应用微阵列初步分析髓母细胞瘤的基因表达谱

目的　应用微阵列技术研究髓母细胞瘤的分子发病机理。方法　收集新鲜髓母细胞瘤 4例及正常脑组织 1份的组织标本 ,提取总 RNA,逆转录成 32 P标记的 c DNA探针 ,与 Atlas人肿瘤芯片杂交 ,通过放射自显影获得基因谱 ,应用 Atlas Image TM1.0 1a分析。结果　与正常脑组织相比 ,髓母细胞瘤下调基因 6个 ,上调基因 35个 ;逆转录 -聚合酶链反应技术验证结果与芯片检测结果相符。除少数基因外 ,大部分基因的表达趋势与肿瘤生物学特性相符。结论　髓母细胞瘤是与星形细胞起源胶质瘤具有不同分子发病机理的多基因病变 ,不同基因之间可能存在复杂的相互作用和联系 ,值得进一步研究。     

用基因芯片研究人肺癌转移相关基因

目的 自制肿瘤转移相关基因芯片研究人肺癌高、低转移细胞系PG和PAa间以及肺癌、淋巴结转移癌与正常肺组织间的基因表达谱差异，筛选与肺癌转移相关的特异基因。方法 提取2种肺癌细胞系、人肺癌、淋巴结转移癌及周围正常肺组织的mRNA后逆转录标记cDNA探针，与自制的含399个肿瘤转移相关基因的微阵列膜杂交，QuantArray软件分析杂交信号强度获得差异表达基因。结果 正常肺组织与肺原发癌的基因表达谱有显著差异。淋巴结转移癌组织与PG高转移细胞系表达基因行聚类分析，共同表达且最具统计学意义的基因有64个，包括上调基因27个，下调基因37个。结论 多基因参与肺癌的转移过程，肺转移癌与高转移细胞系共同差异表达的基因可能与肺癌的高转移特性密切相关。     

胃癌基因表达谱的cDNA微阵列与聚类分析

目的 分析胃癌与非肿瘤胃组织中基因表达特征，探讨其生物学意义。方法 提取18例进展期胃癌患者术前未行治疗的新鲜肿瘤和非肿瘤胃组织总RNA，逆转录标记cy5和cy3制备cDNA探针，与148个基因组成的cDNA微阵列杂交，应用平均联接等级聚类和微阵列数据显著差异分析(significance analysis of microarrays，SAM)方法分析146个符合入选条件基因的实验数据。结果 胃癌与非肿瘤胃组织各被聚为一类，胃癌和非肿瘤胃组织又分别聚为两个亚类。基因在两种组织表达有3个特征，明显基因表达差异表现在特征B和特征C．特征B基因在胃癌组织呈低表达或不表达，特征C基因在胃癌组织呈高表达。在特征A，T2-S2亚类与T1和T2-S1亚类的基因表达存在差异性，然而13例患者的配对胃癌与非肿瘤胃组织有相似基因表达。结合SAM分析，从特征B和特征C分别检出19个和12个在两种组织间呈差异性表达基因。结论 cDNA微阵列实验结果客观地反映了胃癌和非肿瘤胃组织的基因表达特征，可以将胃癌与非肿瘤胃组织各聚为一类．胃癌组织之间基因表达既有相似性，又有异质性，反映了胃癌基因表达变异的复杂性．应用cDNA微阵列技术研究胃癌基因差异性表达特征，有助于阐明胃癌发生、发展的分子基础，为胃癌早期诊断和预后评估的生物标记物研究提供科学依据．     

基因芯片筛选成釉细胞瘤差异表达基因的研究

目的： 用生物芯片技术筛选成釉细胞瘤相关基因。方法: 分别从人成釉细胞瘤和16周人类胚胎的牙胚组织中提取总RNA并纯化为mRNA，2种组织的mRNA分别逆转录合成有荧光分子标记的探针，然后与含有14 000种人类基因的芯片进行杂交，杂交信号用ScanArray4000 Standard Biochip Scanning System扫描仪扫描，结果用芯片图像分析软件（Genepix Pro3.0）进行分析。结果: 经过杂交筛选并与人类胚胎牙胚组织比较，从14 000个基因中筛选出差异表达基因722个，占5.0%，大于2倍的上调基因有240个（33.0%），其中有92个基因表达超过3倍；下调基因482个(67.0%)。结论: 运用基因表达谱芯片可以初步筛选出成釉细胞瘤相关基因。     

重型乙型肝炎与无症状HBsAg携带者免疫相关基因表达差异的研究

目的：应用基因芯片研究重型乙型肝炎（FHB）与无症状HBsAg携带者（ASC）外周血单个核细胞（PBMC）免疫相关基因表达差异，方法：应用含8192条人cDNA的微阵列芯片和来自外周血单个核细胞标记的cDNA，分析了10例重型乙肝和10例无症状HBsAg携带者基因表达谱。通过应用GenePix4000扫描仪和ImaGene3.0分析软件比较Cy5标记的FHB来源cDNA与Cy3标记的ASC来源cDNA的杂交结果，获得个体基因的相对表达比值。结果：在8192个基因中，初筛出21个（0．25％）表达差异2倍以上的免疫相关基因。结论：在乙型肝炎病毒（HBV）感染机体致慢性重型肝炎过程中，全面改变了宿主细胞内免疫相关基因表达，这些差异表达基因可能与慢性重型肝炎的发生有关。     

胶原性关节炎大鼠滑膜基因表达谱研究

cDNA微阵列（又称基因芯片）技术是一种基因表达水平检测的高通量技术,根据遗传学中心法则,利用基因互补配对原理,在同一载体上同时进行多基因检测,能同时准确而且快速地获得成千上万个基因在mRNA水平的表达信息.类风湿关节炎（Rheumatoid arthritis,RA）是一种以关节滑膜炎为主的自身免疫性疾病,致残率极高.滑膜类肿瘤样病变、软骨基质损害是其病理特点,认为是一种多因素参与,多基因改变协同作用的结果.为了获取更多基因表达信息,本研究采用含588个cDNA克隆的大鼠基因表达谱微阵列检测胶原性关节炎大鼠滑膜基因谱表达,并与正常大鼠进行比较.获取差异表达基因,为探讨类风湿关节炎病理机制提供线索.     

钠氢交换体1在星形细胞瘤中的表达及其与恶性程度的关系*

目的：检测人星形细胞瘤和正常脑组织中钠氢交换体1（ NHE1）的表达差异及其与恶性程度的关系,探 讨星形细胞瘤增殖、生长的分子机制。方法：收集人星形细胞瘤标本51 例,低、高级别星形细胞瘤组织分别为 22 例、29 例,以肿瘤周围相对正常脑组织作为对照。用H-E 染色进行诊断和分级,免疫组织化学和免疫印迹检 测肿瘤组织与正常脑组织中NHE1表达变化。结果：NHE1主要分布在对照组神经元和少量星形胶质细胞胞膜上; 在肿瘤组织中,NHE1分布在低级别星形细胞瘤细胞膜上,并强烈表达于高级别星形细胞瘤的胞质和胞膜上。与 对照组相比较,在低级别和高级别星形细胞瘤组织中NHE1表达上调,其中,恶性程度较高的高级别肿瘤相对于 恶性程度低的低级别肿瘤,NHE1的表达更为强烈。结论：NHE1在人星形细胞瘤组织中表达增强,其强度变化 与肿瘤的恶性程度有关。     

脑胶质瘤相关新基因表达的微阵列基因芯片分析

目的用基因芯片技术获取人脑胶质瘤组织和人正常脑组织中差异表达的相关基因，并对部分基因在不同级别胶质瘤中的表达进行初步研究。方法用含有218个与人类神经系统发育相关基因的表达谱芯片，提取正常脑组织及胶质瘤组织总RNA制备探针并杂交芯片，用ScanArray4000扫描芯片，对其中差异表达基因进行生物信息学分析，并用实时定量PCR方法验证smad1、Hmp19和TRIP3的mRNA在不同级别胶质瘤中的表达改变。结果与正常脑组织相比，胶质瘤中明显差异表达基因10个，包括细胞周期相关基因、转录和细胞转导相关基因、增殖和分化相关基因。其中表达下调基因5个，表达上调基因5个，经实时定量PCR验证smad1、Hmp19和TRIP3的表达结果与芯片检测结果相符，且随胶质瘤恶性级别的不同而变化。结论胶质瘤发生发展中存在多类基因表达的改变，表达谱基因芯片技术能快速有效地反映肿瘤发展过程中的基因改变，为胶质瘤的侵袭性和预后判断提供依据以及为导向治疗和基因治疗提供更多的靶基因。     

膀胱移行细胞癌免疫相关基因的研究

目的 探讨人膀胱移行细胞癌组织中免疫功能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，以寻找与免疫功能相关的差异表达的基因。结果以正常膀胱黏膜组织为对照，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。其中与免疫功能相关的基因有17个，明显上调基因8个，明显下调基因9个。结论膀胱肿瘤的发生、发展与多种免疫功能相关基因的异常表达有关。     

肿瘤基因表达谱——肿瘤免疫学研究的新策略

基因表达谱代表了细胞中基因表达的状况。通过比较肿瘤细胞和相应正常组织细胞的基因表达谱所获得的信息 ,就可获得在肿瘤和正常细胞中差异表达的基因 ,进一步研究这些基因的结构和功能 ,对研究肿瘤的发生发展和肿瘤的临床诊断和治疗都具有重要的意义。本文着重介绍了目前常用的研究肿瘤基因表达谱的各种方法 ,包括 m RNA水平和蛋白质水平肿瘤基因表达谱的研究方法 ,国内外的最新研究进展及应用前景     

Caveolins as tumour markers in lung cancer detected by combined use of cDNA and tissue microarrays

To identify new potential diagnostic markers for lung cancer, the expression profiles of 37 lung tumours were analysed using cDNA arrays. Seven samples were from small-cell lung cancer (SCLC), two from large-cell neuroendocrine tumours (LCNEC), and 28 from other non-small-cell lung cancers (mainly squamous cell cancer and adenocarcinoma). Principal component analysis and the permutation test were used to detect differences in the gene expression profiles and a set of genes was found that distinguished high-grade neuroendocrine carcinomas (SCLC and LCNEC) from other lung cancers. In addition, several genes, such as caveolin-1 (CAV1) and caveolin-2 (CAV2), were constantly deregulated in all types of tumour sample, compared with normal tissue. The expression of these two genes was investigated further at the protein level on a tissue microarray containing tumours from 161 patients and normal tissues. Immunostaining for CAV1 was negative in 48% of tumours, whereas 28% of the tumours did not express CAV2. Lack of CAV1 protein expression was not caused by methylation or mutation. In stage I adenocarcinomas, CAV2 protein expression correlated with shorter survival. In conclusion, the present study was able to identify genes that have not previously been implicated in lung cancer by the combined use of two different array techniques. Some of these genes may provide novel diagnostic markers for lung cancer.     

Evaluation of differentially expressed genes by a combination of cDNA array and RAP-PCR using the AtlasImage 2.0 software

Evaluation of genes regulated differentially is essential for the development of therapeutic approaches in multifactorial diseases. To characterize gene expression profiles in multifactorial inflammatory and malignant diseases such as rheumatoid arthritis (RA) or colon adenoma (CA), RNA arbitrarily primed PCR (RAP-PCR) combined with cDNA array hybridization were performed and evaluated using an array-specific software.RNA of synovial fibroblasts from patients with RA and osteoarthritis (OA), and laser microdissected normal and colon adenoma tissue was used. RAP-PCR reactions were hybridized to cDNA array membranes. Arrays were analyzed by phosphor imaging, and the AtlasImage 2.0 software with different normalization settings.The AtlasImage 2.0 software was a useful tool to evaluate differentially expressed genes. However, software settings were needed to be optimized for every experimental approach and should be used without changes for all experiments. To compare RA vs. OA synovial fibroblasts and normal vs. CA expression patterns, global normalization using the sum method is recommended.     

Towards profiling the gene expression of fibroblasts from atopic dermatitis patients: human 8K complementary DNA microarray

BACKGROUND: High-throughput technologies, including DNA-chip array, have been used to search for the genes that are dysregulated in human diseases. The atopic dermatitis (AD)-associated genes are gradually being reported; however, the differentially altered gene expression profiles of atopic fibroblasts have not been well elucidated. OBJECTIVE: We wanted to gain more insights into AD and to find candidate genes, especially in regards to the role of fibroblasts in the pathogenesis of AD. METHODS: cDNA microarray (8K) profiling of the primary cultured AD patients-derived fibroblasts was conducted by a pooling method of the recruited 22 normal controls, the 10 extrinsic type (ADe) patients and the 10 intrinsic type (ADi) patients. SAM analysis of the microarray results (2-fold cut-off) was conducted to select the candidate genes. Quantification by real-time PCRs confirmed the array data in the randomized paired samples (normal vs. ADe n=10; normal vs. ADi n=10). RESULTS: We listed the 22 up-regulated and 95 down-regulated genes in the AD fibroblasts. Real-time PCR results showed that several genes such as hyaluronan synthase 2 (HAS2), TNF-alpha-induced protein 6 (TNFAIP6) and IL-8 were matched with the array results with statistical significance. CONCLUSION: These results suggest gene expression profiles that are associated with AD and this implied that fibroblasts may play important roles in the AD pathogenesis. We provided new insights into three candidate genes such as HAS2, TNFAIP6 and IL-8 with respect to their involvement in AD disease.     

Microarray analysis using amplified mRNA from laser capture microdissection of microscopic hepatocellular precancerous lesions and frozen hepatocellular carcinomas reveals unique and consistent gene expression profiles

The indirect labeling cDNA microarray technique was used to evaluate gene expression profiles of pure cell populations from frozen sections of carcinomas and adenomas harvested from precancerous hepatocellular lesions by using laser capture microdissection (LCM). The levels of differentially expressed genes were investigated using a cDNA microarray with 9,984 features with only 2 ug of two-round amplified aRNA, equivalent to 35 cells from LCM-adenomas and frozen samples of carcinomas from simian virus 40 (SV40) large T antigen transgenic rats. A total of 855 genes were identified as being 3-fold or more differentially expressed in carcinomas or adenomas as compared to normal tissue controls. Among these 855 genes, 71 genes were differentially expressed in both carcinomas and adenomas. Commonly up-regulated genes in both carcinoma and adenomas were 28 while 41 of the 71 genes were commonly down-regulated. Two genes, Igh1 (immunoglobulin heavy chain 1(Serum IgG2a), Image clone ID: 875880) and EST clone (AI893585, Image clone ID: 596604) were more than 7-fold up-regulated in carcinomas and 6-fold down-regulated in adenomas. In Cy5 and Cy3 reciprocal experiments for screening out false positive signals, the amplified carcinomas showed higher Pearson Correlation Coefficient values (-0.94 and -0.92) than the LCM-amplified adenoma samples (-0.79 and -0.84). LCM-amplified samples provided higher signal intensities over backgrounds and a greater average of Cy5:Cy3 ratios. Expression levels of mRNAs from selected genes, determined by using traditional dot blot analysis, revealed that 36 of 40 tested expression profiles were consistent with the microarray data. Thus, amplified aRNA harvested from homogeneous cell types using LCM can be applied to study gene expression profiles by use of microarray analysis.     

食管癌及癌旁组织中基因表达的初步研究

目的　了解食管癌的基因表达概况,寻找在食管癌及癌旁组织中差异表达基因。方法　以癌及癌旁组织ｐｏｌｙ Ａ＋ Ｒ Ｎ Ａ 反转录合成的ｃ Ｄ Ｎ Ａ 为探针,与 Ａｔｌａｓ 微点阵表达分析膜进行差异杂交。结果放射自显影结果显示在所分析的５８８ 种已知基因中,ｃｄｃ２５ Ｂ、 Ｍ Ｍ Ｐ、 Ｍ Ｅ Ｔ 等６１ 个在食管癌组织中表达上调,ｃｙｔｏｋｅｒａｔｉｎ ４ 、 Ｂ Ａ Ｄ、 Ｉ Ｌ１ Ｒ Ｅ Ｃ Ｅ Ｐ Ｔ Ｏ Ｒ Ａ Ｎ Ｔ Ａ Ｇ Ｏ Ｎ Ｉ Ｓ Ｔ、 Ｉ Ｌ６ 等２２ 个表达下调,参与细胞增殖、凋亡、分化和转移调控的多种基因的表达水平发生了明显改变。结论　这些基因的表达改变组成了一个食管癌特异的基因表达谱,首次为食管癌细胞的恶性表型提供了分子遗传学参考数据,一些与肿瘤发生相关的差异表达基因为发展生物标记物或肿瘤早期诊断和治疗提供了线索。 Ａｔｌａｓ 微点阵表达分析滤膜的差异杂交为初步了解某一组织或细胞的表达状况提供了一个较好的方法。     

X chromosome-specific cDNA arrays: identification of genes that escape from X-inactivation and other applications

Mutant alleles are frequently characterized by low expression levels. Therefore, cDNA array-based gene expression profiling may be a promising strategy for identifying gene defects underlying monogenic disorders. To study the potential of this approach, we have generated an X chromosome-specific microarray carrying 2423 cloned cDNA fragments, which represent up to 1317 different X-chromosomal genes. As a prelude to testing cell lines from patients with X-linked disorders, this array was used as a hybridization probe to compare gene expression profiles in lymphoblastoid cell lines from normal males, females and individuals with supernumerary X chromosomes. Measurable hybridization signals were obtained for more than half of the genes represented on the chip. A total of 53 genes showed elevated expression levels in cells with multiple X chromosomes and many of these were found to escape X-inactivation. Moreover, the detection of a male-viable deletion encompassing three genes illustrates the utility of this array for the identification of small unbalanced chromosome rearrangements.     

Gene expression profiles in human nasal polyp tissues studied by means of DNA microarray

BACKGROUND: Nasal polyposis (NP) is a chronic inflammatory disease of the sinuses. Its pathogenesis is unknown. DNA microarray analysis allows simultaneous measurement of expression of thousands of genes in the same tissue sample and might help to identify gene alterations in various disorders. OBJECTIVE: We sought to screen for disease-related genes in NP by using DNA microarrays and to validate the altered expression of selected genes at the mRNA and protein level. METHODS: Expression microarrays containing approximately 10,500 genes were used to compare individual gene profiles of NP samples (n=10) and normal mucosal samples obtained from sphenoid sinuses in patients undergoing pituitary surgery (n=4). Four of the 5 most upregulated, and the single most downregulated, genes were retested by means of quantitative RT-PCR and immunohistochemistry in a different set of NP and normal mucosal samples obtained from the ethmoid and sphenoid sinuses. RESULTS: Compared with normal sinus tissue, 192 genes were upregulated at least 2-fold, and 156 genes were downregulated by at least 50% in NP samples (approximately 3% of genes evaluated). Four of the top 5 overexpressed genes (statherin, 48.0-fold; prolactin-induced protein [PIP] , 24.9-fold; lactoferrin, 26.6-fold; and deleted in malignant brain tumor 1 [DMBT1] , 30.3-fold) and the most underexpressed gene (Clara cell 10-kd protein [CC10] , -20.1-fold) were selected and retested by means of quantitative RT-PCR and immunohistochemical staining. Quantitative RT-PCR and immunohistochemical staining confirmed the differential expression of all except statherin in NP tissue. CONCLUSION: DNA microarrays can provide new insight into the possible pathophysiologic processes involved in NP.