应用基因芯片技术筛选肝细胞癌相关基因

目的:探讨基因表达谱芯片技术在筛查肝细胞癌(HCC)相关基因群表达中的作用。方法:TRIzol法抽提2例HCC及正常肝脏组织总RNA,分离纯化两种组织的mRNA;逆转录合成掺入生物素标记的cDNA合成探针,与基因芯片(涵盖了18400个转录本,代表了14 500个明晰的基因)杂交,扫描芯片荧光信号图像,计算机分析,比较2种组织基因表达谱差异。结果:2例HCC组织与正常肝脏组织相比,有2756条基因(19.01%)共同表达差异,其中共同上调基因1772条和共同下调基因984条,对2756条共同差异表达基因作了初步功能分类,这些基因与HCC的发病机制存在相关性。结论:基因表达谱芯片技术可以筛选出HCC表达异常的相关基因群,有助于认识肝癌的发病机制。     

应用基因芯片技术筛选肝硬化相关基因

目的:探讨基因表达谱芯片技术在筛查肝硬化相关基因群表达中的作用.方法:按TRIzol法抽提肝硬化及正常肝脏组织总RNA,分离纯化两种组织的mRNA.经逆转录合成掺入生物素标记的cDNA合成探针,与基因芯片(涵盖18 400个转录本,代表14 500个明晰的基因)杂交,扫描芯片荧光信号图像,计算机分析,比较二种组织基因表达谱差异.结果:2例肝硬化组织与正常肝脏组织相比,有1424条基因(9.82%)共同表达差异,其中共同上调基因980条和共同下调基因444条.对1424条共同差异表达基因作了初步功能分类,这些基因与肝硬化的发病机制存在相关性.结论:基因表达谱芯片技术可以筛选出肝硬化表达异常的相关基因群,对其进一步研究有助于认识肝硬化的发病机制.     

应用基因芯片研究肝细胞癌组织差异基因表达谱

目的：应用基因芯片技术研究原发性肝细胞癌组织中的差异基因表达谱改变，以寻找肝细胞癌相关基因。方法：抽提正常肝组织和肝癌组织中的mRNA来制备探针，经杂交、洗涤后，通过计算机扫描分析正常肝组织和肝癌组织基因表达谱的差异情况。结果：在10000个候选基因中，筛选出102条差异表达基因，表达上调的有42条，表达下调的有60条。未知基因12条。结论：基于DNA微阵矩技术的肿瘤基因表达谱分析能够高通量筛选与肝癌发生发展相关的基因。     

应用基因表达谱芯片筛选胃腺癌相关基因

目的:利用基因芯片技术筛选胃腺癌组织和癌旁正常组织间的差异表达基因．方法:分别抽取胃腺癌组织和癌旁正常组织的总RNA．采用逆转录的方法,制成cDNA链, 并以两种荧光Cy5和Cy3标记后作为探针,与含有14 784条人类14KcDNA基因表达谱芯片进行杂交．以Agilent荧光扫描仪扫描芯片上两种荧光信号,并用计算机处理和分析．结果:在14 784条基因中,4例胃腺癌组织和癌旁正常组织共同差异表达基因29条,其中上调基因10条,下调基因19条,上调的基因中有2 条功能信息不明．结论:胃腺癌发生过程中有多基因的参与,胃腺癌与癌旁正常组织共同差异表达的29条基因可能与胃癌的发生、发展有关．     

骨髓形成肝细胞的基因表达谱分析

目的：观察小鼠骨髓形成肝细胞的基因表达谱的变化规律，研究骨髓形成肝细胞的分子机制。方法：采用交叉性别骨髓移植模型，雌性BABL／C小鼠接受9．0Gy 60^Co源γ射线照射后，经尾静脉注射输入同种系雄性小鼠骨髓细胞，6个月后处死动物，取肝组织标本。选用小鼠基因表达谱寡核苷酸芯片，观察实验小鼠肝组织基因表达谱的变化。结果：实验小鼠在骨髓移植6个月后的肝组织的基因表达谱与正常雌性小鼠比较有显著不同，差异表达基因有865条，已知功能基因有447条，其中上调基因92条、下调基因355条。结论：交叉性别骨髓移植小鼠肝脏的基因表达谱变化规律为进一步研究骨髓形成肝细胞的分子机制奠定了实验基础。     

移植雄性骨髓的雌性小鼠肝组织肝再生相关基因的信号通路

目的:探讨骨髓形成肝细胞的分子机制.方法:采用移植♂骨髓的♀小鼠模型,♀Balb/c小鼠随机分为模型组和正常对照组,选用小鼠基因表达谱寡核苷酸芯片,利用反转录酶合成掺有荧光标记的cDNA探针,将探针与基因表达谱芯片杂交观察小鼠肝组织基因表达谱的变化,筛选样本之间杂交信号比值有差异表达的基因.采用生物信息学方法分析模型组中小鼠肝组织再生相关基因的信号通路变化规律.结果:♀小鼠在移植♂骨髓6 mo后肝组织的基因表达谱显著不同,对照组相对于正常组的差异表达基因有865条,已知功能基因有447条,其中上调基因92条,下调基因355条.与肝再生相关基因信号通路涉及HGF,TGF-β,Focal Adhesion,JAK-Stat,VEGF等信号通路,他们相关基因的上调表达促进肝细胞的增殖分化.TGF-β信号通路中相关基因下调,抑制信号通路的激活,减弱肝再生的负性作用,利于肝细胞的增殖分化.结论:♀小鼠移植♂骨髓后的肝组织基因表达谱显著变化,有可能通过其肝再生相关基因的信号通路激活或抑制调节肝再生.     

基因表达谱芯片在胰腺癌组织差异表达相关基因筛选中的应用

目的：探讨基因表达谱芯片技术筛选胰腺癌组织和癌旁正常组织基因表达谱差异的可行性。方法应用Agilent公司生产的包含27958条DNA的基因芯片检测4例胰腺癌患者胰腺癌组织和癌旁正常组织的基因表达谱,筛选差异表达基因,并用半定量RT-PCR法检测差异基因CD151、S100A4、TIMP-3和NME3表达情况。结果共筛选出46条基因表达谱差异基因,其中26条表达上调、20条表达下调。差异基因CD151、S100A4、TIMP-3和NME3的表达情况与基因表达谱芯片检测结果一致。结论基因表达谱芯片技术可以筛选出胰腺癌组织差异表达相关基因,为胰腺癌分子标志物研究提供了可靠依据。     

应用基因芯片技术筛选胰腺癌相关基因

目的应用基因芯片技术筛选胰腺癌相关基因。方法将14000种人类基因PCR产物按微矩阵排列点样于化学涂层的载玻片上，制成基因芯片。按一步法抽提4例胰腺癌和癌旁正常胰腺组织的总RNA，将等量的RNA分别逆转录合成荧光分子掺人的cDNA一链作探针，混合后杂交上述基因芯片。经严格洗片后用ScanArray 4000扫描仪扫描芯片荧光信号图像，每点上两种荧光信号的强度分别代表Cy5-dCTP和Cy3-dCTP的量，获得的荧光信号图像用计算机分析。结果按差异显著性标准，从14000个基因中筛选出在胰腺癌组织中共同差异表达基因189条，其中已知基因101条，新基因88条。在筛选出的已知基因中，有50条表达上调，51条表达下调。结论基因芯片技术的肿瘤基因表达谱分析能够高通量筛选胰腺癌相关基因。并高效对基因功能进行研究。胰腺癌基因表达谱的分析有助于认识肿瘤发病机制。     

应用寡核苷酸芯片技术探索原发性肝细胞癌发生发展中的差异表达基因

目的应用寡核苷酸芯片技术进行高通量分析原发性肝细胞癌与癌旁组织以及正常肝组织的差异表达基因,探索与肿瘤进展相关的靶基因。方法对原发性肝细胞癌患者的癌组织、癌旁组织以及正常肝组织,应用Trizol-步法进行总RNA抽提,10g/L琼脂糖凝胶电泳和芯片实验室进行RNA质量检测。总RNA纯化后进行逆转录cRNA合成、荧光标记和纯化,将癌组织和正常肝组织、癌组织和癌旁肝组织的cRNA探针分别与Agilent寡核苷酸芯片(21 074探针)进行杂交,洗涤后应用Agilent扫描仪获取图像,特征提取软件进行定量分析处理。挑选明显差异表达的基因进行SYBR Green I染料掺入的荧光实时逆转录聚合酶链反应验证。结果(1)配对组织总RNA质量高,反转录cRNA及荧光标记质量好；(2)2倍差异表达基因中,上调基因共420个,下调基因共552个,其中包括5倍上调基因DKK1；(3)以β-肌动蛋白为内对照的实时逆转录聚合酶链反应结果提示,DKK1在癌、癌旁和正常肝组织中的2~(-ΔCt)值分别为0．089 504、0．007 65和0．000 631。结论应用Agilent寡核苷酸芯片高通量、高效率地分析原发性肝细胞癌发生发展过程中的基因表达差异,可以筛选新的治疗靶点；原发性肝细胞癌的发生发展涉及多基因、多步骤,是一个复杂的过程；DKK1可能是原发性肝细胞癌发展过程中的新的分子靶点,其表达变化涉及肿瘤进展。     

采用cDNA芯片对人胃腺癌凋亡相关基因表达谱的研究

背景：细胞凋亡及其调控基因在肿瘤（如胃癌）的发生、发展中的作用是当今研究的热点，有必要全面筛查胃癌相关凋亡基因。目的：研究胃癌发病过程中凋亡基因表达谱的变化。方法：利用人细胞凋亡相关基因cDNA芯片研究胃腺癌和癌旁组织中相关基因的改变。采用荧光定量聚合酶链反应（PCR）和组织芯片技术验证IGFBP4基因的表达情况。结果：在458个凋亡相关基因中，有17个基因在癌组织和癌旁组织中出现2倍以上的明显改变；4个基因表达增强，13个基因表达下调。荧光定量PCR和组织芯片验证结果与cDNA芯片相符。结论：发生改变的17个凋亡相关基因可能与胃腺癌的发病机制有关，并涉及整个凋亡途径，值得进一步深入研究。     

Molecular features of non-B, non-C hepatocellular carcinoma: a PCR-array gene expression profiling study

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) usually develops following chronic liver inflammation caused by hepatitis C or B virus. Through expression profiling in a rare type of HCC, for which the causes are unknown, we sought to find key genes responsible for each step of hepatocarcinogenesis in the absence of viral influence. METHODS: We used 68 non-B, non-C liver tissues (20 HCC, 17 non-tumor, 31 normal liver) for expression profiling with PCR-array carrying 3072 genes known to be expressed in liver tissues. To select the differentially expressed genes, we performed random permutation testing. A weighted voting classification algorithm was used to confirm the reliability of gene selection. We then compared these genes with the results of previous expression profiling studies. RESULTS: A total of 220 differentially expressed genes were selected by random permutation tests. The classification accuracies using these genes were 91.8, 92.0 and 100.0% by a leave-one-out cross-validation, an additional PCR-array dataset and a Stanford DNA microarray dataset, respectively. By comparing our results with previous reports on virus-infected HCC, four genes (ALB, A2M, ECHS1 and IGFBP3) were commonly selected in some studies. CONCLUSIONS: The 220 differentially expressed genes selected by PCR-array are potentially responsible for hepatocarcinogenesis in the absence of viral influence.     

Cloning and expression of MXR7 gene in human HCC tissue

AIM To clone and identify the whole cDNA of MXR7 gene and to find out its expression in human HCC, and normal tissues.METHODS The DNA primers were designed and synthesized according to the whole cDNA sequence of MXR7 gene. The cDNA of human HCC was taken as the template while the cDNA of MXR7 gene was synthesized by polymerase chain reaction (PCR). Recombinant DNA conforming to reading frame was constructed by connecting purified PCR product of the cDNA of MXR7 gene with expression vector pGEX-5X-1 of fusion protein. The plasmid MXR7/pGEX-5X-1 was identified by sequencing. Using 32P labeled MXR7 cDNA as probe, MXR7 mRNA expression was detected by Northern blot analysis in 12 different human normal tissues, 7 preoperatively untreated non-liver tumor tissues, 30 preoperatively untreated HCC, the paracancerous liver tissues and 12 normal liver tissues samples.RESULTS Restriction enzyme and sequence analysis confirmed that the insertion sequence in vector pGEX-5X-1 was the same as the cDNA sequence of MXR7 gene. Northern blot analysis showed no expression of MXR7 mRNA in 12 kinds of normal human tissues including liver, 7 tumor tissues in other sites and 12 normal liver tissues, the frequencies of MXR7 mRNA expression in HCC and paracancerous liver tissues were 76.6% and 13.3%, respectively.The frequency of MXR7 mRNA expression in HCC without elevation of serum AFP and in HCC ＜5cm was 90% (9/10) and 83.3% (5/6),respectively.CONCLUSION MXR7 mRNA is highly expressed in human HCC, which is specific and occurs at an early stage of HCC, suggesting MXR7 mRNA can be a tumor biomarker for HCC. The detection of MXR7 mRNA expression in the biopsied liver tissue is helpful in discovering early subclinical liver cancer in those with negative serum AFP.     

DNA微阵矩技术研究肝纤维化差异基因表达谱变化

应用DNA微阵矩技术研究乙型肝炎肝硬化组织差异基因表达谱改变,以寻找肝纤维化相关基因,探讨肝纤维化的发病机理。抽提正常肝组织和肝硬化组织中的mRNA来制备探针,经杂交、洗涤后,通过计算机扫描分析正常肝组织和肝硬化组织基因表达谱的差异情况。在10000个候选基因中,筛选出99条差异表达基因,表达上调的有45条,表达下调的有54条。其中未知基因9条。因此基于DNA微阵矩技术的肝纤维化基因表达谱分析能够高通量筛选与肝纤维化发生发展相关的基因。     

用cDNA阵列技术研究黄曲霉毒素B1诱发树鼩肝癌形成过程中的基因变化

目的 对树鼩肝癌形成过程中的基因表达差异进行动态分析,探讨肝癌发生的分子机制。 方法用cDNA阵列技术,将2例黄曲霉毒素B1诱发的树鼩肝癌组织分别与其癌旁组织和其肝癌形成前的活检肝组织、实验前对照和同期对照肝组织进行基因表达水平的6种比较分析。结果 不同的比较方式所显示的差异表达的基因谱不同,可归为4类:癌组织表达高于癌旁组织、癌旁组织表达高于癌发生前肝的组织;癌与癌旁表达水平相仿,但高于癌发生前的肝组织;癌组织下调,低于癌旁组织;癌发生前表达上调,在癌发生后表达下调。 结论 对肝癌形成过程中不同时期的肝组织基因表达水平进行动态对比分析,有助于阐明肝癌发生的分子机制并最终筛选出与肝癌发生有关的关键基因。     

人肝癌父系表达基因10的遗传印记状态

目的 研究人肝癌组织及肝癌细胞株中父系表达基因10(PEG10)的遗传印记状态.方法 从40例肝癌及其癌旁组织、15例正常肝组织、5株肝癌细胞(PLC/PRF/5、SMMC 7721、HepG2、Hep3B、SK-HEP-1)、2株正常肝细胞(changliver、HL7702)中提取基因组DNA,针对PEG10基因单核苷酸多态性位点设计引物进行PCR,扩增片段经测序分析基因型;从杂合样本中提取总RNA进行RT-PCR,对扩增产物测序以检测等位基因表达状态,同时进行实时荧光定量RT-PCR检测PEG10表达水平.计量资料以均数±标准差(-x±s)表示,组间比较用t检验与方差分析;两组率的比较用x2检验.结果 40例肝癌及其癌旁组织中16例呈杂合状态,15例正常肝组织中3例呈杂合状态,肝癌细胞HepG2扩增片段测序检测到一杂合突变位点,其余组织及细胞株均为纯合状态.杂合样本中,82.4%(14/17)肝癌样本(包括组织及肝癌细胞株)中PEG10基因呈双等位基因表达,发生印记丢失;17.6%(3/17)肝癌样本呈单等位基因表达,提示印记存在.PEG10在癌旁及正常肝组织中几乎不表达.发生印记丢失的肝癌组织与印记存在的肝癌组织相比,PEG10表达水平的差异无统计学意义(t=1.311,P＞0.05).结论 大多数肝癌组织中存在PEG10印记丢失现象,PEG 10印记状态与其在肝癌组织中的表达水平无明确关系.     

Expression of cytochrome P4502E1 gene in hepatocellular carcinoma

AIM: To investigate cytochrome P4502E1 (CYP2E1) gene expression in occurrence and progression of hepatocellular carcinoma (HCC). METHODS: The human liver arrayed library was spotted onto the nylon membranes to make cDNA array. Hybridization of cDNA array was performed with labeled probes synthesized from RNA isolated from HCC and adjacent liver tissues. Sprague-Dawley rats were administrated diethylnitrosamine (DENA) to induce HCC. CYP2E1 expression was detected by the method of RT-PCR and Northern blot analysis. RESULTS: CYP2E1 was found by cDNA array hybridization to express differently between HCC and liver tissues. CYP2E1 only expressed in liver, but did not express in HCC tissues and expressed lowly in cirrhotic tissues. In the progression of cirrhosis and HCC, the expression level of CYP2E1 was gradually decreased and hardly detected until the late stage of HCC. CONCLUSION: Using arrayed library to make cDNA arrays is an effective method to find differential expression genes. CYP2E1 is a unique gene expressing in liver but did not express in HCC. CYP2E1 expression descended along with the initiation and progression of HCC, which is noteworthy further investigations in its significance in the development of HCC.     

Expression of the midkine gene in human hepatocellular carcinomas

BACKGROUND/AIMS: Aberrant expression of Midkine (MK) has been found in various human carcinomas including hepatocellular carcinoma (HCC). The aim of study is to identify the incidence of MK expression in tumor and surrounding non-tumor tissues of the liver, and to find the correlation of MK expression with other tumor markers. METHODOLOGY: Liver tissues were obtained from 16 patients with HCC and 4 with metastatic liver cancer. Background diseases of the HCC patients include liver cirrhosis and chronic hepatitis of type B or C. RNA was prepared from both cancerous and surrounding non-cancerous tissues, and analyzed for the presence of MK mRNA by RT-PCR, PCR-Southern blot, and Northern blot analysis. RESULTS: MK expression was detected in 12 (75%) of 16 HCCs by PCR-Southern blot analysis, the most sensitive of the 3 methods. Three of 9 surrounding cirrhotic tissues were weakly positive for MK expression, and none of chronic hepatitis and 4 normal tissues were negative. No significant difference was found in clinical and pathological parameters between MK negative and positive cases. Among metastatic cancers, 1 of gastric origin was positive for MK expression, but 1 each of chorangiocellular, gall bladder, and gastrinoma origin was negative. CONCLUSIONS: These results suggest that MK is expressed in the majority of HCC tissues and rarely in surrounding tissues in chronic liver diseases.     

A comprehensive gene expression analysis of human hepatocellular carcinoma cell lines as components of a bioartificial liver using a radial flow bioreactor.

BACKGROUND/AIMS: The cells constituting a bioartificial liver are crucial for an effective liver support system. We compared global gene expression profiles in a radial flow bioreactor or a monolayer culture of three functional liver cell lines previously established from human hepatocellular carcinoma. METHODS: The expressions of 60,000 genes of the FLC-4, FLC-5, and FLC-7 cell lines were analyzed by the microarray technique with the Affymetrix GeneChip system. Global gene expression profiles were compared with two-way cluster analysis. Several liver function-related genes were compared between the bioreactor and culture conditions. RESULTS: Cluster analysis revealed that gene expression profiles of bioreactor-grown cells resembled those of the normal liver. Genes related to cellular structure were highly expressed in the bioreactor-grown cells, while genes involved in proliferation or carcinogenesis were suppressed. In the bioreactor-grown cells, some genes for liver functions were expressed at a level similar to that in normal liver, although none of the cell lines expressed the complete set of genes encoding ammonium metabolism or cytochrome P450 species. CONCLUSION: The high-density three-dimensional culture in the radial flow bioreactor prompted differentiation of the cells. These data may be useful for improving the cells by genetic or pharmacological reinforcement and for monitoring bioartificial livers.