人腹主动脉瘤与正常腹主动脉基因表达谱差异的研究

目的 运用基因芯片研究人腹主动脉瘤与正常腹主动脉的基因表达谱,筛选差异表达基因.方法 选取腹主动脉瘤标本及正常腹主动脉标本各5例.抽提总RNA,转录并以生物素标记后,与芯片杂交,并对结果进行分析.结果 腹主动脉瘤组与正常组织组比较差异表达的基因共1962个,其中上调基因554个,下调基因1408个.功能分析发现与炎症反应、免疫反应及某些化学趋化因子有关的基因在腹主动脉瘤组表达上调.结论 表达谱芯片筛选腹主动脉瘤组与正常腹主动脉组的差异基因,为研究腹主动脉瘤的发病机制提供了新思路.     

表达谱芯片筛选粥样硬化性腹主动脉瘤的差异表达基因

目的运用基因芯片研究粥样硬化性腹主动脉瘤中基因表达谱的变化,筛选差异表达基因。方法选取粥样硬化性腹主动脉瘤(AAA)标本5例,以5例正常腹主动脉作对照。抽提总RNA,纯化mRNA后逆转录制备杂交探针,采用含有4096种人类基因全长cDNA的芯片进行差异表达谱分析。结果在粥样硬化性AAA的基因表达谱中差异表达基因共有186条,其中上调的基因102条,下调的基因84条;共存性基因有37条(上调基因26条,下调基因11条),其中有细胞凋亡相关基因、原癌基因和抑癌基因、细胞信号和传递蛋白等多种基因。反转录聚合酶链反应(RTPCR)及蛋白印迹验证了Caspase9及周期素依赖蛋白激酶(CDK)4在腹主动脉瘤中的差异表达。结论表达谱芯片筛选差异表达基因,为AAA发病机制的研究提供新思路;凋亡/增殖相关基因的表达失衡可能是粥样硬化性AAA的重要发病机制。     

应用基因表达谱芯片研究人腹主动脉瘤细胞周期和细胞凋亡相关基因

目的：筛选腹主动脉瘤发生过程中与正常组织差异表达的细胞周期和细胞凋亡相关基因并作初步功能分析。方法：应用Trizol一步法抽提2块腹主动脉瘤壁及2块正常主动脉组织总RNA后,Oligotex mRNA Spin-Column操作法分离纯化组织mRNA;经逆转录合成荧光分子（Cy3/Cy5）标记cDNA探针,与微矩阵排列的含有4096种cDNA基因的表达谱芯片杂交,利用GenePix Pro3.0软件分析动脉瘤与正常组织中差异表达的基因,对所获得的基因进行分子生物信息学分析。结果：腹主动脉瘤与正常组织间差异表达的细胞周期和细胞凋亡基因有18条,占芯片基因总数的0．44％。其中与细胞周期相关的基因11条,与细胞凋亡相关的基因7条。在动脉瘤组织中表达上调的基因9条,平均Ratio值为3．860,表达下调的9条,平均Ratio值为0．294。生物信息学分析显示,该18条差异表达细胞周期和细胞凋亡基因与腹主动脉瘤中平滑肌细胞的生长和凋亡有关。结论：腹主动脉瘤的发生、发展过程中存在多基因表达调控的改变,细胞周期和细胞凋亡基因与腹主动脉瘤中平滑肌细胞等的生长和凋亡有相关性,对于该相关基因群的进一步研究有助于阐释腹主动脉瘤的发病机制。     

微小RNA let-7基因表达在膀胱癌生物学行为中的作用

目的 探讨膀胱癌组织中微小RNA(miRNA)表达在膀胱癌发生发展中的作用. 方法 Trizol法抽提9例膀胱癌患者癌组织及癌旁正常膀胱组织标本总RNA,分离与富集miRNA,随机选取2组miRNA标本与miRNA表达谱芯片杂交,采用LuxScan 3.0和SAM 2.1软件对芯片结果 进行图像数据分析.在差异性表达的miRNA中筛选出感兴趣的let-7基因,采用印迹杂交技术对9组miRNA标本中let-7基因进行验证. 结果 膀胱癌组织和正常膀胱组织标本中差异性表达有显著性意义的miRNA 71个,其中表达上调33个,表达下调38个;差异性表达有极显著性意义的miRNA(上调或下调≥4倍)26个,其中显著上调12个,显著下调14个.let-7基因验证结果 与芯片表达结果 一致,癌组织let-7基因表达吸光度值为479.6±228.2,正常膀胱组织为694.6±152.9,组间差异有统计学意义(P<0.05). 结论 膀胱癌组织和正常膀胱组织中存在差异表达的miRNA,let-7基因在膀胱癌的发生中可能起着抑癌基因的作用.     

腹主动脉瘤信号传导相关基因的研究

目的：研究细胞信号传导基因在腹主动脉瘤和正常主动脉的差异表达，探讨其与腹主动脉瘤发生的关系。方法：应用基因芯片技术检测腹主动脉瘤和正常主动脉信号传导相关基因的表达，筛选出差异表达显著的基因，从RNA和蛋白质水平作进一步的分子生物学实验。结果：发现腹主动脉瘤差异表达的信号传导基因45条，在动脉瘤组织中表达上调基因为28条，表达下调基因17条。对其中MAPK信号系统的进一步实验发现ASK1、ERK1等基因异常表达，均在主动脉中层有蛋白质表达。结论：信号传导相关基因的差异表达可能与腹主动脉瘤的发生有关。     

DNA芯片技术筛选膀胱移行细胞癌信号转导相关基因

目的探讨人膀胱移行细胞癌信号转导相关基因的表达变化。方法使用人肿瘤基因表达谱芯片检测11例膀胱移行细胞癌组织基因表达谱的变化，以寻找与细胞信号转导相关的差异表达基因。结果以正常膀胱黏膜组织为对照，11例膀胱肿瘤组织中有87个基因表达明显下调，102个基因表达明显上调。其中与细胞信号转导相关的差异表达基因有35个，明显上调基因13个，明显下调基因22个。结论膀胱肿瘤的发生与发展与多种细胞信号转导相关基因的异常表达有关。     

全基因组寡核苷酸表达谱筛选哈萨克族食管癌相关靶基因

目的 筛选哈萨克族食管癌与正常食管黏膜的差异表达基因.方法 新鲜标本取自哈萨克族食管鳞癌患者.采用RNA保护技术保护组织标本,分离癌组织、正常食管黏膜组织标本,提取RNA,线性扩增获得足量cRNA,利用基因芯片分别检测癌组织和正常食管黏膜组织基因表达谱,并利用生物信息学方法对检测结果进行分析.结果 癌组织和正常食管黏膜组织比较差异10倍以上共有170个基因,其中表达上调(信号比的对数值>3)有39个,表达下调(信号比的对数值<-3)有131个.表达异常的基因与细胞周期调节、细胞凋亡、细胞骨架、细胞外基质、细胞内信号传递、蛋白质的翻译合成及免疫功能等相关.结论 利用全基因组寡核苷酸芯片可准确、高效地筛选出哈萨克族食管癌相关的候选靶基因170个,这些基因与哈萨克族食管癌的发生、发展有关.     

皮肤恶性黑色素瘤与良性痣基因表达差异的研究

目的 通过癌症相关基因芯片比较中国人皮肤恶性黑色素瘤与良性痣及正常皮肤间的基因表达谱差异,探讨中国人皮肤恶性黑色素形成相关的差异表达基因.方法 收集我科收治的皮肤恶性黑色素瘤标本及肿瘤旁正常皮肤组织6例、良性痣标本10例.抽提出总RNA用生物素标记后与美国SUPERARRY公司的OHS802癌症相关基因芯片结合,通过计算机扫描,数据分析筛选差异表达基因,并针对差异表达基因采用RT-PCR法验证.结果 经比较440条癌症相关基因中31条基因存在明显表达差异,其中与正常皮肤及良性痣标本相比,恶性黑色素瘤中表达上调的有24条,下调的有7条.结论 基因表达谱芯片技术可以有效地筛选出皮肤恶性黑色素瘤和良性痣及正常皮肤组织的差异表达基因,这些差异表达的基因包括多种癌症相关的基因,其中ITGA3及ILK基因在癌旁正常皮肤、良性痣及恶性黑色素瘤中表达水平依次升高,可应用于对恶性黑色素发病机制的进一步研究.     

慢性胰腺炎与细胞外基质代谢失衡的关系

目的　研究慢性胰腺炎与细胞外基质 (ECM )代谢失衡的关系 ,探讨慢性胰腺炎的发病机制。方法　应用含有 40 96条人类全长基因的cDNA表达谱芯片 ,分别对 6例慢性胰腺炎及正常胰腺组织标本的基因表达谱进行分析。结果　在 6例慢性胰腺炎组织中 ,均有差异表达的基因2 0 7条 ,占芯片基因数的 5 .0 1%。从中筛选出与ECM代谢有关的显著表达差异基因 18条 ,其中表达上调基因 10条 ,下调基因 8条。结论　转化生长因子 (TGF) β1持续性高表达造成的ECM代谢紊乱 ,可能是导致慢性胰腺炎胰腺纤维化发生、发展的重要因素。     

应用基因微矩阵芯片筛选前列腺癌的相关基因

目的　应用基因微矩阵芯片筛选前列腺癌的相关基因。　方法　按一步法提取前列腺癌及正常前列腺组织总RNA ,并纯化mRNA ,以包含 4 0 96个cDNA的基因微矩阵芯片对前列腺癌及正常前列腺基因表达谱进行分析。　结果　前列腺癌及正常前列腺组织中表达差别有显著性意义的基因共 3 4 1条 ,其中前列腺癌下调基因 12 8条 ,上调基因 2 13条 ;表达差别有极显著性意义的基因有15条 ,其中显著下调基因 6条 ,显著上调基因 9条。　结论　基因微矩阵芯片可有效筛查出前列腺癌的相关基因 ;前列腺癌发生发展由多种类型的基因参与调控 ,而非单一因素所致     

用基因表达谱芯片筛选原发性下肢静脉曲张相关基因的研究

目的运用基因表达谱芯片研究原发性下肢静脉曲张基因表达谱的变化。方法选取原发性下肢静脉曲张患者隐 股交界瓣膜区静脉 5条 ,取 5条正常静脉作对照。抽提总RNA、纯化mRNA、反转录制备杂交探针 ,应用含有 4 0 96种人类基因全长cDNA的表达谱芯片进行差异表达谱分析 ,随后应用反转录PCR验证部分差异表达基因。结果曲张大隐静脉瓣膜区表达谱中差异表达基因共有 16 8条 ,上调基因 96条 ,下调基因 72条 ;5例标本均差异表达的基因共 39条 ,其中上调2 8条 ,下调 11条 ;多条细胞凋亡相关基因、细胞信号和传递蛋白基因、原癌基因和抑癌基因等均有差异表达 ;反转录PCR证实caspase 9及MAP3K基因及其蛋白产物在曲张静脉中差异表达。 结论应用基因表达谱芯片筛选致病相关基因 ,可能为下肢静脉曲张的发病机制研究提供新思路。     

类表皮生长因子域7在动脉硬化闭塞症和腹主动脉瘤中的表达

目的 探讨类表皮生长因子域7(epidermal growth factor-like domain 7,egfl 7)在动脉硬化闭塞症(arteriosclerosis obliterans,ASO)和腹主动脉瘤(abdominal aortic aneurysm,AAA)中的表达特点及其在动脉粥样硬化(atherosclerosis,AS)发病过程中的作用.方法收集正常动脉8例,下肢ASO标本11例,AAA标本34例,应用免疫组织化学技术和原位杂交技术检测egfl7蛋白和egfl7mRNA.应用qRT-PCR技术检测egfl 7 mRNA的表达水平.应用RNA干扰技术检测egfl 7的作用.结果 在ASO和AAA中,egfl 7蛋白主要表达于动脉中膜;egfl7 mRNA的表达部位与egfl 7蛋白一致.在ASO早期、AAA与正常动脉相比,egfl 7 mRNA的表达水平显著上调(325%±120%比100%±36%.P＜0.01,216%±133%比100%±36%,P＜0.05).干扰egfl 7后,平滑肌细胞(smooth muscle cells,SMCs)的增殖能力与对照组相比明显降低(0.85±0.05比1.34±0.04,P＜0.01),吸光度值降低(0.85±0.05比1.32±0.09,P＜0.01).结论 egfl 7可能是调控AS发病过程的一个重要基因,可能通过促进血管平滑肌细胞的增殖起作用,是早期ASO的标志.

Abstract：

Objective To investigate the expression characteristics of epidermal growth factor-like domain 7 ( egfl 7 ) in arteriosclerosis obliterans ( ASO) tissues and abdominal aortic aneurysm ( AAA ) tissues, and it's role in atherosclerosis (AS).Methods In this study, 8 normal artery, 11 lower extremity ASO and 34 AAA samples were collected.Immunohistochemistry and in situ hybridization were performed in artery sections to investigate the expression characteristics of egfl 7 at protein and mRNA levels.The relative quantitative detection of egfl 7 mRNA was detected by qRT-PCR.The effect of egfl 7 was examined by RNA interference.Results In ASO and AAA samples, the expression of egfl 7 protein was mainly in the tunica media; The expression site of egfl 7 mRNA was the same as that of egfl 7 protein.Compared with normal artery samples, the expression of egfl 7 mRNA was significantly upregulated in the early stage of ASO samples and AAA samples (325 ± 120 vs.100 ± 36, P ＜ 0.01, 216 ± 133 vs.100 ± 36, P ＜ 0.05 ).Compared with the control group, the proliferative ability of smooth muscle cells was significantly down regulated after egfl 7 interference (0.85 ± 0.05 vs.1.34 ± 0.04, P ＜ 0.01).Conclusions egfl 7 might be a gene that regulates the pathogenic process of atherosclerosis through promoting vascular smooth muscle cell proliferation.     

Differential gene expression in human abdominal aorta: aneurysmal versus occlusive disease

OBJECTIVE: Inflammation and atherosclerosis are present in both abdominal aortic aneurysm (AAA) and arterial occlusive disease (AOD). Changes in gene expression that underlie the development of AAA versus AOD are poorly defined. This study evaluated differences in gene expression in AAA, AOD, and control aortic tissue with human gene array technology. METHODS: RNA was isolated from human aortic specimens (seven AAA, five AOD, and five control), and complementary DNA (cDNA) probes were generated. The cDNA probes were hybridized to a human cell interaction array of 265 genes and quantitated with phosphorimaging. The data were corrected for background and were standardized to housekeeping genes. Statistical differences in individual gene expression were determined with the Kruskal-Wallis test. RESULTS: Of 265 genes studied, 11 showed statistically different expression in diseased aorta as compared with control. The following three genes were downregulated in AAA: collagen VI alpha1 (P <.037), glycoprotein IIIA (P <.006), and alpha2-macroglobulin (P <.020). The following two genes were upregulated in AOD: laminin alpha4 (P <.034) and insulin-like growth factor 2 receptor (P <.049). The following three genes were upregulated in both AAA and AOD: matrix metalloproteinase-9 (MMP-9; P <.005), intercellular adhesion molecule-1 (P <.012), and tumor necrosis factor--beta receptor (P <.022). The following three genes were downregulated in both AAA and AOD: integrin alpha5 (P <.012), ephrin A5 (P <.037), and rho/rac guanine nucleotide exchange factor (P <.028). Of 16 MMPs evaluated, only MMP-9 was significantly (P <.005) upregulated in both AAA and AOD. Evaluation results of four tissue inhibitors of metalloproteinases showed no significant difference in expression for all tissue types, although tissue inhibitor of metalloproteinase-1 trended toward upregulation in AAA (P =.053). Eight of the fifteen most highly expressed genes in all the groups were extracellular matrix or secreted proteins. Of these, only collagen VI alpha1 (P <.037) showed a significant change, although biglycan trended toward downregulation in AAA (P =.076). CONCLUSION: This study used cDNA array technology in the comparison of human control and pathologic aortic tissue. Six genes had similar differential expression in both AAA and AOD as compared with control. Even more interesting were differences between AAA and AOD in the expression of five genes. These data suggest a similarity in genetic expression for both AAA and AOD, with altered expression of several genes playing a role in disease differentiation.     

腹主动脉瘤中细胞黏附分子差异表达的研究

目的研究腹主动脉瘤 (AAA)中细胞黏附分子的差异表达与AAA发病的关系。方法利用基因芯片技术筛查AAA和正常腹主动脉中差异表达的细胞黏附分子基因 ,再利用分子生物学方法在基因、蛋白质水平检测。结果AAA中有 3种细胞黏附分子存在差异表达 ,它们是VCAM 1,PECAM 1,TSP ,上调比率分别达到 5 7,3 6及 5 7 4倍。结论AAA中有细胞黏附分子的表达差异 ,差异表达基因可能在AAA的发病过程中起作用     

基质金属蛋白酶及抑制因子与腹主动脉瘤临床病理特点的相关性研究

目的：研究基质金属酶（MMP）-2、MMP-9及抑制因子TIMP-1在腹主动脉瘤中的表达及与临床病理特征之间的关系。方法：应用免疫组化PV-9000通用型二步法对70例腹主动脉瘤和15例正常腹主动脉标本中的MMP-2、MMP-9及TIMP-1表达进行检测。结果：腹主动脉瘤组织中MMP-2和MMP-9蛋白表达阳性率明显高于正常腹主动脉组织，TIMP-1蛋白表达阳性率和正常腹主动脉没有统计学差异，（X^2=0.103，P=0.991）；MMP-2蛋白的表达与腹主动脉瘤的直径呈负相关（X^2=13.785，P=0.032），MMP-9蛋白的表达与患者临床症状，腹主动脉瘤直径、破裂有相关性，（P〈0.05），TIMP-1蛋白表达阳性率与临床病理特征无相关性（X^2=0.103，P=0.991）。结论：腹主动脉瘤组织中MMP高表达和TIMP的相对弱表达在腹主动脉瘤发生、发展过程中起重要作用，MMP-9可以预测腹主动脉瘤的自然病程从而作为腹主动脉瘤手术治疗的指征之一。     

Differential expression of immunoglobulin kappa chain constant region in human abdominal aortic aneurysm

BACKGROUND: A number of the research into the pathogenesis of the abdominal aortic aneurysm (AAA) have focused on the alteration of gene expression. The current technique for elucidating alterations of gene expression has a setback in that many artifact complementary DNA (cDNA) products present abnormal polymerase chain reaction (PCR) amplification. Our study was designed to identify differentially expressed genes in AAA using the annealing control primer (ACP) system, which was recently developed to identify only authentic genes. MATERIALS AND METHODS: The tissues of the human abdominal aorta were obtained from the patients of AAA and aortic occlusive disease (AOD), and normal abdominal aorta (NA) from brain death donors. Total RNAs were isolated from three groups of human abdominal aorta (10 AAA, five NA, three AOD) and then reverse transcribed into complementary DNA (cDNA). The ACP method was done to screen the difference in the expression pattern of the mRNA (mRNA). RESULTS: One differentially expressed cDNA band was detected in AAA but not in NA and AOD. This cDNA was sequenced and computer searching against the GenBank revealed that the cDNA had more than 90% identity with the immunoglobulin kappa chain constant region (Ig kappa-C). DISCUSSION: Our finding suggests that differentially expressed Ig kappa-C gene only in AAA is a candidate gene that may play a pivotal role in the pathogenesis of AAA formation. The correlation of mRNA level and protein level is, however, not clear. Thus, to directly identify the role of Ig light chains in the pathogenic event of AAA, the further study comparing the level and kinds of expressed protein with the corresponding Ig kappa-C gene will be required.