本文選題：人有機陰離子轉(zhuǎn)運體1 + MDCK細(xì)胞��； 參考：《浙江大學(xué)》2014年碩士論文

【摘要】：本研究利用穩(wěn)定轉(zhuǎn)染的方法構(gòu)建了穩(wěn)定表達(dá)人有機陰離子轉(zhuǎn)運體OAT1和/或藥物代謝酶CYP1A2的MDCK細(xì)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(xì)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(xì)胞模型的毒性差異。 目的：構(gòu)建穩(wěn)定表達(dá)人有機陰離子轉(zhuǎn)運體OAT1和/或藥物代謝酶CYP1A2的MDCK細(xì)胞模型,應(yīng)用該系列細(xì)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(xì)胞毒性中所發(fā)揮的作用。 方法：構(gòu)建重組質(zhì)粒pcDNA3.1(+)-hOAT1,將其轉(zhuǎn)染MDCK細(xì)胞,經(jīng)G418篩選后采用有限稀釋法挑選單克隆細(xì)胞,通過OAT1經(jīng)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(xì)胞中hOAT1轉(zhuǎn)運活性,通過熒光定量PCR和Western-blot驗證mRNA和蛋白表達(dá)情況,從中篩選出穩(wěn)定高表達(dá)hOAT1的MDCK-OAT1單克隆細(xì)胞。再將得到的hOAT1的單克隆細(xì)胞和MDCK細(xì)胞采用pcDNA3.1(+)/Hygro-CYP1A2質(zhì)粒進行轉(zhuǎn)染,通過考察功能活性、mRNA表達(dá)水平篩選出高活性的MDCK-CYP1A2和MDCK-OAT1/CYP1A2細(xì)胞株。利用構(gòu)建的MDCK-OAT1細(xì)胞模型考察若干中藥化學(xué)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(xì)胞模型上的毒性差異。 結(jié)果：采用含OAT1基因的質(zhì)粒轉(zhuǎn)染MDCK細(xì)胞后獲得兩株高表達(dá)OAT1的細(xì)胞株,通過RT-PCR. Western-blot驗證了OAT1在mRNA和蛋白水平顯著高表達(dá)。OAT1經(jīng)典底物對氨基馬尿酸、6-羧基熒光素在單克隆細(xì)胞內(nèi)的積聚顯著高于空白細(xì)胞。將上述細(xì)胞進一步采用含CYP1A2基因的質(zhì)粒轉(zhuǎn)染后,在mRNA水平可檢測到CYP1A2基因的高表達(dá)。轉(zhuǎn)染后的單克隆細(xì)胞可顯著代謝CYP1A2的熒光底物。應(yīng)用MDCK-OAT1細(xì)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(xì)胞模型中表現(xiàn)出不同程度的細(xì)胞毒性,OAT1和CYP1A2均可增加馬兜鈴酸的細(xì)胞毒性。 結(jié)論：本研究成功構(gòu)建了表達(dá)OAT1和/或CYP1A2的細(xì)胞模型。該細(xì)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(guān)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，

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