稳定表达人寡肽转运体1的细胞模型构建及应用  

Establishment and Application of Cell Model with Stable Expression of hPepT1

作　　者：戴燕青[1] 涂美娟[1] 孙冬黎[1] 石爱琴[1] 曾苏[1] 蒋惠娣[1] 

机构地区：[1]浙江大学药学院药物分析与药物代谢研究室,杭州310058

出　　处：《中国药学杂志》2013年第1期13-17,共5页Chinese Pharmaceutical Journal

基　　金：国家自然科学基金资助项目(81072689);浙江省钱江人才项目(J20100471)资助

摘　　要：目的构建稳定表达人寡肽转运体1(hPepT1)的马丁-达比犬肾细胞模型(MDCK-hPepT1),并将其应用于中药单体中人寡肽转运体1抑制剂的筛选。方法构建重组质粒pcDNA3.1(+)-hPepT1,将其转染马丁-达比犬肾系细胞,经G418筛选后以有限稀释法挑选单克隆细胞。以人寡肽转运体1荧光底物β-Ala-Lys(AMCA)和经典底物Glysar对单克隆细胞转运活性进行验证;通过反转录聚合酶链式反应验证人寡肽转运体1在转录水平的表达。以中药单体对Glysar摄取的影响实现人寡肽转运体1抑制剂的筛选。结果获得的两株单克隆细胞(D19、A11)对β-Ala-Lys(AMCA)摄取均显著高于转染空载体的马丁-达比犬肾系细胞,对Glysar的摄取分别为转染空载体细胞的24、11倍;D19、A11中hPepT1的mRNA水平亦显著高于转染空载体的细胞;已知的人寡肽转运体1抑制剂(底物)显著减少单克隆细胞D19和A11对Glysar的摄取;部分中药单体对Glysar摄取产生不同程度的抑制。结论本实验成功构建了稳定高表达人寡肽转运体1的马丁-达比犬肾系细胞模型,并应用该模型筛选出对人寡肽转运体1具有潜在抑制作用的中药单体。OBJECTIVE To establish a cell model with stable-expressed human oligopeptide transporter 1 （ hPepT1 ） in MDCK, then apply the cell model to screen inhibitors of hPepT1 from monomers of Chinese herbs. METHODS Recombinant plasmid pcD- NA3. 1 （ ＋ ）-hPepT1 was constructed and transfected into MDCK cells. After G418 screening, monoclones were obtained by limiting dilution. Functional monoclones were selected and validated by uptake of fluorescent substrate β-Ala-Lys（AMCA） and Glysar, respectively, while the reverse transcription-polymerase chain reaction（RT-PCR） was employed to validate the expression of hPepT1 at transcription level. Screening of hPepT1 inhibitors from monomers of Chinese herbs was achieved by investigating their effects on the uptake of Glysar. RESULTS Two monoclones named as D19 and A11 possessed significantly higher uptake of 13-Ala-Lys （AMCA）than that mock cells ; the uptake of Glysar in D19 and A11 were found to be increased up to 24 and 11-fold of mock cells, respectively. The mRNA level of hPepT1 in DI9 and A11 were significantly higher than that in mock cells; the uptake of Glysar in D19 was obviously inhibited by known hPepT1 inhibitors （substrates）. Some monomers from Chinese herbs inhibited the uptake of Glysar to different extent. CONCLUSION A cell model with stable-expressed hPepT1 is successfully established. Using this model, a few of potential inhibitots of hPepT1 from monomers of Chinese herbs are found.

关 键 词：人寡肽转运体1 马丁达比犬肾上皮细胞 细胞模型 中药 单体 

分 类 号：R965[医药卫生—药理学]