Studies on the metabolism and toxicity of drugs using mammalian cells expressed in drug metabolism enzymes

利用表达药物代谢酶的哺乳动物细胞研究药物的代谢和毒性

• 批准号: 05454575
• 负责人: SATOH Tetsuo
• 金额: $ 4.48万
• 依托单位: Chiba University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05454575/
• 关键词: carboxyl esterase; glutathion S-transferase; CYP 450; CO57 cells; esterase; drug metabolism; 発現; 培養細胞; P450; グルタチオン S-トランスフェラーゼ

Drugs and naturally occurring products are metabolized and detoxified by various drug metabolizing enzymes. Recently, gene technology has been progressively developed and these techniques are used for the studies on the metabolism and toxicity of drugs. In this regard, we tried to incorporate the genes of the drug metabolizing enzymes into the cells to express their enzymatic functions. The drug metabolizing enxyme which we used in these studies are carboxylesterases, glutathione S-transferase a dn cytochrome P-450, and we had carried out the cDNA cloning of these enzymes. We had extracted and purified the RNA from rats, mice, monkeys and human livers, and synthesized cDNA of each enzyme. Then, we prepared the libraries using ramda gt 11, ramda gt 10 and ramda gt Zap as the expression vectors. Next, we have used the respective antibodies against each enzyme as the probes to screen the cDNA libraries, and determine the DNA sequences of the positive clones of each enzyme. As the results of these experiments, we had six cDNA clones of the drug metabolizing enzymes mentioned above. Among these cDNA,we have expressed the cDNA of esterases using mammalian cells. In fact, we treated the cDNAs of liver esterases of rat RL1 and RH1 and mouse MH1 with pCR3 as an expression vector, and incorporated the genes of these esterases into COS 7 cells. As the consequence of these experiments, the activities of esterase isozymes were expressed. The novel mammalian cells possessing the genes of several drug metabolizing enzymes we have developed here are extremely useful for new drug development in future.

药物和自然产生的产物被各种药物代谢酶代谢和解毒。近年来，基因技术逐渐得到发展，这些技术被用于研究药物的代谢和毒性。为此，我们尝试将药物代谢酶的基因导入细胞，表达其酶功能。我们在这些研究中使用的药物代谢酶是羧酸酯酶、谷胱甘肽s -转移酶和细胞色素P-450，我们对这些酶进行了cDNA克隆。我们从大鼠、小鼠、猴子和人的肝脏中提取并纯化了RNA，并合成了每种酶的cDNA。然后，以ramda gt 11、ramda gt 10和ramda gt Zap为表达载体，制备了相应的文库。接下来，我们使用针对每种酶的相应抗体作为探针筛选cDNA文库，并确定每种酶的阳性克隆的DNA序列。通过这些实验，我们获得了上述药物代谢酶的6个cDNA克隆。其中，我们利用哺乳动物细胞表达了酯酶的cDNA。事实上，我们用pCR3作为表达载体处理大鼠RL1和RH1肝酯酶的cdna和小鼠MH1肝酯酶的cdna，并将这些酯酶的基因导入COS 7细胞。通过这些实验，测定了酯酶同工酶的活性。我们在这里开发的具有几种药物代谢酶基因的新型哺乳动物细胞对未来的新药开发非常有用。