路的调控。     

cDNA微阵列技术研究NaHCO3胁迫下星星草基因表达谱

为研究NaHCO3胁迫下星星草基因的表达,分别将荧光染料Cy5-dCTP和Cy3-dCTP用反转录方法标记在处理和对照星星草cDNA上制成探针,并与载有星星草基因的cDNA芯片进行杂交。通过对芯片的杂交信号强度分析来研究基因的表达情况。分析结果显示,共有25个基因在NaHCO3胁迫处理前后差异表达,其中17个基因在NaHCO3胁迫下表达下调,8个基因在NaHCO3胁迫下表达上调。生物信息学分析表明这些基因的功能涉及了信号传导与转录调控、细胞防御、细胞代谢等多个方面。从而获得了NaHCO3胁迫下星星草的基因表达谱,定量地阐述了NaHCO3胁迫和非胁迫条件下星星草基因的差异表达情况。     

基因芯片技术研究柽柳NaHCO3胁迫下基因的表达

运用基因芯片技术研究了NaHCO3胁迫下柽柳(Tamarix androssowii)基因的表达.将Cy5和Cy3两种荧光染料分别标记在NaHCO3处理和对照的柽柳cDNA上,将两种荧光探针混合,与载有柽柳基因的高密度芯片进行杂交并用芯片扫描系统进行扫描,通过Cy5与Cy3信号强度比值的计算研究基因的差异表达.共获得了89个差异表达的基因,其中,27个下调表达,62个上调表达.BlastX分析表明这些基因按功能可以分为光合作用、活性氧清除、渗透调节、信号传导与表达调控、代谢、发育相关、核糖体蛋白、蛋白质的分解与再生、转运类蛋白、水通道蛋白等几大类别.同时,发现了一些与盐胁迫相关的功能未知基因或未有任何功能信息的基因,这些基因可能在柽柳抗盐过程中具有重要作用.揭示了柽柳的抗盐胁迫涉及的几种重要途径,并获得了NaHCO3胁迫前后柽柳基因表达谱.     

鼻咽癌组织中基因差异表达及蛋白互作网络分析

目的:利用生物信息学方法从鼻咽癌组织基因芯片中筛选差异表达基因,并分析它们之间的互相作用。方法:利用R语言程序包等相关软件分析鼻咽癌组织和正常组织中差异表达的基因,并对其进行在线GO分析、KEGG富集分析及蛋白互作分析。结果:从25例鼻咽癌组织样本和3例正常组织对照样本中共分析出1103个差异表达基因,包括600个上调基因和503个下调基因(P0.05)。GO分析结果显示,差异表达基因在生物过程中显著富集,包括细胞分裂、DNA复制、G1/S有丝分裂细胞周期的转变和有丝分裂核分裂;在分子功能中,包括与蛋白质结合、与ATP结合、蛋白同二聚化活性、与DNA复制起点结合以及与受损的DNA结合;在细胞组分中,包括细胞质、核质、胞外体和船体中部。KEGG通路分析显示,差异表达基因在细胞周期、DNA复制、癌症途径、p53信号通路、错配修复、小细胞肺癌、卵母细胞减数分裂、氨基糖和核苷酸糖代谢、基部切除修复和人T淋巴细胞病毒(HTLV-Ⅰ)感染等信号通路中显著富集。在蛋白互作分析网络中筛选出10个节点度最高核心基因CDC45、MCM10、MCM3、MCM5、MCM2、CDC7、CDT1、CDC6、SMC2和NCAPG。结论:通过鼻咽癌组织和正常组织的对比筛选,发现多个基因表达发生变化,为相关临床研究提供了重要的信息基础。     

汉坦病毒感染细胞整合素受体信号转导相关差异表达基因筛选

通过基因芯片技术观察汉坦病毒感染细胞后整合素受体信号转导相关基因的差异表达,以探讨汉坦病毒进入细胞的分子机制.用汉滩病毒76-118株感染原代人胚肺成纤维细胞,同时设立正常对照细胞,于感染后第6h、24h和96h同时抽提感染组与正常对照组细胞总RNA,利用荧光标记dUTP逆转录制备cDNA探针,与人类受体及信号转导类表达谱芯片杂交,并对Cy3、Cy5荧光信号做扫描分析.结果显示有37项基因出现差异表达,其中31项基因在感染后6h出现差异表达,13项下调、18项上调;但在感染后24h和96h分别只有5项和4项基因出现表达差异;而且整合素信号转导通路中的重要基因如一些蛋白激酶相关基因在感染6h表达增强,丝化增强子1在感染6h和96h表达下调,微管相关蛋白1A在感染6h表达上调、感染24h和96h表达下调.这些进一步说明汉坦病毒感染细胞与整合素有关.     

肾透明细胞癌Caki-1细胞系差异表达基因的生物信息学分析

目的比较肾透明细胞癌Caki-1细胞系与正常肾上皮细胞系ASE-5063中的差异表达基因（DEGs）,寻找潜在的肾透明细胞癌特异性分子标志物。 方法利用GEO数据库自带的GEO2R在线分析工具分析基因芯片GSE78179,将筛选出的DEGs分别导入Metascape、STRING以及Cytoscape进行综合分析并筛选出核心基因。最后使用FunRich等软件对筛选出的核心基因进行GO和KEGG富集分析。 结果共筛选出562个DEGs,其中上调基因345个,下调基因217个。进一步使用MCODE筛选出36个关键基因,GO功能分析发现这些基因与细胞粘附分子活性、趋化因子活性、细胞通讯和信号转导等密切相关;KEGG通路富集结果则表明差异基因主要集中在趋化因子信号通路、TNF信号通路以及NF-κB信号通路等多种与肿瘤相关的通路上。 结论运用生物信息学方法筛选出肾透明细胞癌Caki-1细胞系中DEGs,其中数个核心基因广泛参与多种肿瘤的病理进程,但尚未在肾透明细胞癌有相关研究报道,提示其可能是治疗肾透明细胞癌的潜在靶点。     

Gene expression profile in immunologically injured liver cell of mice

To study the gene expression profiles between immunologically injured liver cell and normal liver cell of mice and to screen on a large scale the differentially expressed genes associated with the formation of liver injury, the experimental mice were randomly divided into the normal group for controlling and the immunologically liver-injured group induced by BCG and LPS. The liver mRNA of the two groups were extracted respectively and reversely-transcribed to cDNA with the incorporation of different fluorescence (Cy3, Cy5) labeled dUTP as the hybridization probes. The mixed probes were hybridized to the cDNA microarray chips. The fluorescent signal results were acquired by scanner ScanArray 4000 and analyzed with software GenePix Pro 3.0. Among the 14112 target genes, 293 genes were found to be significantly differentially expressed, in which 188 genes were up-regulated and 105 genes were down-regulated. Based on the analysis of biological functions of those differentially expressed genes, it was indicated that the occurrence and development of mouse liver damage induced by BCG and LPS were highly correlated with the processes of immune reactions, cell synthesis, metabolism, apoptosis and transportation in liver cell, which might be quite important for elucidating the regulatory network of gene expression associated with the liver damage, also important for finally discovering the pathogenic mechanisms of immunological liver damage.     

Gene expression profile in immunologically injured liver cell of mice

To study the gene expression profiles between immunologically injured liver cell and normal liver cell of mice and to screen on a large scale the differentially expressed genes associated with the formation of liver injury,the experimental mice were randomly divided into the normal group for controlling and the immunologically liver-injured group induced by BCG and LPS.The liver mRNA of the two groups were extracted respectively and reversely-transcribed to cDNA with the incorpora-tion of different fluorescence(Cy3,Cy5) labeled dUTP as the hybridization probes.The mixed probes were hybridized to the cDNA microarray chips.The fluorescent signal results were acquired by scanner ScanArray 4000 and analyzed with software GenePix Pro 3.0.Among the 14112 target genes,293 genes were found to be significantly differentially expressed,in which 188 genes were up-regulated and 105 genes were down-regulated.Based on the analysis of biological functions of those differentially expressed genes,it was indicated that the occurrence and development of mouse liver damage induced by BCG and LPS were highly correlated with the processes of immune reac-tions,cell synthesis,metabolism,apoptosis and transportation in liver cell,which might be quite im-portant for elucidating the regulatory network of gene expression associated with the liver damage,also important for finally discovering the pathogenic mechanisms of immunological liver damage.     

肾透明细胞癌预后相关miRNAs的生物信息学分析

目的寻找可作为肾透明细胞癌（ccRCC）生物标志物的miRNA,以及ccRCC与正常组织间miRNA差异表达情况。 方法利用TCGA数据库下载ccRCC中miRNA表达数据,分析肿瘤与正常组织间差异表达miRNA。使用Kaplan-Meier曲线对患者进行生存分析,筛选出表达情况与临床预后相关的miRNA。通过生物信息学对miRNA的靶基因进行预测,然后运用FunRich软件和ClueGO对靶基因进行GO和KEGG富集分析。 结果通过TCGA数据库分析发现,ccRCC较正常组织差异表达miRNA共54个,其中上调33个,下调21个。通过生存分析发现hsa-miR-21和hsa-miR-155与患者预后相关,P≤0.05。进一步通过Perl软件在Targetscan、miRDB、miRTarBase、miRPath这四个数据库中预测miRNA靶基因并将结果取交集,共发现129个靶基因。GO和KEGG分析结果表明,这些靶基因主要与转录因子活性、信号转导以及FoxO、TNF等信号通路密切相关。 结论通过生物信息学分析发现了ccRCC与正常组织的差异表达miRNA;其中hsa-miR-21和hsa-miR-155与患者总体生存率相关,并通过调控靶基因参与相关的信号通路进而影响ccRCC的发生发展进程,提示hsa-miR-21和hsa-miR-155可能是ccRCC潜在的生物标志物。     

用基因表达谱芯片研究人正常肝和肝细胞癌中差异表达的基因

以基因表达谱芯片对人正常肝及肝癌组织基因表达的差异性进行了研究比较。奖４０９６条人ｃＤＮＡ用点样仪点在特制玻片上制备成表达谱芯片;利用肝和肝癌组织的ｍＲＮＡ通过逆转录方法,将Ｃｙ３和Ｃｙ５２种荧光分别标记到两种组织的ｃＤＮＡ上,制备成ｃＤＮＡ探针,并与表达谱芯片进行杂交及扫描,重复４次实验,通过计算机数据处理判定基因是否在上述２种组织中有表达差异,筛选出差异表达的基因共９０３条。基因芯片技术可同时     

Expression profiling by whole-genome microarray hybridization reveals differential gene expression in breast cancer cell lines after lycopene exposure

The correlation between diet and variation in gene-expression is an important field which could be considered to approach cancer pathways comprehension. We examined the effects of lycopene on breast cancer cell lines using pangenomic arrays. Lycopene is derived predominantly from tomatoes and tomato products and there is some epidemiologic evidence for a preventive role in breast cancer. Previously, we investigated lycopene in breast cancer using a dedicated breast cancer microarray. To confirm these results and explore pathways other than those implicated in breast cancer, for this study we used pangenomic arrays containing 25,000 oligonucleotides. This in vitro study assayed two human mammary cancer cell lines (MCF-7 and MDA-MB-231), and a fibrocystic breast cell line (MCF-10a) treated or not with 10 microM lycopene for 48 h. A competitive hybridization was performed between Cy3-labeled lycopene treated RNA and Cy5-labeled untreated RNA to define differentially expressed genes. Using t-test analysis, a subset of 391 genes was found to be differentially modulated by lycopene between estrogen-positive cells (MCF-7) and estrogen-negative cells (MDA-MB-231, MCF-10a). Hierarchical clustering revealed 726 discriminatory genes between breast cancer cell lines (MCF-7, MDA-MB-231) and the fibrocystic breast cell line (MCF-10a). Modified gene expression was observed in various molecular pathways, such as apoptosis, cell communication, MAPK and cell cycle as well as xenobiotic metabolism, fatty acid biosynthesis and gap junctional intercellular communication.