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Development of early elucidaion of chemical substance biotransformation with DMA chip

DMA芯片开发早期阐明化学物质生物转化

基本信息

批准号：
12307010
负责人：
OMAE Kazuyuki
金额：
$ 22.71万
依托单位：
Keio University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

项目摘要

In 2000 and 2001, trichloroethylene (TCE) or dichloromethane (DCM) was exposed to rats or mice, expression profiles of liver were obtained with DNA chip (Affymetrix), which contains ESTs as well as genes. TCE or DCM causes hepatoma to mice, but not to rats or human. Whereas intraperitoneal injection of TCE to rats caused activation of lipid metabolism, that to mice seemed to disturb cell proliferation system. Consecutive TCE oral administration up to 2 weeks revealed up-regulation of genes which are induced by peroxisome proliferator. We verified the results of DNA chip by real-time PCR, and sequenced EST clones which showed significant change. Inhalation exposure of DCM to rat up-regulated P450 2E1 gene.A homologue search program was developed to compare expression profiles between species. Homologues were searched for probes that showed significant change on TCE exposure. New expression profile, in which mouse genes correspond with those of rat, was subjected to cluster analysis. Expression profiles of mouse and those of rat were almost separated.Our goal is to know the plausibility of expression profile when we determine which animal is suit for extrapolation. We had planned comparison experiments between mouse, rat and human with flesh primary culture hepatocytes. We had established a culture system of which hepatocytes retain inducibility of P450. Mouse primary culture hepatocytes were exposed to TCE after a series of preliminary tests. The exposure condition was determined by real-time PCR, being based on the results of in vivo experiments. We are waiting for human hepatocytes to be delivered from United States.

2000年和2001年，将三氯乙烯(TCE)和二氯甲烷(DCM)分别暴露于大鼠和小鼠，用含有EST和基因的DNA芯片(Affymetrix)获得肝脏的表达谱。TCE或DCM会引起小鼠肝癌，但不会引起大鼠或人的肝癌。大鼠腹腔注射TCE可引起脂代谢的激活，而小鼠腹腔注射TCE则可干扰细胞增殖系统。连续口服TCE至2周，发现由过氧化物酶增殖物诱导的基因表达上调。用实时荧光定量聚合酶链式反应对DNA芯片的结果进行了验证，并对有显著变化的EST克隆进行了测序。吸入DCM暴露于大鼠上调的P450 2E1基因。开发了一个同源搜索程序来比较物种间的表达谱。搜索同源物以寻找显示TCE暴露显著变化的探针。对小鼠基因与大鼠基因相对应的新的表达谱进行了聚类分析。小鼠和大鼠的表达谱几乎是分开的，我们的目的是在确定哪种动物适合外推时，了解表达谱的可信程度。我们计划在小鼠、大鼠和人之间进行原代培养肝细胞的对比实验。我们建立了一套肝细胞保持P450诱导性的培养体系。小鼠原代培养肝细胞在一系列初步试验后暴露于三氯乙烷。根据体内实验结果，通过实时荧光聚合酶链式反应(Real-Time-PCR)确定暴露条件。我们正在等待从美国运来的人类肝细胞。