【摘要】：為構(gòu)建穩(wěn)定表達(dá)人多藥及毒素外排轉(zhuǎn)運(yùn)體1(h MATE1)的轉(zhuǎn)基因細(xì)胞模型,提取人腎總m RNA,經(jīng)逆轉(zhuǎn)錄PCR獲得h MATE1 c DNA,借助HindⅢ、KpnⅠ兩個(gè)酶切位點(diǎn)與pc DNA3.1(+)重組獲得重組質(zhì)粒。將pc DNA3.1(+)-h MATE1重組質(zhì)粒轉(zhuǎn)染至MDCK、MDCK-h OCT1和MDCK-h OCT2細(xì)胞中,經(jīng)潮霉素B抗性篩選后,以4',6-二脒基-2-苯基吲哚(DAPI)和N-甲基-4-苯基吡啶(MPP+)的積聚實(shí)驗(yàn)篩選獲得具有良好h MATE1功能的單克隆。測定篩選獲得的細(xì)胞中轉(zhuǎn)運(yùn)體m RNA的表達(dá)量,并表征其對(duì)二甲雙胍的積聚或?qū)ξ鬟涮娑〉霓D(zhuǎn)運(yùn)能力。結(jié)果表明,本研究構(gòu)建的MDCK-h MATE1、MDCK-h OCT1/h MATE1、MDCK-h OCT2/h MATE1細(xì)胞模型均高表達(dá)h MATE1 m RNA,MDCK-h MATE1細(xì)胞對(duì)二甲雙胍的積聚為轉(zhuǎn)染空載體細(xì)胞的17.6倍;MDCK-h OCT1/h MATE1和MDCK-h OCT2/h MATE1細(xì)胞對(duì)西咪替丁的凈外排率分別為17.5和3.65。因此,本研究成功構(gòu)建了穩(wěn)定表達(dá)h MATE1及共表達(dá)h MATE1與h OCT1或h OCT2的細(xì)胞模型,可用于h MATE1及其與h OCT1或h OCT2共同參與的藥物轉(zhuǎn)運(yùn)或藥物-藥物相互作用的體外研究。
[Abstract]:In order to construct a transgenic cell model expressing human multidrug and toxin efflux transporter 1 (h MATE1) stably, h MATE1 c DNA, was obtained by reverse transcription PCR from total human kidney RNA,. The recombinant plasmid was obtained by recombination of two restriction sites of Kpn 鈪，

本文編號(hào)：2329050