本文選題：人有機陰離子轉運體1 + MDCK細胞　； 參考：《浙江大學》2014年碩士論文

【摘要】：本研究利用穩(wěn)定轉染的方法構建了穩(wěn)定表達人有機陰離子轉運體OAT1和/或藥物代謝酶CYP1A2的MDCK細胞模型,并且從mRNA水平、蛋白水平和活性水平進行了驗證,利用該細胞模型進行了潛在的OAT1底物/抑制劑的篩選,并考察了馬兜鈴酸對不同細胞模型的毒性差異。 目的：構建穩(wěn)定表達人有機陰離子轉運體OAT1和/或藥物代謝酶CYP1A2的MDCK細胞模型,應用該系列細胞模型進行OAT1底物/抑制劑的篩選,考察OAT1和CYP1A2在馬兜鈴酸細胞毒性中所發(fā)揮的作用。 方法：構建重組質粒pcDNA3.1(+)-hOAT1,將其轉染MDCK細胞,經G418篩選后采用有限稀釋法挑選單克隆細胞,通過OAT1經典底物6-羧基熒光素、對氨基馬尿酸的攝取實驗驗證單克隆細胞中hOAT1轉運活性,通過熒光定量PCR和Western-blot驗證mRNA和蛋白表達情況,從中篩選出穩(wěn)定高表達hOAT1的MDCK-OAT1單克隆細胞。再將得到的hOAT1的單克隆細胞和MDCK細胞采用pcDNA3.1(+)/Hygro-CYP1A2質粒進行轉染,通過考察功能活性、mRNA表達水平篩選出高活性的MDCK-CYP1A2和MDCK-OAT1/CYP1A2細胞株。利用構建的MDCK-OAT1細胞模型考察若干中藥化學成分對OAT1功能的抑制效果,評估馬兜鈴酸在MDCK-OAT1、MDCK-CYP1A2和MDCK-OAT1/CYP1A2細胞模型上的毒性差異。 結果：采用含OAT1基因的質粒轉染MDCK細胞后獲得兩株高表達OAT1的細胞株,通過RT-PCR. Western-blot驗證了OAT1在mRNA和蛋白水平顯著高表達。OAT1經典底物對氨基馬尿酸、6-羧基熒光素在單克隆細胞內的積聚顯著高于空白細胞。將上述細胞進一步采用含CYP1A2基因的質粒轉染后,在mRNA水平可檢測到CYP1A2基因的高表達。轉染后的單克隆細胞可顯著代謝CYP1A2的熒光底物。應用MDCK-OAT1細胞模型從若干中藥活性成分中篩選出阿魏酸、二氫丹參酮Ⅰ、丹酚酸A、甘草次酸、隱丹參酮、馬兜鈴酸等6種成分可顯著抑制OAT1對6-羧基熒光素的攝取。馬兜鈴酸在MDCK、MDCK-OAT1、MDCK-OAT1/CYP1A2細胞模型中表現(xiàn)出不同程度的細胞毒性,OAT1和CYP1A2均可增加馬兜鈴酸的細胞毒性。 結論：本研究成功構建了表達OAT1和/或CYP1A2的細胞模型。該細胞模型可用于OAT1潛在底物和抑制劑的篩選,也可用于OAT1和CYP1A2相關的藥物毒理研究。
[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，

本文編號：1844548