Differential gene expression between chronic hepatitis B and C hepatic lesion

BACKGROUND & AIMS: Complementary DNA (cDNA) microarray technology allows simultaneous expression analysis of hundreds to thousands of genes. We applied the cDNA microarray technique to clarify gene expression profiles in chronic viral hepatitis tissue lesions. METHODS: We made cDNA microarrays consisting of 1080 human cDNAs and analyzed gene expression using labeled cDNAs prepared from 6 normal, 12 chronic hepatitis B, and 14 chronic hepatitis C liver tissues. Relative expression ratios of individual genes were obtained by comparing hybridization of Cy5-labeled cDNAs from chronic hepatitis lesions and Cy3-labeled cDNA from normal liver tissue. RESULTS: Hierarchical clustering analysis of the gene expression profiles in 26 patients showed that the patients were clustered into 2 groups with respect to similarities in differentially expressed genes. Hepatitis B and C virus infection, but not age, sex, or histology of hepatitis, were significant factors determining clustering (P < 0.05). In hepatitis B tissue lesions, genes involved in inflammation were predominant, whereas in hepatitis C, expression of anti-inflammatory response genes was relatively dominant. CONCLUSIONS: These findings shed new light on the possible differential molecular mechanisms in the pathogenesis of hepatitis caused by hepatitis B virus and hepatitis C virus infection, from which hepatocellular carcinoma frequently develops.     

Differential expression genes analyzed by cDNA array in the regulation of rat hepatic fibrogenesis.

PURPOSE: To analyze the gene expression pattern in rat hepatic fibrogenesis and further assess the role of some key genes during the pathological process. METHODS: Hepatic fibrosis was induced by intraperitoneal injection of dimethylnitrosamine or carbon tetrachloride (CCl(4)) injection subcutaneously in rats, and identification of the hepatic fibrosis related genes with cDNA microarray was performed. After some key genes up-regulated during the development of hepatic fibrosis were screened and confirmed, their effects on the function of the activated rat hepatic stellate cells (HSC) were assessed using the small interfering RNA (siRNA) technique. RESULTS: Using an Atlas rat cDNA array, a number of differentially expressed genes in fibrotic liver tissues were identified compared with non-diseased control. A total of 15 genes predominantly associated with the mitogen-activated protein kinase (MAPK) signal transduction pathway were upregulated in the fibrotic liver. Immunohistochemical study revealed that the expressions of both extracellular signal-regulated kinases (ERK) and ribosomal protein S6 kinase (RSK), two of the key genes in the MAPK pathway, were remarkably induced, which was closely correlated to that of collagen types I and III during the development of hepatic fibrosis. Transfection of siRNA targeting ERK1 mRNA (siERK1) into HSC led to a 66% and 72% reduction of ERK1 mRNA and protein expression, respectively. Furthermore, siERK1 exerted the inhibition of the proliferation of HSC, accompanied by the induction of HSC apoptosis and reduction of collagen types I and III. In addition, siERK1 abolished the effect of platelet-derived growth factor-BB on the proliferation of HSC. CONCLUSIONS: The present study provided strong evidence for the participation of the MAPK pathway in the pathogenesis of hepatic fibrosis. Selective targeting of ERK1 inhibitors to HSC might present as a novel strategy for the treatment of hepatic fibrosis.     

基因芯片技术研究肝纤维化相关基因

目的通过对大鼠肝纤维化发生过程中基因表达差异性的分析，探讨肝纤维化的发生机制。方法采用四氯化碳((CC14)诱导大鼠肝纤维化，于用药2周、4周、6周、8周分别取肝组织提取总RNA，分离纯化poly(A)^ RNA，逆转录并用[α-^33P]dATP制备探针，与含1176个基因的大鼠cDNA微阵列杂交及洗涤，信号检测，微阵列图像分析和计算机软件处理数据。结果用药4周时，筛选出的166条差异表达基因中，上调基因156条，主要为细胞骨架和动力蛋白基因、激素受体基因、细胞内蛋白磷酸化酶基因和ras相关蛋白基因等；下调基因主要为P450相关基因、核糖体蛋白基因、脂肪酸结合蛋白基因和生长因子受体基因等。用药8周时，筛选出差异表达基因中上调基因246条，主要为细胞外信息传导基因、生长因子相关基因、细胞周期相关基因、凋亡相关蛋白基因和激素受体基因等；下调基因主要为脂质代谢基因、激素受体基因和氨基酸受体基因等。其中部分差异表达基因用RT—PCR和Northern印迹法加以证实。结论肝纤维化形成过程中存在多个表达水平不同的基因，涉及丝裂原活化蛋白激酶(MAPK)信号通路，同一基因在不同时期表达水平也不一样，值得进一步研究。     

B7-H4 mediates inhibition of T cell responses by activated murine hepatic stellate cells

Liver fibrosis is mediated by the transformation of hepatic stellate cells (HSC) from a quiescent to an activated state. To understand the role of HSC in liver immunity, we investigated the effect of this transition on T cell stimulation in vitro. Unlike quiescent HSC, activated HSC did not induce proliferation of antigen-specific T cells. Phenotypic analysis of quiescent and activated HSC revealed that activated HSC expressed the coinhibitory molecule B7-H4. Silencing B7-H4 by small interfering RNA (siRNA) in activated HSC restored the ability of T cells to proliferate, differentiate, and regain effector recall responses. Furthermore, expression of B7-H4 on HSC inhibits early T cell activation and addition of exogenous interleukin (IL)-2 reversed the T cell anergy induced by activated HSC. Conclusion: These studies reveal a novel role for activated HSC in the attenuation of intrahepatic T cell responses by way of expression of the coinhibitory molecule B7-H4, and may provide fundamental insight into intrahepatic immunity during liver fibrogenesis.     

应用抑制性消减杂交技术筛选转化生长因子β1刺激LX02细胞的反式调节基因

目的 构建转化生长因子(TGF)β1刺激大鼠肝星状细胞(LX02)反式调节基因的cDNA消减文库,筛选并克隆TGF β1反式调节相关基因,以阐明TGF β1介导肝纤维化的分子生物学机制.方法 以TGF β1刺激LX02细胞,同时以磷酸盐缓冲液刺激的LX02细胞作为对照.提取mRNA并逆转录为cDNA,经Rsa Ⅰ酶切后,将实验组cDNA分成两组,分别与两种不同的接头衔接,再与对照组cDNA进行两次消减杂交及两次抑制性多聚酶链反应.将产物与pGEM-Teasy载体连接,构建cDNA消减文库,并转染大肠杆菌进行文库扩增;随机挑选克隆经PCR扩增后进行测序及同源性分析. 结果成功构建了TGF β1刺激LX02细胞反式调节基因的cDNA消减文库.文库扩增后得到146个200～1000bp插入片段的阳性克隆;随机挑取其中35个克隆进行测序,30个列序成功,并通过生物信息学分析发现有28个与已知基因序列和2个与未知功能基因序列高度同源.结论 应用抑制性消减杂交技术成功构建了TGF β1刺激LX02细胞反式调节基因的cDNA消减文库,筛选到一些与细胞生长调节、蛋白质合成,信号传导、细胞外基质代谢、扰脂质过氧化等密切相关的蛋白质编码基因,为进一步阐明TGF β1介导肝纤维化的分子生物学机制提供了线索.     

筛选转化生长因子β1刺激肝星状细胞差异表达基因的研究

目的 筛选转化生长因子β 1(TGF β 1)刺激大鼠肝星状细胞(Hsc)的差异表达基因,以揭示TGF β1介导肝纤维化的分子发病机制. 方法分别用Trizol法抽提TGF β1刺激的HSC及磷酸盐缓冲液刺激为对照的HSC总RNA,逆转录合成双链cDNA,制备掺入生物素标记的cDNA探针,与人基因表达谱芯片杂交,用Agilent扫描仪对芯片结果进行扫描,利用软件对差异表达基因进行生物信息学分析. 结果 从13824条目的 基因中筛选出177条差异表达基因,其中123条基因表达上调,其中包括:结缔组织生长因子,微管蛋白ε 1,V型胶原α2,连环蛋白6 2,钙粘蛋白6,2型,Smad3,丝裂源活化蛋白激酶4,生长因子受体结合蛋白7,丝裂原活化蛋白激酶相互作用/丝氨酸/苏氨酸激酶1等;54条基因表达下调,包括:肿瘤坏死因子受体相关因子4,干扰素调节因子7,干扰素诱生蛋白p78,骨形态发生蛋白7,基质gLa蛋白,人类丝氨酸蛋白酶抑制剂进化支B成员8,干扰素刺激基因2.0×104,死亡相关蛋白6,金属硫蛋白1H,超氧化物歧化酶2等;同时筛选到8个未知功能蛋白. 结论 应用基因表达谱芯片技术成功筛选了TGF β 1刺激HSC的差异表达基因,初步揭示了TGF β1致肝纤维化的分子机制是诸多因素共同作用的结果,为进一步寻找新的基因治疗靶点奠定了基础.     

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。     

Inhibitory effects of microRNA 19b in hepatic stellate cell-mediated fibrogenesis

Hepatic stellate cell (HSC) activation is a pivotal event in initiation and progression of hepatic fibrosis and a major contributor to collagen deposition driven by transforming growth factor beta (TGF-β). MicroRNAs (miRs), small noncoding RNAs modulating messenger RNA (mRNA) and protein expression, have emerged as key regulatory molecules in chronic liver disease. We investigated differentially expressed miRs in quiescent and activated HSCs to identify novel regulators of profibrotic TGF-β signaling. miR microarray analysis was performed on quiescent and activated rat HSCs. Members of the miR-17-92 cluster (19a, 19b, 92a) were significantly down-regulated in activated HSCs. Because miR 19b showed the highest fold-change of the cluster members, activated HSCs were transfected with miR 19b mimic or negative control and TGF-β signaling and HSC activation assessed. miR 19b expression was determined in fibrotic rat and human liver specimens. miR 19b mimic negatively regulated TGF-β signaling components demonstrated by decreased TGF-β receptor II (TGF-βRII) and SMAD3 expression. Computational prediction of miR 19b binding to the 3' untranslated region of TGF-βRII was validated by luciferase reporter assay. Inhibition of TGF-β signaling by miR 19b was confirmed by decreased expression of type I collagen and by blocking TGF-β-induced expression of α1(I) and α2(I) procollagen mRNAs. miR 19b blunted the activated HSC phenotype by morphological assessment and decreased smooth muscle α-actin expression. Additionally, miR 19b expression was markedly diminished in fibrotic rat liver compared with normal liver; similarly, miR 19b expression was markedly down-regulated in fibrotic compared with normal human livers. CONCLUSION: miR 19b is a novel regulator of TGF-β signaling in HSCs, suggesting a potential therapeutic approach for hepatic fibrosis.     

乙型肝炎病毒相关的肝硬化患者外周血单个核细胞基因表达谱的研究

目的分析乙型肝炎病毒（HBV）相关的肝硬化患者外周血单个核细胞的基因表达谱，探索慢性HBV相关的肝硬化形成的分子机制。方法应用含14 000条人体cDNA的微阵列芯片和来自外周血单个核细胞的标记cDNA，分析了10例慢性HBV相关肝硬化患者和10例健康人基因表达谱。通过应用GenePix 4000B扫描仪和ImaGene3．0分析软件比较Cy5标记的慢性HBV相关肝硬化来源cDNA与Cy3标记的健康人来源cDNA的杂交结果，获得个体基因的相对表达比值。结果和14 000条基因中，差异表达的基因有199条，占1．42％。其中108条基因表达水平上调，91条基因表达水平下调。这些差异表达的基因主要为细胞信号转导．细胞周期和代谢、凋亡及炎症相关类基因。结论HBV相关肝硬化发病过程中，发观众多基因的差异表达，为进一步阐明慢性HBV相关的肝硬化形成的分子机制提供基础。     

重型乙型肝炎外周血单核细胞基因表达谱研究

目的 应用基因芯片技术建立慢性重型乙型肝炎外周血单核细胞基因表达谱。方法 应用含8192条人体cDNA的微阵列芯片和来自外周血单核细胞的标记cDNA，分析了10例慢性重型乙型肝炎和10例无症状HBsAg携带者（ASC）基因表达谱。通过应用GenePix 4000B扫描仪和ImaGene3.0分析软件比较Cy5标记的慢性重型肝炎来源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249个（3％）表达差异达2倍以上的基因，其中主要是细胞信号和传递，细胞周期和代谢，凋亡及炎症相关类基因（占79％）。结论 这些结果提示了乙型肝炎病毒感染机体致慢性重型肝炎过程中，全面改变了宿主细胞内基因表达，并为进一步对这些差异表达基因的功能研究提供了一个框架。     

Gene expression profile analyses of mice livers injured by Leigongteng

AIM： To analyze the gene expression profiles of mice livers injured by Leigongteng and explore the relationship between the differentially expressed genes and liver damage. METHODS： The experimental mice were randomly divided into a control group and a liver-injured group in which the mice were administrated 33 μγ, of triptolide/ kg per day for 30 d. Liver mRNAs were extracted from animals in both groups and were reverse-transcribed to cDNA with dUTP labeled by different fluorescence （Cy3, Cy5） as hybridization probes. The mixed probes were hybridized with oligonucleotide microarray chips. The fluorescent signal results were acquired by scanner and analyzed with software. RESULTS： Among the 35852 target genes, 29 genes were found to be significantly differentially expressed, with 20 genes up-regulated and 9 genes down-regulated. The reliability of the differentially expressed genes was validated by RT-PCR experiments of 5 randomly selected differentially expressed genes. CONCLUSION： Based on the biological functions of the differentially expressed genes, it is obvious that the occurrence and development of liver damage induced by Leigongteng in mice are highly associated with immune response, metabolism, apoptosis and the cell skeleton of liver cells. This might be important for elucidating the regulatory network of gene expression associated with liver damage and it may also be important for discovering the pathogenic mechanisms of liver damage induced by Leigongteng.     

Gene expression analysis of primary normal human hepatocytes infected with human hepatitis B virus

AIM: To find the relationship between hepatitis B virus (HBV) and hepatocytes during the initial state of infection by cDNA microarray. METHODS: Primary normal human hepatocytes (PNHHs) were isolated and infected with HBV. From the PNHHs, RNA was isolated and inverted into complement DNA (cDNA) with Cy3- or Cy5- labeled dUTP for microarray analysis. The labeled cDNA was hybridized with microarray chip, including 4224 cDNAs. From the image of the microarray, expression profiles were produced and some of them were confirmed by RT-PCR, immunoblot analysis, and NF-κB luciferase reporter assay. RESULTS: From the cDNA microarray, we obtained 98 differentially regulated genes. Of the 98 genes, 53 were up regulated and 45 down regulated. Interestingly, in the up regulated genes, we found the TNF signaling pathway-related genes: LT-α, TRAF2, and NIK. By using RT-PCR, we confirmed the up-regulation of these genes in HepG2, Huh7, and Chang liver cells, which were trans-fected with pHBV1.2x, a plasmid encoding all HBV messages. Moreover, these three genes participated in HBV-mediated NF-κB activation. CONCLUSION: During the initial state of HBV infection, hepatocytes facilitate the activation of NF-κB through up regulation of LT-α, TRAF2, and NIK.     

Lhx2-/- mice develop liver fibrosis

Liver fibrosis is a wound-healing response to chronic injury of any type and is characterized by a progressive increase in deposition of extracellular matrix (ECM) proteins, the major source of which are activated hepatic stellate cells (HSCs). Because the LIM homeobox gene Lhx2 is expressed in HSCs and liver development in Lhx2(-/-) mice is disrupted, we analyzed liver development in Lhx2(-/-) embryos in detail. Lhx2(-/-) embryos contain numerous activated HSCs and display a progressively increased deposition of the ECM proteins associated with liver fibrosis, suggesting that Lhx2 inhibits HSC activation. Transfection of Lhx2 cDNA into a human HSC line down-regulates expression of genes characteristic of activated HSCs. Moreover, the Lhx2(-/-) liver display a disrupted cellular organization and an altered gene expression pattern of the intrahepatic endodermal cells, and the increased deposition of ECM proteins precedes these abnormalities. Collectively these results show that Lhx2 negatively regulates HSC activation, and its inactivation in developing HSCs appears therefore to mimic the signals that are triggered by the wound-healing response to chronic liver injury. This study establishes a spontaneous and reproducible animal model for hepatic fibrosis and reveals that Lhx2 expression in HSCs is important for proper cellular organization and differentiation of the liver.     

肝星状细胞激活与ICAM-1的表达

目的探讨肝星状细胞（ＨＳＣ）的活化与细胞间粘附分子１（ＩＣＡＭ１）表达的关系．方法用链酶蛋白酶和胶原酶原位灌流，Ｎｙｃｏｄｅｎｚ密度梯度离心分离大鼠ＨＳＣ，并进行体外培养，应用免疫组织化学方法观察静息或活化状态下的ＨＳＣ中ＩＣＡＭ１表达．结果静息的ＨＳＣ不表达ＩＣＡＭ１，而活化的ＨＳＣ表达ＩＣＡＭ１，且随着培养时间的延长ＩＣＡＭ１表达量逐渐增加．结论ＩＣＡＭ１表达与ＨＳＣ活化及肝纤维化的发生有关．     

采用人基因表达谱芯片筛选肺癌转移相关基因的初步研究

目的应用基因芯片技术分析人高、低转移肺巨细胞癌细胞株的基因差异表达谱，筛选与肺癌转移相关基因。方法提取人高转移肺巨细胞株95D和人低转移肺巨细胞株95C的mRNA，通过逆转录，制备分别用cy5-dUTP和cy3-dUTP进行标记的cDNA探针，并与芯片杂交。经过ScanArray3000扫描仪扫描，获取图象及对杂交信号进行数据分析，筛选出95D和95C细胞表达差异的基因谱，并对其中部分基因进行RT—PCR分析、验证。结果在所检测95C和95D细胞中，466条基因有表达差异，其中具有同-Genebank量的有108对，上调基因47对，下调基因61对。对其中KIAA1108、PGR1、JWA、S182、Jab1部分差异表达基因，经RT-PCR技术验证，结果与基因芯片基本符合。结论肺癌转移过程与多种基因作用有关，基因芯片技术可为肺癌转移相关基因的筛选，提供有效方法。     

Role of the receptor for advanced glycation end products in hepatic fibrosis

AIM: To study the role of advanced glycation end products (AGE) and their specific receptor (RAGE) in the pathogenesis of liver fibrogenesis. METHODS: In vitro RAGE expression and extracellular matrix-related gene expression in both rat and human hepatic stellate cells (HSC) were measured after stimulation with the two RAGE ligands, advanced glycation end product-bovine serum albumin (AGEBSA) and Nε-(carboxymethyl) lysine (CML)-BSA, or with tumor necrosis factor-α (TNF-α). In vivo RAGE expression was examined in models of hepatic fibrosis induced by bile duct ligation or thioacetamide. The effects of AGE-BSA and CML-BSA on HSC proliferation, signal transduction and profibrogenic gene expression were studied in vitro. RESULTS: In hepatic fibrosis, RAGE expression was enhanced in activated HSC, and also in endothelial cells, inflammatory cells and activated bile duct epithelia. HSC expressed RAGE which was upregulated after stimulation with AGE-BSA, CML-BSA, and TNF-α. RAGE stimulation with AGE-BSA and CML-BSA did not alter HSC proliferation, apoptosis, fibrogenic signal transduction and fibrosis- or fibrolysis-related gene expression, except for marginal upregulation of procollagen α1(Ⅰ) mRNA by AGE-BSA. CONCLUSION: Despite upregulation of RAGE in activated HSC, RAGE stimulation by AGE does not alter their fibrogenic activation. Therefore, RAGE does not contribute directly to hepatic fibrogenesis.