DEVELOPMENT OF EVALUATION SYSTEM FOR IMMUNOTOXICITY BY DIOXIN COMPOUNDS

二恶英类化合物免疫毒性评价系统的研制

• 批准号: 11558068
• 负责人: KIKUCHI Hideaki
• 金额: $ 8.83万
• 依托单位: Tohoku University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11558068/
• 关键词: Dioxin; T cell; Immunotoxicity; Apoptosis; カスパーゼ3

Our aim of this project is a development of assay system for immunotoxicity by dioxin compounds. We tried to find a method for a large number of samples, high sensitivity and low background for the detection of T cell apoptosis. We tested caspase 3 assay system as a possible candidate method. In the process of apoptosis by TCDD, caspase 3 was activated in L-MAT cells. Therefore, we utilized fluorescence dye conjugated to peptide of PARP as a substrate to improve the sensitivity of apoptosis assay. The apoptosis of L-MAT cells by TCDD can be detected as low as 10^5 cells by this method. Using fluorometric-measuring equipment with 96 well culture dish, we developed a mass-screening system.Furthermore, we analyzed the pathway of apoptotic signal to caspase 3. A specific inhibitor of Protein kinase C-theta (PKC-θ) blocked the apoptosis of L-MAT cells by TCDD. 1,2-Diacylglycerol (DAG) is known to activate PKC-θ, therefore, DAG producing enzyme, phospholipase Cγ(PLCγ), was blocked by a specific inhibitor, U73122. The blockade of PLCγ suppressed the apoptosis of L-MAT cells by TCDD. We concluded that TCDD activates PLCγ by some mechanism.

本计画的目的是发展二英化合物免疫毒性的检测系统。我们试图寻找一种样本量大、灵敏度高、本底低的T细胞凋亡检测方法。我们测试了半胱天冬酶3测定系统作为可能的候选方法。在TCDD诱导L-MAT细胞凋亡的过程中，caspase 3被激活。因此，我们利用与PARP肽缀合的荧光染料作为底物来提高凋亡测定的灵敏度。该方法可检测到10^5个细胞的凋亡。利用96孔培养皿的荧光检测装置，建立了一套细胞凋亡的筛选系统，并对凋亡信号转导途径进行了分析。蛋白激酶C-θ（PKC-θ）特异性抑制剂可阻断TCDD诱导的L-MAT细胞凋亡。已知1，2-二酰基甘油（DAG）可激活PKC-θ，因此，DAG产生酶磷脂酶Cγ（PLCγ）可被特异性抑制剂U 73122阻断。阻断PLCγ可抑制TCDD诱导的L-MAT细胞凋亡。TCDD通过一定的机制激活PLCγ。

项目成果

Endo Y. et al.: "Enhancement by galactosamine of lipopolysaccharide (LPS)-induced tumour mecrosis fantor production and lethality: its suppression by LPS pretreatment"Br.J. Pharmacol.. 128. 5-12 (1999)

Endo Y. 等人：“半乳糖胺增强脂多糖 (LPS) 诱导的肿瘤坏死因子的产生和致死率：LPS 预处理对其的抑制作用”Br.J.

Kikuchi,H. et.al.: "Method for evaluation of immunotoxicity of dioxin compounds using human T-lymphoblastic cell line, L-MAT."Chemosphere. (in press).

菊池，H.

Kikuchi, H.: "Mechanism of ligand independent Activation of Ah receptor"Seikagaku. 72(4). 194-198 (2000)

Kikuchi, H.：“Ah 受体的配体独立激活机制”Seikagaku。

菊池英明: "生体防御に関わる蛋白因子 シトクロームp-450(CYP)遺伝子の核内受容体による調節"衛生学雑誌. 56. 622-628 (2002)

Hideaki Kikuchi：“核受体对细胞色素 p-450 (CYP) 基因（一种参与生物防御的蛋白质因子）的调节”《卫生杂志》56. 622-628 (2002)。

Kikuchi, H.: "Presence on human chromosome 10 of omeprazole-sensitivity gene whose product mediates CYP1A1 induction"Cyt. Genome Res.. (in press). (2002)

Kikuchi, H.：“人类 10 号染色体上存在奥美拉唑敏感性基因，其产物介导 CYP1A1 诱导”Cyt。