摘要
为建立表达乳腺癌耐药蛋白(breast cancer resistance protein,BCRP)的猪肾小管上皮细胞系LLC-PK1/BCRP,利用脂质体转染的方法将表达载体质粒pcDNA3.1(+)-BCRP转入LLC-PK1细胞。G418筛选后,采用细胞群体倍增试验、流式细胞术和Western blotting等方法鉴定构建的细胞系。利用体外细胞毒实验检测转基因细胞系对米托蒽醌和阿霉素的耐药指数,荧光倒置显微镜检测其对荧光染料Hoechst 33342的外排作用并以GF120918为BCRP抑制剂观察抑制剂对BCRP外排荧光染料的逆转作用。结果表明,LLC-PK1/BCRP细胞表达BCRP蛋白升高;细胞群体倍增时间较亲本细胞略有延长;对米托蒽醌和阿霉素的耐药指数分别提高了51.95倍和6.09倍;对荧光染料Hoechst 33342的外排作用明显增强,且可被抑制剂GF120918逆转。因此,表达BCRP的猪肾小管上皮细胞系LLC-PK1/BCRP构建成功,可作为进一步研究BCRP生物学特征的模型。
To establish a pig kidney cell line LLC-PK1/BCRP in which human breast cancer resistance protein was highly expressed, the expression vector pcDNA3.1（＋）-BCRP which contained BCRP gene was constructed and transfected into LLC-PK1 cells via liposomes. After selecting with G418, population doubling time, flow cytometry and Western blotting analysis were used to evaluate the cell line. MTT assays were employed to determine the drug resistance index of mitoxantrone and doxorubicin. Invert fluorescent microscope was used to observe the efflux of fluorescence dye Hoechst 33342 by BCRP, furthermore, the BCRP＇s inhibitor GF120918 was applied to reverse the efflux of Hoechst 33342. The experiment results showed that the expression of BCRP protein increased in LLC-PK1/BCRP cell. The population doubling time of LLC-PK1/BCRP cell was a little longer than that of the parental cell LLC-PK1. The resistance indexes to mitoxantrone and doxorubicin were 51.95 and 6.09 times, respectively, higher than LLC-PK1 cell. The efflux of Hoechst 33342 was significantly enhanced and could be reversed by GF120918. So a LLC-PK1/BCRP cell line was established, which highly expressed BCRP protein successfully. This cell line could be a valuable model to further investigate the biological profile of BCRP and select the substrate and inhibitor of BCRP.
出处
《药学学报》
CAS
CSCD
北大核心
2012年第12期1599-1604,共6页
Acta Pharmaceutica Sinica
基金
国家"重大新药创制"科技重大专项(2009ZX09304-003
2012ZX09506001-004)
