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

虽然尚未发现转运共面Pgb的P-糖蛋白(Pgp)(Co-PCB)，但发现Co-PCb抑制Pgp的功能。以下是这项实验的发现。从亚马逊利什曼原虫中克隆到药物转运泵mdr1和mdr2，La-mdr1具有多重耐药性，La-mdr2转运5FU。在表达人Pgp的转化型人癌细胞KB3-1中检测到药物蓄积。与野生型KB3-1相比，表达野生型Pgp的KB3-1细胞和用第61位氨基酸取代丝氨酸或苯丙氨酸的突变型Pgp细胞的药物积累减少。与KB3-Ser；61&gt；相比，KB3-Phe；61&gt；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61个氨基酸侧链的体积与底物的相对分子质量呈负相关。将突变的人Pgp基因(His61被Ser^&lt；61&gt；或Phe^&lt；61&gt；)导入猪肾细胞LLC-PK1，制备了转化细胞LLC-His^&lt；61&gt；、LLC-Phe^&lt；61&gt；和LLC-Ser^&lt；61&gt；在转化子细胞中，耐药能力和积聚减少与表达KB3-1的Pgp细胞相同，跨皮转运增加。在表达LLC-PK1的PGP中，co-PCb不能通过上皮单层转运。但毒性最大的同系物3，3‘，4，4’，5-五氯联苯抑制了PGP对其他药物的转运，且在KB3-Phe和LLC-Phe；61&gt；从犬肿瘤细胞中筛选出耐药细胞系(耐药100倍)。免疫印迹和RT-PCR分别检测到细胞内Pgp蛋白和mRNA的清晰表达。

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.