U230A芯片动态观察非酒精性脂肪性肝病大鼠肝脏基因表达

目的探讨大鼠非酒精性脂肪性肝病（NAFLD）发生过程中肝脏基因表达谱的改变。方法通过持续24周高脂饮食诱导大鼠NAFLD模型，应用U230A芯片检测不同造模时期肝脏基因表达，并设普通饮食饲养大鼠作对照。结果与对照大鼠相比，造模4周和8周时差异表达基因数分别为426条和540条，上调基因主要为细胞内磷酸化酶基因、代谢酶基因、脂肪酸结合蛋白基因，细胞色素P450基因以及细胞转录和分化基因等，下调基因主要为离子通道基因、激素受体基因、细胞黏附基因以及细胞骨架基因等；12周时差异表达基因有501条，其中表达上调352条，除上述基因外，还包括白细胞介素，Toll样受体4等炎症和凋亡相关基因；16周时差异表达的基因有665条，其中上调基因430条，炎症和凋亡相关基因表达进一步增加，且Ⅰ型胶原等纤维化相关基因出现表达上调，而细胞再生相关基因表达下调；24周时差异表达的基因有663条，其中上调基因512条，除上述基因表达差异外，主要包括成纤维细胞生长因子，转化生长因子和胰岛素样生长因子等纤维化相关基因。在所有表达差异的基因中，随着时间进展表达持续上调的基因共128条，其中成脂相关基因10条，代谢酶基因46条，炎症相关基因15条、凋亡相关基因10条，纤维化相关基因16条；持续下调的基因有52条，包括激素受体相关基因6条，细胞再生相关基因5条，电子转运基因11条等。结论高脂饮食大鼠肝脏基因谱呈动态改变，并与NAFLD的组织学进展一致。     

应用基因芯片技术筛选大鼠再生肝中差异表达基因

目的应用基因芯片技术筛选大鼠再生肝中差异表达基因,为探讨急性肝功能衰竭的发病机制提供基础。方法雄性SD大鼠行95％肝部分切除术,用含1176个基因的大鼠尼龙膜微阵列筛选大鼠再生肝组织中差异表达的基因。RT—PCR随机验证若干差异表达基因在微阵列中的表达。结果再生肝组织有138个基因明显较对照组织表达高,它们主要是生长因子基因、核受体基因、核糖体蛋白基因、应急反应蛋白基因、细胞周期调节基因、神经递质基因等;下调基因共50个,主要为代谢酶基因、激素受体基因及表面抗原基因等。结论cDNA微阵列技术有助于研究急性肝功能衰竭的发病机制,并提供诊断和治疗的潜在靶点。     

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.     

二甲双胍对非酒精性脂肪性肝病大鼠肝脏基因表达谱的影响

目的 探讨二甲双胍对非酒精性脂肪性肝病(NAFLD)大鼠造模过程中肝脏基因表达谱的影响.方法 雄性Sprague-Dawley大鼠随机分为3组:对照组6只,模型组和治疗组各12只.治疗组在给予高脂饮食的同时,于实验开始第4周起按每天250 mg/kg加入二甲双胍干预.于实验第24周时处死,应用大鼠U230A芯片检测肝脏基因表达的改变.结果 与模型组相比,二甲双胍治疗组出现差异表达的基因共483条,其中上调基因133条,主要为激素受体基因、细胞周期相关基因及离子通道基因等,胰岛素受体及其底物、瘦素受体基因均有不同程度的表达上调,与模型组比较上升约3～7倍;下调基因350条,包括代谢酶相关基因、脂肪酸结合蛋白基因、细胞色素P450相关基因、炎症和凋亡相关基因,与纤维化相关的基因也略有下降.结论 从基因学角度分析显示二甲双胍可减轻肝脂肪变,改善肝脏炎症损伤和纤维化程度.     

基因芯片研究甘草酸对大鼠肝星状细胞转化生长因子-β信号通路的影响

目的应用基因芯片技术探讨甘草酸对大鼠肝星状细胞（HSC）转化生长因子（TGF）-β信号转导通路相关基因表达的作用。方法体外分离、培养大鼠HSC，分为对照组、TGF-β1组（5ng／ml）、TGF-β1（5ng／ml）＋甘草酸（100μmol／L）组，10h后分别收集细胞抽提总RNA。应用针对于TGF-β/BMP信号转导通路的GEArray^TM Q基因芯片技术，筛选出甘草酸作用后HSC中TGF-β信号通路表达发生明显改变的相关基因。结果经TGF-β1作用后上调，再经甘草酸作用后下调的基因有16项，占16．7％（如Smad蛋白2，Smad蛋白3，Smad蛋白7，a1Ⅲ型前胶原，a2 Ⅰ型前胶原，纤溶酶原激活物抑制剂1）；经TGF-β1作用后下调，再经甘草酸作用后上调的基因有5项，占5．2％（如骨成型蛋白7，胰岛素生长因子结合蛋白3等）；经TGF-β1作用后上调，再经甘草酸作用后上调更显著的基因有2项，占2．1％（转化生长因子2型受体，转化生长因子受体3）。并用RT-PCR法验证了部分基因（Smad蛋白2，Smad蛋白3，Smad蛋白7）mRNA的表达与基因芯片中变化趋势的一致性。结论甘草酸可能通过干预大鼠HSC中TGF-β信号通路，减少胶原合成，促进细胞外基质降解而发挥抗纤维化的分子机制。     

Wnt信号通路相关分子在肝纤维化中的表达水平变化及其意义

目的 观察Wnt信号通路相关分子在实验性肝纤维化组织中表达水平的变化.探讨其在肝纤维化发生中的作用.方法 建立大鼠肝纤维化动物模型.用Wnt信号通路PCR array功能基因芯片观察Wnt信号通路相关分子在肝纤维化模型中表达水平的变化.以模型组较正常组表达上调或下调2倍为差异表达基因;利用免疫组化及Western印迹观察平滑肌肌动蛋白、Wnt4、Frizzled2、β-钙粘蛋白在肝纤维化组织表达水平的变化.结果 芯片检测发现共有36条基因发生了显著改变,模型组较正常组上调2倍的基因有25个,上调基因主要包括Wnt5a类基因,如Wnt4、Wnt5a、Wnt11等,分别上调了13.9、16.5和2.17倍;较正常组下调2倍的基因有11个,主要为Wnt1、Wnt3等.分别下调了2.32、2.15倍.免疫组化及Western印迹检测发现模型组肝组织Wnt4、Frizzled2的表达水平较正常组显著上升,而磷酸化的β-钙粘蛋白的表达水平下降.结论 经典及非经典Wnt信号通路均可能参与了实验性肝纤维化的发生机制.     

己酮可可碱对非酒精性脂肪性肝炎大鼠肝脏基因表达谱的影响

己酮可可碱（PTX）可减轻高脂饮食大鼠脂肪性肝炎和肝纤维化的程度,但其作用机制尚未明确.目的：探讨PTX对非酒精性脂肪性肝炎（NASH）大鼠肝脏基因表达谱的影响.方法：24只Spragu-Dawley大鼠在高脂饮食4周后随机分为模型组（n=12）和PTX干预组（n=12,PTX每天100 mg/kg）,并继续予高脂饮食;6只普通饮食饲养大鼠作为对照组.于实验第24周处死大鼠,应用含15 650条基因的大鼠U230A芯片检测肝脏基因表达的改变.结果：与模型组相比,PTX干预组共出现370条差异表达基因,其中模型组较对照组上调而PTX干预后下调的基因57条,主要包括炎症/免疫反应相关基因、细胞信号转导相关基因、细胞外基质和细胞黏附分子基因、代谢酶和生物转化相关基因、离子通道/运输蛋白基因等;模型组较对照组下调而PTX干预后上调的基因25条,其功能涉及细胞信号转导、脂质代谢、生物转化等.结论：PTX可能通过影响NASH大鼠肝脏多种结构和功能基因的表达而有助于NASH和肝纤维化的防治.     

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微矩阵研究肝纤维化基因表达的变化

目的：筛选在肝纤维化过程中发生异常表达的基因。方法：建立四氯化碳诱导肝纤维化大鼠模型：选取正常和造模大鼠肝脏各1只进行cDNA微矩阵（microarray)研究；从cDNA微矩阵研究结果中选取一个在造模大鼠肝脏中表达明显异常的基因（smurf 2)，用半定量RT－PCR检测此基因在不同实验阶段（第1、2、4、8）两组大鼠中的表达变化。结果：通过cDNA微矩阵发现在肝纤维化过程中，smurf 2、PTAFR、CYP2D6、FGG等许多和炎症、代谢有关的基因表达均上调。通过半定量RT－PCR研究发现经四氯化碳处理等1周时smurf 2基因在造模大鼠肝脏中表达与正常大鼠表达无明显变化，第2周时表达下调，第4周时表达明显上调，第8周时该基因表达又趋于下降。结论：smurf 2基因转录水平的变化，影响smad-TGFβ信号传导的调控，可能参与了四氯化碳诱导大鼠肝纤维化的形成过程。     

沉默结缔组织生长因子对鼠转化生长因子β/Smads信号的影响

目的 研究小干扰RNA(siRNA)沉默结缔组织生长因子(CTGF)对大鼠转化生长因子(TGF)β/Smads信号通路的影响.方法 将化学合成CTGF siRNA转染肝星状细胞(HSC)T6和经门静脉注入CCl4诱导6周的肝纤维化大鼠,设空白及随机siRNA对照,抽提HSC T6及大鼠肝组织总RNA和蛋白质,应用Western blot和RT-PCR检测HSC T6及肝组织CTGF及TGF β1,Smad2、3、7蛋白质和基因表达. 结果与空白对照组相比,siRNA能显著下调HSC T6 CTGF蛋白表达,以48 h最明显,CTGF蛋白表达下调94%±4%(t=46.196,P<0.01),而TGF β1、Smad2,3,7 mRNA表达差异无统计学意义;模型组及对照siRNA组,CCl4诱导的大鼠肝组织CTGF和TGF β1蛋白表达明显上调,与模型组相比,CTGF siRNA组大鼠肝组织CTGF及TGF β1蛋白表达分别下调95%±2%(F=21.234,P<0.01)和74%±8%(F=13.464,P<0.05),但Smad2和Smad7蛋白表达无明显改变. 结论沉默CTGF基因表达对大鼠肝TGF β/Smads信号具有阻抑作用.     

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.     

Halofuginone, an inhibitor of collagen synthesis by rat stellate cells, stimulates insulin-like growth factor binding protein-1 synthesis by hepatocytes

BACKGROUND/AIMS: Halofuginone, an inhibitor of collagen synthesis, prevented and caused resolution of established hepatic fibrosis. A genomic approach in vivo was used to search for additional genes responsible for halofuginone mode of action. METHODS: Fibrosis was induced in rats by thioacetamide (TAA) and evaluated by collagen type I gene expression and the levels of collagen, tissue inhibitors of metalloproteinases-2 and smooth-muscle actin. Halofuginone was given in the diet. cDNA from liver biopsies was hybridized on Atlas arrays comprising of 588 genes. The results were confirmed by Northern blots and in situ hybridization. RESULTS: Insulin-like growth factor binding protein-1 (IGFBP-1) was one of the 13 genes differentially expressed in the fibrotic liver after halofuginone treatment. After 2 and 4 weeks, halofuginone prevented the TAA-induced down-regulation of IGFBP-1 gene expression. Halofuginone also prevented the TAA-dependent changes in IGFBP-3 gene expression. Halofuginone affected IGFBP-1 synthesis in rat hepatocytes and cells of hepatocyte origin and caused time- and dose-dependent increases in the IGFBP-1 gene expression and synthesis by HepG2 cells. The IGFBP-1 secreted by HepG2-inhibited stellate cell motility. CONCLUSIONS: Halofuginone is an anti-fibrotic drug that inhibits collagen synthesis by stellate cells and preventing alteration in the synthesis of IGFBPs by hepatic cells.     

Hepatic gene expression in patients with obesity-related non-alcoholic steatohepatitis.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is among the most common causes of chronic liver disease. NAFLD includes a spectrum of clinicopathologic syndromes that includes non-alcoholic steatohepatitis (NASH) that has potential for progression. The pathogenesis of NASH is poorly characterized. AIM: This study was designed to identify differences in hepatic gene expression in patients with NASH and to relate such differences to their clinical characteristics. DESIGN: Consecutive patients undergoing bariatric surgery were prospectively recruited. Extensive clinical data and two liver biopsy specimens were obtained at the time of enrollment. A single hepatopathologist reviewed and classified the liver biopsies. Patients with excessive alcohol use and other causes of liver disease were excluded. A group of 29 NASH patients, 12 with steatosis alone, seven obese controls and six non-obese controls were selected for further investigation. Customized cDNA microarrays containing 5220 relevant genes were designed specifically for this study. Microarray experiments were run in triplicate for each sample and a selected group of genes were confirmed using real-time PCR. OUTCOME MEASURE: Differential hepatic gene expressions in patients with NASH as compared with controls. Results: Thirty-four genes with significant differential expression were identified in patients with NASH when compared with non-obese controls. Moreover, 19 of these genes showed no significant expression differences in obese vs. non-obese controls, suggesting a stronger association of these genes to NASH. CONCLUSIONS: Several differentially expressed genes in patients with NASH are related to lipid metabolism and extracellular matrix remodeling. Additionally, genes related to liver regeneration, apoptosis, and the detoxification process were differentially expressed. These findings may help clarify the molecular pathogenesis of NASH and identify potential targets for therapeutic intervention.