Molecular pro filing of genes specifically up-regulated during

为了寻找在癌变促进阶段稳定上调的基因，抑制性PCR消减杂交和随后的Northern杂交使用基于中期肝脏生物测定的苯巴比妥(PB)促进模型进行筛选。用二乙基亚硝胺(DEN)灌胃2周后，给大鼠灌胃PB，最长可达周。为了进行比较，在促进阶段的饮食中给予乙炔雌二醇(EE)或丁基羟基甲苯(BHT)的动物也包括在内。大鼠于第3周行肝部分切除(PH)，并在第8周检测PB的剂量依赖关系及对典型非遗传毒性致癌物的反应性。在PB处理的第10天，从肝脏中上调的基因群中，通过单独从DEN+PH的基础表达中减去，总共分离到67个不同的基因片段。通过Northern杂交筛选信号可检测到的48个基因，有16个基因在TH…中表达上调在促进的第8周，与未促进的肝脏的基础表达相比，PB-和EE-处理组常见的更多的肝脏。这些基因中的大多数在8周时也被BHT处理上调，并且在DEN(-)，PH(-)未处理的大鼠肝脏中也有结构性表达。在PB和EE启动子共同上调且对非遗传毒性致癌物无反应的非启动肝细胞癌基因中，有6个基因在周PBCAP中高表达，哺乳动物ABC半转运蛋白、载脂蛋白A4、核受体结合因子-2、CD81、假设蛋白HSPC014和1个未知基因普遍表达。通过对两阶段致癌模型的分析，这些基因可能成为筛选非遗传毒性肝癌致癌物的候选生物标志物。此外，在硫代乙酰胺促进的肝脏中，APOA4mRNA信号针对GST-P阳性灶进行了原位定位。尽管APOA4在肝癌发生中的作用还有待进一步研究，但APOA4可能与肿瘤病变的形成有关。较少

In search of genes steadily up-regulated during the promotion stage in carcinogenesis, suppression PCR subtractive hybridization and following Northern blot screening were performed using a phenobarbital (PB)-promotion model based on a medium-term liver bioassay. Two weeks after an initiation with diethylnitrosamine (DEN), rats were given PB for up to 64 weeks. For comparison, animals given ethinylestradiol (EE) or butylated hydroxytoluene (BHT) in the diet at promotion stage were also included. Rats were subjected to partial hepatectomy (PH) at week 3.In addition, dose-dependence of PB at week 8 of promotion and responsiveness to representative non-genotoxic carcinogens without DEN-initiation were examined. Fragments of a total of 67 different genes were isolated from the up-regulated gene population in the liver at day 10 of PB-treatment by subtracting from basal expression of DEN+PH alone. By Northern blot screening for signal-detectable 48 genes, 16 genes showed up-regulation in th … More e livers at week 8 of promotion, common to the PB-and EE-treatments as compared to the basal expression of unpromoted liver. The majority of these genes were also up-regulated at week 8 by BHT-treatment, and were also constitutively expressed in the DEN(-), PH(-)-untreated rat livers. Among the up-regulated genes common to the PB- and EE-promotion, and not responding to the non-genotoxic carcinogens in uninitiated liver, the following 6 genes showed overexpression in PBpromoted hepatocellular carcinomas at week 64 ; ubiquitously expressed mammalian ABC half transporter, apolipoprotein A4 (APOA4), nuclear receptor binding factor-2, CD81, hypothetical protein (HSPC014), and one unidentified gene. These genes might be candidates for biomarkers in screening of non-genotoxic hepatocarcinogens by analysis in two-stage carcinogenesis models. In addition, in situ localization of APOA4mRNA-signal was observed specific to GST-P-positive foci in the thioacetamide-promoted livers. Although further study is required on the role in the hepatocarcinogenesis, APOA4 may link to the formation of neoplastic lesions. Less