稳定表达人药物转运体OAT1和药物代谢酶CYP1A2的细胞模型的构建和应用

发布时间：2018-05-04 20:31

  本文选题：人有机阴离子转运体1 + MDCK细胞　； 参考：《浙江大学》2014年硕士论文

【摘要】：本研究利用稳定转染的方法构建了稳定表达人有机阴离子转运体OAT1和/或药物代谢酶CYP1A2的MDCK细胞模型,并且从mRNA水平、蛋白水平和活性水平进行了验证,利用该细胞模型进行了潜在的OAT1底物/抑制剂的筛选,并考察了马兜铃酸对不同细胞模型的毒性差异。 目的：构建稳定表达人有机阴离子转运体OAT1和/或药物代谢酶CYP1A2的MDCK细胞模型,应用该系列细胞模型进行OAT1底物/抑制剂的筛选,考察OAT1和CYP1A2在马兜铃酸细胞毒性中所发挥的作用。 方法：构建重组质粒pcDNA3.1(+)-hOAT1,将其转染MDCK细胞,经G418筛选后采用有限稀释法挑选单克隆细胞,通过OAT1经典底物6-羧基荧光素、对氨基马尿酸的摄取实验验证单克隆细胞中hOAT1转运活性,通过荧光定量PCR和Western-blot验证mRNA和蛋白表达情况,从中筛选出稳定高表达hOAT1的MDCK-OAT1单克隆细胞。再将得到的hOAT1的单克隆细胞和MDCK细胞采用pcDNA3.1(+)/Hygro-CYP1A2质粒进行转染,通过考察功能活性、mRNA表达水平筛选出高活性的MDCK-CYP1A2和MDCK-OAT1/CYP1A2细胞株。利用构建的MDCK-OAT1细胞模型考察若干中药化学成分对OAT1功能的抑制效果,评估马兜铃酸在MDCK-OAT1、MDCK-CYP1A2和MDCK-OAT1/CYP1A2细胞模型上的毒性差异。 结果：采用含OAT1基因的质粒转染MDCK细胞后获得两株高表达OAT1的细胞株,通过RT-PCR. Western-blot验证了OAT1在mRNA和蛋白水平显著高表达。OAT1经典底物对氨基马尿酸、6-羧基荧光素在单克隆细胞内的积聚显著高于空白细胞。将上述细胞进一步采用含CYP1A2基因的质粒转染后,在mRNA水平可检测到CYP1A2基因的高表达。转染后的单克隆细胞可显著代谢CYP1A2的荧光底物。应用MDCK-OAT1细胞模型从若干中药活性成分中筛选出阿魏酸、二氢丹参酮Ⅰ、丹酚酸A、甘草次酸、隐丹参酮、马兜铃酸等6种成分可显著抑制OAT1对6-羧基荧光素的摄取。马兜铃酸在MDCK、MDCK-OAT1、MDCK-OAT1/CYP1A2细胞模型中表现出不同程度的细胞毒性,OAT1和CYP1A2均可增加马兜铃酸的细胞毒性。 结论：本研究成功构建了表达OAT1和/或CYP1A2的细胞模型。该细胞模型可用于OAT1潜在底物和抑制剂的筛选,也可用于OAT1和CYP1A2相关的药物毒理研究。
[Abstract]:In this study, a stable MDCK cell model expressing human organic anion transporter (OAT1) and / or drug metabolizing enzyme CYP1A2 (CYP1A2) was constructed by stable transfection, and was verified by mRNA level, protein level and activity level. The cell model was used to screen potential OAT1 substrates / inhibitors and the toxicity of aristolochic acid to different cell models was investigated. Aim: to construct a MDCK cell model expressing human organic anion transporter (OAT1) and / or drug metabolizing enzyme (CYP1A2) stably, and to screen OAT1 substrate / inhibitor by using this series of cell models. To investigate the role of OAT1 and CYP1A2 in the cytotoxicity of aristolochic acid. Methods: the recombinant plasmid pcDNA3.1 (hOAT1) was constructed and transfected into MDCK cells. After G418 selection, monoclonal cells were selected by limited dilution method, and 6-carboxyl fluorescein was obtained by OAT1 classic substrate. The hOAT1 transport activity was confirmed by the uptake of p-aminohippuric acid. The expression of mRNA and protein was confirmed by fluorescence quantitative PCR and Western-blot. The stable and high expression of hOAT1 in MDCK-OAT1 cells was screened. The hOAT1 monoclonal cells and MDCK cells were transfected with pcDNA3.1 (/ Hygro-CYP1A2) plasmid, and the highly active MDCK-CYP1A2 and MDCK-OAT1/CYP1A2 cell lines were screened by investigating the expression level of functional active MDCK-CYP1A2 mRNA. MDCK-OAT1 cell model was used to study the inhibitory effect of some traditional Chinese medicines on the function of OAT1 and to evaluate the toxicity of aristolochic acid on MDCK-OAT1 MDCK-CYP1A2 and MDCK-OAT1/CYP1A2 cell models. Results: two cell lines with high expression of OAT1 were obtained by transfection of MDCK cells with plasmid containing OAT1 gene. Western-blot confirmed that OAT1 was significantly overexpressed at the mRNA and protein levels. The accumulation of 6-carboxylfluorescein in Monoclonal cells was significantly higher than that in blank cells. After the above cells were transfected with plasmid containing CYP1A2 gene, the high expression of CYP1A2 gene could be detected at mRNA level. The transfected monoclonal cells could significantly metabolize the fluorescent substrates of CYP1A2. Ferulic acid, dihydrotanshinone 鈪，

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