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Preparation of Co-PCB free animal by transfection with drug transporter gene

转染药物转运蛋白基因制备无Co-PCB动物

基本信息

批准号：
11839027
负责人：
FUJISE Hiroshi
金额：
$ 2.3万
依托单位：
Azabu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
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

项目摘要

Though P-glycoprotein (PGP) which transport coplanar PGB (Co-PCB) has not been found, it was revealed that Co-PCB inhibited the function of PGP. Followings were the findings in this experiment. Drug transport pump, mdr1 and mdr2, were cloned from leishmania amazonensis (La) ; La-mdr1 exerted multi drug resistance, and La-mdr2 transported 5FU. Drug accumulation was examined in the transformant human carcinoma cell, KB3-1, expressed human PGP. Drug accumulation was decreased in the KB3-1 cells expressing wild PGP (KB3-His^<61>) and mutant PGP in which the 61st amino acid was substituted for serine (KB3-Ser^<61>) or phenylalanine (KB3-Phe^<61>), compared to wild KB3-1. Drug tolerance and transport ability for small molecule chemical, colchicine, were greater in KB3-Phe^<61> compared to KB3-Ser^<61>, thus inverse correlation was indicated between the bulk of the side chain of 61st amino acid and the molecular weight of the substrates. Mutant human PGP gene, in which His61 was substituted with Ser^<61> or Phe^<61>, were transfected in porcine kidney cell, LLC-PK1 , and prepared transformant cells, LLC-His^<61>, LLC-Phe^<61> and LLC-Ser^<61>. In the transformant cells, the abilities of the drug tolerance and the decrease of the accumulation were same as in PGP expressing KB3-1 cells, and transepithelial transport was increased. Co-PCB was not transported through the epithelial monolayer in the PGP expressing LLC-PK1. However, the most toxic congenor of Co-PCBs, 3, 3', 4, 4', 5-pentachlorobiphenyl, inhibited transport of the other drugs by PGP, and the inhibiton was remarkable in KB3-Phe^<61> and LLC-Phe^<61>. Drug tolerance cell lines (100 times tolerance) were selected from canine tumor cells. In the cells, clear appearance of the protein and mRNA of PGP were detected by western blot and RT-PCR, respectively.

虽然尚未发现转运共面PGB （Co-PCB）的p -糖蛋白（PGP），但发现Co-PCB可抑制PGP的功能。以下是这次实验的发现。从亚马逊利什曼原虫（leishmania amazonensis, La）中克隆了药物输送泵mdr1和mdr2；La-mdr1多药耐药，La-mdr2转运5FU。在表达人PGP的转化人癌细胞KB3-1中检测药物蓄积。与野生的KB3-1相比，在表达野生PGP （KB3-His^<61>）和突变PGP(其中第61个氨基酸被丝氨酸（KB3-Ser^<61>）或苯丙氨酸（KB3-Phe^<61>）的KB3-1细胞中，药物积累减少。与KB3-Ser^<61>相比，KB3-Phe^<61>对小分子化学物质秋水仙碱的耐受性和转运能力更强，因此第61个氨基酸侧链的体积与底物的分子量呈负相关。将人PGP突变基因His61替换为Ser^<61>或Phe^<61>，转染猪肾细胞LLC-PK1，制备转化细胞LLC-His^<61>、LLC-Phe^<61>和LLC-Ser^<61>。在转化细胞中，与表达PGP的KB3-1细胞相比，PGP的耐药能力和积累减少的程度相同，经上皮转运增加。在表达LLC-PK1的PGP中，Co-PCB不通过上皮单层运输。而Co-PCBs毒性最大的同系物3,3 ',4,4 '，5-五氯联苯可抑制PGP对其他药物的转运，且对KB3-Phe^<61>和LLC-Phe^<61>的抑制作用显著。从犬肿瘤细胞中筛选耐药细胞系（100倍耐药）。western blot和RT-PCR检测细胞中PGP蛋白和mRNA的清晰表达。