CAJ | 학술논문

目的：建立乳腺癌耐药蛋白BCRP高效稳定表达COS-7细胞系,为相关药物的药物转运及其耐药性研究奠定基础。方法采用RT-PCR技术从MCF-7/ADR乳腺癌细胞中扩增出乳腺癌耐药蛋白基因BCRP的cDNA序列,插入真核表达载体pcDNA3.1(+),获重组质粒pcDNA3.1(+)-BCRP,将重组质粒pcDNA3.1(+)-BCRP转染COS-7细胞,经G418筛选后获得抗性细胞克隆。运用基因组PCR、SDS-PAGE鉴定抗性细胞中外源BCRP基因的整合及表达,并以BCRP底物mitoxantrone对鉴定阳性的细胞克隆进行转运活性验证。结果成功克隆BCRP的cDNA并构建真核细胞表达载体pcDNA3.1(+)-BCRP。转染COS-7细胞后获得抗性克隆10个,PCR鉴定阳性有4个,其中2个克隆能高效表达BCRP蛋白。经活性验证,2个高表达的细胞克隆中有一个能显著高效外排BCRP底物mitoxantrone,显示良好的生物活性功能。结论成功建立一株BCRP高效稳定表达的COS-7细胞系,且具有特异性生物活性,可用于BCRP相关药物转运及耐药性研究。
목적：건립유선암내약단백BCRP고효은정표체COS-7세포계,위상관약물적약물전운급기내약성연구전정기출。방법채용RT-PCR기술종MCF-7/ADR유선암세포중확증출유선암내약단백기인BCRP적cDNA서렬,삽입진핵표체재체pcDNA3.1(+),획중조질립pcDNA3.1(+)-BCRP,장중조질립pcDNA3.1(+)-BCRP전염COS-7세포,경G418사선후획득항성세포극륭。운용기인조PCR、SDS-PAGE감정항성세포중외원BCRP기인적정합급표체,병이BCRP저물mitoxantrone대감정양성적세포극륭진행전운활성험증。결과성공극륭BCRP적cDNA병구건진핵세포표체재체pcDNA3.1(+)-BCRP。전염COS-7세포후획득항성극륭10개,PCR감정양성유4개,기중2개극륭능고효표체BCRP단백。경활성험증,2개고표체적세포극륭중유일개능현저고효외배BCRP저물mitoxantrone,현시량호적생물활성공능。결론성공건립일주BCRP고효은정표체적COS-7세포계,차구유특이성생물활성,가용우BCRP상관약물전운급내약성연구。
Objective To establish the human BCRP-COS-7 cell line and provide the platform for the study of BCRP-mediated drug transport and resistance. Methods cDNA of BCRP was cloned from MCF-7/ADR cell line and constructed into pcDNA3.1(+) . Then recombinant vector was transfected into COS-7 cells. The resulting colonies treated with G418 were selected,and the expression of BCRP was identified by genome PCR and SDS-PAGE. The transport activity of high-stably expressing human BCRP was confirmed by efflux of BCRP substrate mitoxantrone. Results The expression vector pcDNA3. 1 (+)-BCRP was successfully constructed. COS-7 cells were transfected with pcDNA3. 1 (+)-BCRP and ten G418-resistant colonies were selected. PCR and SDS-PAGE were used to confirm that four colonies are positive and two of them were positive expression, respectively. One colony displayed high efficiency to efflux mitoxantrone with the comparison of wild type COS-7 cells. Conclusion The high-stably expressing BCRP-COS-7 cell line has been successfully established. The BCRP efflux function to mitoxantrone is confirmed. The resulting cell line could be applied to further study of BCRP-mediated drug transport and resistance.