人主动脉夹层与正常主动脉基因表达谱的差异

目的:利用基因芯片技术研究人主动脉夹层组织与正常主动脉组织基因表达谱的差异。方法:选取主动脉夹层病例标本5例,正常主动脉标本4例。提取总RNA,并反转录成cDNA、体外转录合成aRNA后与芯片进行杂交,对结果进行分析。同时对表达谱筛选出MYLK、PKD1、MYH11、SOD3、FLNA、TAGLN 6个差异基因进行基因转录水平的定量验证。结果:人主动脉夹层组与正常主动脉组比较基因表达差异倍数大于2的基因共有1 661个,其中有997个基因上调表达,664个基因下调表达。6个基因RT-qPCR验证结果表明,与正常组相比,6个基因都下调表达,其中MYLK、PKD1、MYH11、SOD3、TAGLN基因下调表达是有显著性差异的(P≤0.05),FLNA基因没有显著性差异(P0.05)。结论:通过全基因组表达谱芯片可以筛选主动脉夹层与正常主动脉表达差异基因,同时结合RT-qPCR验证,为研究主动脉夹层提供新的思路。     

应用基因芯片技术研究原发性胆汁性肝硬化患者外周血单个核细胞基因表达谱特征

目的 应用基因芯片技术分析原发性胆汁性肝硬化患者外周血单个核细胞的基因表达谱特征.方法 选取9例原发性胆汁性肝硬化患者、9名健康对照为研究对象,提取其外周血单个核细胞总RNA,进行人类全基因组寡核苷酸芯片(约22000个基因)检测,筛选出差异表达基因及相关信号通路.结果 原发性胆汁性肝硬化患者外周血单个核细胞中差异表达的基因共有79个,其中21个表达上调,58个表达下调.这些基因对应着27条信号通路,其中有6条通路参与了免疫调控及细胞凋亡过程,分别是自然杀伤细胞介导的细胞毒通路、Toll样受体信号通路、抗原加工提呈通路、细胞因子相互作用通路、T细胞受体信号通路、细胞凋亡通路.结论 原发性胆汁性肝硬化患者外周血单个核细胞中存在特有的差异表达基因,针对这些基因及相关信号通路的进一步研究,将为探索发病机制和寻找生物标志物提供新的方向.     

基因组水平筛选冠状动脉病变部位参与急性心肌梗死急性期的信号通路

目的利用基因芯片技术筛选冠状动脉病变部位参与急性心肌梗死急性期过程的信号通路,探讨急性冠脉综合征斑块破裂病变的发生机制。方法收集急性心肌梗死患者急性期和冠状动脉造影结果正常者的冠状动脉血,用人类基因组U133A寡核苷酸芯片筛选差异表达基因,然后采用DAVID功能注释生物信息学芯片分析系统对2倍差异表达基因进行基因富集分析,并用定量多聚酶连反应（RT-qPCR）验证化学因子信号通路部分筛选基因的表达。结果与冠状动脉造影正常者相比,急性心肌梗死患者在急性期的2倍上调基因有2897个,2倍下调基因有1974个。GO信号通路富集分析显示96个信号通路参与急性心肌梗死急性期过程（BenjaminiP〉0.01）。KEGG信号通路富集分析显示6个信号通路参与急性心肌梗死急性期过程（BenjaminiP〉0.01）。选择化学因子信号通路6个上下游基因进行验证,与芯片结果高度一致。其中CXCR1、CXCR2、Src、Raf和ERK1/2基因在急性期表达显著增高,与对照组比较,差异有统计学意义（P〈0.001）,而PI3K表达也有增高的趋势。结论在急性心肌梗死急性期过程中有许多信号通路参与,其中Chemokine信号通路与其发病机制密切相关。     

基因芯片联合荧光定量PCR筛查主动脉夹层肺损伤标志物

目的:使用人表达谱基因芯片筛查主动脉夹层外周血白细胞差异基因表达,用荧光定量PCR进行验证主动脉夹层肺损伤标志基因。方法:分别抽取主动脉夹层患者和主动脉瘤患者外周血白细胞RNA,用基因芯片筛查差异表达基因,选取与肺损伤相关的基因。抽取主动脉夹层肺损伤和非肺损伤患者外周血白细胞RNA,用荧光定量PCR进行验证。结果:差异表达基因134个,其中78个为上调基因,56个为下调基因,差异基因主要涉及炎症反应、免疫反应、宿主防御、胶原蛋白调节、应激反应以及造血和细胞凋亡等多个生物学过程,用荧光定量PCR验证肺损伤相关基因,发现基质金属蛋白酶(MMP)-9在主动脉夹层肺损伤组明显升高(P<0.05)。结论:基因表达谱cDNA芯片是筛查主动脉夹层肺损伤差异基因的有效方法,差异基因可以进一步在荧光定量PCR中验证,MMP-9可用于主动脉夹层肺损伤的诊断和治疗。     

慢性乙型肝炎患者的差异表达基因筛选及相关信号通路预测

目的:研究慢性乙型肝炎发病的分子机制.方法:通过基因表达谱芯片,运用Gene Spring软件筛选慢性乙型肝炎患者的差异表达基因;运用Gene Trail软件,对差异表达基因进行信号通路富集分析.结果:发现417个差异表达基因,其中表达上调的基因数目为205个,表达下调的基因数目为212个;下调基因的显著性信号通路为ErbB、非小细胞肺癌、mTOR、RNA降解、T细胞受体、慢性粒细胞白血病、肾细胞癌信号通路,上调基因的显著性信号通路为趋化因子、溶酶体、霍乱弧菌感染、IgGFc受体介导的吞噬作用信号通路.结论:PI3K/AKT下调可能是乙型肝炎患者持续感染的主要分子机制之一.     

沉默MCM7基因后肝癌SMMC-7721细胞差异表达基因的筛选

目的:探究微小染色体维持蛋白7(minichromosome maintenance protein 7,MCM7)基因在调节肝癌细胞生长的相关机制.方法:通过s i R N A干扰技术沉默人肝癌SMMC-7721细胞中的MCM7基因表达,采用人全基因组表达谱芯片,筛查MCM7沉默后差异表达基因,进行生物信息学分析,并对部分差异表达基因进行蛋白免疫印迹法(Western blot)验证.结果:芯片筛选出在人肝癌SMMC-7721细胞中沉默MCM7基因后差异表达基因1010个,其中上调基因391个,下调基因619个.这些基因主要涉及生物大分子代谢、细胞周期调控、细胞增殖、细胞凋亡等方面,参与胞吞、肿瘤相关通路、P53信号通路、mTOR信号通路等通路的改变.Western blot对部分差异基因表达验证的结果显示CCND1、SKP2和JUP蛋白表达均下调,与芯片检测结果一致.结论:应用人基因表达谱芯片,成功筛选出沉默MCM7基因后人肝癌细胞SMMC-7721差异表达基因,为探究MCM7基因影响肝癌细胞生长提供了有效线索.     

非小细胞肺癌差异基因的筛选及预后分析

目的利用生物信息学方法分析非小细胞肺癌(NSCLC)基因表达谱芯片,筛选差异基因,分析其与预后的关系。方法从GEO数据库下载芯片数据(GSE19804、GSE27262、GSE18842),利用GEO2R软件筛选差异基因,通过基因本体(GO)和京都基因与基因组百科全书(KEGG)进行差异基因的功能及通路富集分析,用STRING数据库构建蛋白-蛋白相互作用网络,Cytoscape筛选出核心基因。Kaplan-Meier plotter数据库进行预后分析,采用实时荧光定量PCR(QPCR)检测目的基因在NSCLC组织中的表达。结果共筛选出401个差异表达基因,GO分析结果表明差异基因主要参与血管生成、细胞粘附、调节细胞增殖等生物学过程。通过Cytoscape软件筛选出29个核心基因。预后分析显示ASPM低表达患者的总生存期较高表达患者明显延长。QPCR显示ASPM在NSCLC组织中高表达,差异有统计学意义。结论ASPM在NSCLC组织中高表达,与患者的预后相关,可能是NSCLC潜在的治疗靶点。     

慢性血栓栓塞性肺动脉高压基因表达谱的研究

目的 利用基因表达谱芯片研究慢性血栓栓塞性肺动脉高压病变组织中差异表达的基因,从多基因角度研究疾病发生的分子机制。 方法 选取5例慢性血栓栓塞性肺动脉高压病人的肺动脉内膜组织作为实验组,并选择年龄和性别相匹配的5例肺移植供体的正常肺动脉内膜组织为对照组。分别提取、纯化RNA,反转录为cDNA,与Affymetrix 2.0 ST基因芯片进行体外杂交,通过扫描信号及数据处理,分析出CTEPH肺动脉内膜的差异基因表达谱,并选择部分基因进行反转录聚合酶链反应(RT-PCR)验证。 结果 通过对两组基因表达谱比较分析,筛选出有统计学差异的1614个基因。其中880个基因表达上调,734个基因表达下调。筛选出差异最显著的5个上调基因和5个下调基因, RT-PCR验证TBX15、FMO3、ITIH3、CHRDL1、ACADL表达与芯片结果符合。 结论 慢性血栓栓塞性肺动脉高压存在显著的差异基因表达谱,筛选出来的差异基因涉及炎症反应、细胞粘附、血栓形成、平滑肌增殖、血管生成等功能,而完整的基因功能信息、传导通路、信号网络等,尚需进一步研究。     

永久性心房颤动患者左心房差异基因表达谱的建立与分析

目的:探讨与永久性心房颤动(p AF)发生、发展相关的基因和信号通路,为研究p AF发生的分子机制提供基础。方法:用Agilent 4x44K基因芯片分析p AF患者(n=7例)与窦性心律健康者(n=4例)的左心房组织m RNA,寻找差异表达的基因。以Gene Ontology(GO)和KEGG、Biocarta数据库为基础,分析差异表达基因参与的功能和信号通路。选取差异显著的部分基因应用实时定量-聚合酶链式反应(q RT-PCR)在5例p AF患者与5例窦性心律健康者标本进行验证。结果:表达谱结果显示,共有987个差异表达基因,其中567个基因表达下调,420个基因表达上调。选取9个差异显著的基因在新一组的病例标本上应用q RT-PCR验证,结果显示均有统计学差异,且改变与芯片结果一致。这些基因通过参与左心房组织纤维化、电重构、炎症、细胞应激、代谢调节、转录调节等与p AF的发生密切相关。GO和Pathway分析表明下调的基因主要参与调节代谢,表达上调的基因影响细胞应激反应、免疫应答、血小板活化等过程。结论:通过基因芯片技术发现与p AF发生相关的重要基因,通过影响细胞代谢、炎症、免疫应答、血栓形成等参与左心房的结构、功能重构。     

应用基因芯片筛选HBV 对肝癌细胞脂代谢相关基因影响的初步研究

目的通过基因芯片技术探测HBV感染对肝癌细胞脂代谢相关基因表达的影响,为深入研究HBV在肝癌脂代谢中的作用及机制提供新的依据。方法本研究以HepG2细胞作为对照,用稳定表达HBV的肝癌细胞HepG2.2.15来模拟HBV感染的肝癌细胞,利用基因芯片对肝癌细胞HepG2和HepG2.2.15进行分析,筛选出脂代谢相关的差异表达基因,对差异基因参与的信号传导通路进行分析。RT-PCR检测显著差异表达基因CYP1A1、CYP1B1和AHR。结果基因芯片共筛选出肝癌细胞HepG2和HepG2.2.15中差异表达基因共1263个,其中涉及脂代谢的基因有92个,具有表达上调的基因53个,表达下调的基因39个,涉及细胞生长、增殖、分化、运动和耐药等多种分子功能。RT-PCR检测脂代谢差异表达基因,其结果与芯片结果一致,确定芯片检测结果可靠。结论应用人类表达芯片成功筛选出HBV转染的人肝癌细胞后的脂代谢相关差异表达基因,为HBV感染在肝癌中的作用提供线索。     

Differential expression of microRNAs in aortic tissue and plasma in patients with acute aortic dissection

Background Biomarker-assisted diagnosis of acute aortic dissection (AAD) is important for diagnosis and treatment. However, identification of biomarkers for AAD in blood is a challenging task. The aim of this study is to search for new potentially microRNA (miRNAs) biomarkers in AAD. Methods The miRNAs expression profiles in ascending aortic tissue and plasma were examined by microarray analysis in two sets or groups. The tissue group was composed of four patients with AAD and four controls of healthy male organ donors. The plasma group included 20 patients with AAD and 20 controls without cardiovascular disease. Bioinformatics was used to analyze the potential targets of the differentially expressed miRNAs. Results Our study revealed that in AAD patients, the aortic tissue had 30 differentially expressed miRNAs with 13 up-regulated and 17 down-regulated, and plasma had 93 differentially expressed miRNAs, of which 33 were up-regulated and 60 were down-regulated. Four miRNAs were found to be up-regulated in both aortic tissue and plasma in AAD patients. The predicted miRNA targets indicated the four dysregulated miRNAs mainly targeted genes that were associated with cell-cell adhesion, extracellular matrix metabolism, cytoskeleton organization, inflammation, and multiple signaling pathways related to cellular cycles. Conclusions Four miRNAs, which are up-regulated both in aortic tissue and in plasma in AAD patients, have been identified in this study. These miRNAs might be potential diagnostic biomarkers for AAD. Larger sample investigations are needed for further verification.     

Broadly altered gene expression in blood leukocytes in essential hypertension is absent during treatment

We assessed whether large-scale expression profiling of leukocytes of patients with essential hypertension reflects characteristics of systemic disease and whether such changes are responsive to antihypertensive therapy. Total RNA from leukocytes were obtained from untreated (n=6) and treated (n=6) hypertensive patients without apparent end-organ damage and from normotensive controls (n=9). RNA was reverse-transcribed and labeled and gene expression analyzed using a 19-K oligonucleotide microarray using dye swaps. Samples of untreated and of treated patients were pooled for each sex and compared with age- and sex-matched controls. In untreated patients, 680 genes were differentially regulated (314 up and 366 down). In the treated patients, these changes were virtually absent (4 genes up, 3 genes down). A myriad of changes was observed in pathways involved in inflammation. Inflammation-dampening interleukin receptors were decreased in expression. Intriguingly, inhibitors of cytokine signaling (the PIAS family of proteins) were differentially expressed. The expression of several genes that are involved in regulation of blood pressure were also differentially expressed: angiotensin II type 1 receptor, ANP-A receptor, endothelin-2, and 3 of the serotonin receptors were increased, whereas endothelin-converting enzyme-1 was decreased. Strikingly, virtually no changes in gene expression could be detected in hypertensive patients who had become normotensive with treatment. This observation substantiates the long-standing idea that hypertension is associated with a complex systemic response involving inflammation-related genes. Furthermore, leukocytes display differential gene expression that is of importance in blood pressure control. Importantly, treatment of blood pressure to normal values can virtually correct such disturbances.     

基因芯片技术对哮喘病人相关基因的筛选和分析

目的 利用基因芯片技术寻找哮喘病患者与正常人外周血单核细胞之间差异表达基因,拟为哮喘的早期诊断及预防提供分子标记.方法 用淋巴细胞分离液分别提取16例哮喘病患者与16例正常人外周血单核细胞,用QIAGEN Rneasy Kit提取纯化样本总RNA,并合成用荧光标记的cRNA,分别与含有41 000条基因序列的全基因芯片杂交,以基因表达倍数值≥2.0和基因表达倍数值≤-2.0为阈值来确定差异表达基因,然后用Genespring软件利用生物信息学方法对差异表达基因进行功能分类分析.结果 按P＜0.05差异显著性标准,从34 183条表达基因谱中,筛选出哮喘患者与正常对照差异表达2倍以上的基因有4177条,差异表达2倍以上已知与哮喘相关的基因有19条.经代谢途径分析发现这些差异基因主要涉及到炎症反应、免疫反应、防御反应、创伤反应、外部刺激反应等8大功能分类.结论 哮喘的发生涉及众多基因表达的改变,芯片技术可以有效地筛选出哮喘患者与正常对照的差异表达基因,对进一步探索哮喘的发病机制、有效的干预或逆转哮喘具有重要意义.     

Gene expression profiling of mantle cell lymphoma cells reveals aberrant expression of genes from the PI3K-AKT, WNT and TGFbeta signalling pathways

Microarray studies have revealed the differential expression of several genes in mantle cell lymphoma (MCL), but it is unknown which of these differences are dependent on the transformed MCL cell itself or on the tumour microenvironment. To investigate which genes and signalling pathways are aberrantly expressed in MCL cells we used oligonucleotide microarrays to perform gene expression profiling of both purified leukaemic MCL cells and their normal counterparts, the naive B cells. A total of 106 genes were differentially expressed at least threefold in MCL cells compared with naive B cells; 63 upregulated and 43 downregulated. To validate the microarray results in a larger set of samples, we selected 10 differentially expressed genes and quantified their expression by real-time polymerase chain reaction in peripheral blood of MCL patients (n=21), purified MCL cells (n=6) and naive B cells (n=4), obtaining fully concordant results. A computer-assisted approach was used to procure specific molecular signalling pathways that were aberrantly expressed in MCL cells. Several genes related to apoptosis and to the PI3K/AKT, WNT and tumour growth factor beta signalling pathways were altered in MCL cells when compared with naive B cells. These pathways may play a significant role in the pathogenesis of MCL and deserve further investigation as candidates for new therapeutic targets.     

应用高通量基因表达谱芯片研究替米沙坦对成熟脂肪细胞的影响

目的应用基因表达谱芯片技术筛查替米沙坦独立干预下成熟脂肪细胞的差异表达基因,进而对替米沙坦作用下成熟脂肪细胞的功能变化进行预测。方法将3T3-L1前脂肪细胞诱导为成熟脂肪细胞并进行替米沙坦（0．1mg／L）干预,Trizol一步法提取总RNA并纯化,反转录合成荧光分子（Cy3／Cy5）标记的cDNA探针,分别与含有36000个基因或基因片段的高通量小鼠全基因组寡核苷酸微阵列芯片杂交,杂交信号经扫描和数字化处理,筛选替米沙坦干预组与对照组成熟脂肪细胞间的差异表达基因,进而通过生物信息学数据分析推测替米沙坦独立作用下对于成熟脂肪细胞功能的影响以及可能信号途径。结果共筛选157个差异表达基因,其中表达上调的基因86个,表达下调的基因71个,这些基因涉及脂质代谢、细胞分化及成脂过程以及脂肪细胞分泌,其基因功能可能参与替米沙坦对于脂肪细胞功能的独立影响。通过信号通路的生物信息学分析,包括细胞-细胞受体相互作用、细胞黏附分子、脂肪细胞因子信号途径、氧化应激等与脂肪细胞分泌有关的途径均受到影响。此外,替米沙坦还可能作用于脂肪细胞增殖、分化与成脂过程相关途径如Wnt信号途径、β-catenin相关途径以及Notch信号途径等。结论替米沙坦对成熟脂肪细胞可能具有独立于AT受体的特殊作用,并对细胞脂质合成、代谢产生影响。     

脂多糖诱导实验性急性水肿性胰腺炎演变为急性坏死性胰腺炎的基因表达谱变化

背景：既往对急性坏死性胰腺炎（ANP）的研究一般仅限于个别炎症相关基因的阐明,其发展过程中的基因表达谱变化尚未明确。目的：应用基因芯片技术分析脂多糖（LPS）诱导小鼠实验性急性水肿性胰腺炎（AEP）演变为ANP的基因表达谱变化,探讨该过程的基因变化机制。方法：分别以腹腔内注射雨蛙肽和雨蛙肽＋LPS诱导小鼠AEP和ANP模型,组织病理学检查鉴定建模是否成功。以Affymetrix小鼠寡核苷酸芯片检测AEP组和ANP组血白细胞基因表达谱,筛选差异表达基因。结果：ANP组/AEP组表达显著上调的基因包括转录因子、信号转导、炎症反应、黏附分子、急性反应蛋白、蛋白酶、细胞凋亡、氧自由基代谢等基因;表达显著下调的基因包括信号转导、黏附分子、细胞凋亡、防御反应、转录因子、代谢酶等基因。结论：AEP演变为ANP的基因变化模式可能为LPS等炎症信号激活酪氨酸蛋白激酶（TPK）信号通路和G蛋白介导的信号转导途径,活化C/EBPβ、Klf5等转录因子,从而促发Tnf、Il1b、Icam1等相关基因表达。     

Intrahepatic gene expression in human alcoholic hepatitis

BACKGROUND/AIMS: Alcoholic hepatitis remains an important cause of morbidity and mortality. Treatment remains unsatisfactory, in part, due to limited understanding of the pathogenesis. The aim of this study is to define the global intrahepatic expression profile of human alcoholic hepatitis. METHODS: Gene expression was analysed by DNA microarray on RNA isolated from liver of patients with alcoholic hepatitis (AH, n = 8), alcoholic steatosis (AS, n = 9) and explants from non-diseased donor liver controls (ND, n = 7). Differential expression of selected genes was confirmed by real-time RT-PCR and immunohistochemistry. RESULTS: Cluster analysis allowed differentiation of alcoholic hepatitis from alcoholic steatosis. The gene expression profile of AH revealed 586 genes differentially expressed from AS and 211 genes differentially expressed from that of ND liver. In comparison, only 98 genes were differentially expressed in AS from ND. Novel differentially expressed genes in AH in comparison to ND and AS included claudins, osteopontin, CD209, selenoprotein and genes related to bile duct proliferation. Real-time RT-PCR confirmed up-regulation of IL-8, osteopontin, and TNFRSF14 and down-regulation of SAMeS and CD209. CONCLUSIONS: This study has defined the intrahepatic gene expression profile of human alcoholic hepatitis and revealed a number of novel differentially expressed genes.     

血浆微RNA在骨关节炎患者中的表达谱特征及生物信息学分析

目的 探讨OA患者血浆微RNA(miRNA)表达谱特征,并对差异表达的miRNA进行生物信息学分析,寻找与OA相关的血浆生物标记物.方法 收集20例OA患者及15名健康体检者静脉血,通过Agilent miRNA芯片表达谱检测OA血浆miRNA的表达谱.对差异表达的miRNA进行靶基因预测,聚类分析(GO)功能分析及KEEG通路聚类分析等生物信息学分析.最后选取独立验证样本采用方差分析对差异表达miRNA进行实时荧光定量-PCR验证,并评估效能.组间差异采用t检验分析.结果 ①与健康对照组相比,OA组筛选获得74个差异表达血浆miRNA,其中45个表达上调,29个表达下调.②预测差异性表达miRNA潜在靶基因2 731个,参与到462个KEGG通路条目中,主要富集于环磷酸腺苷(cAMP)信号通路、丝裂原活化蛋白激酶(MAPK)信号通路、破骨细胞分化等通路,主要功能集中在细胞间黏附作用、胶原合成作用、细胞内信号转导等生物学功能上.③RT-PCR显示OA患者血浆miR-20a-5p、miR-320c表达水平明显升高(t=-6.142,P<0.05;t=-3.854,P<0.05),而miR-940表达明显下调(t=2.767,P<0.05),变化趋势与芯片结果一致.受试者工作特征曲线(ROC)显示这3个miRNA的曲线下面积(AUC)分别为0.864,0.851和0.818.结论 OA患者有着独特的血浆miRNA表达谱,差异性表达的miRNA可能是OA诊断的生物标志物和潜在治疗靶点.     

D-二聚体和高敏C反应蛋白对急性主动脉夹层的早期诊断价值

目的探讨D-二聚体(D-dimer)和高敏C反应蛋白(hs-CRP)对急性主动脉夹层(AAD)早期诊断的价值。方法选取2010—2012年急性胸背痛患者382例,根据多层螺旋CT结果分为AAD组(206例)和对照组(176例),比较两组间D-dimer和hs-CRP水平的差异,应用受试者工作特征曲线(ROC)分析D-dimer和hs-CRP对AAD诊断的敏感度、特异度、阳性预测值和阴性预测值。结果 AAD组D-dimer和hs-CRP水平明显高于对照组[(9.40±18.79)mg/L比(0.31±0.28)mg/L,t=3.868,P<0.01;(31.88±40.05)mg/L比(5.98±16.16)mg/L,t=4.237,P<0.01];应用ROC分析,当D-dimer取值0.56 mg/L、hs-CRP取值5.94 mg/L时,对AAD诊断的敏感度、特异度、阳性预测值和阴性预测值分别为98.1%、75.2%、85.7%、98.3%和61.9%、71.8%、75.9%、69.3%。结论在AAD早期诊断中,D-dimer和hs-CRP是有效、重要的筛选指标。