Objective: To establish human embryonic kidney (HEK293) cell line stably expressing human OATP1A2, and to explore whether the uptake of fentanyl can be mediated by OATP1A2 in vitro. Methods: HEK293 cells were transfected with the plasmid pIRES2-ZsGreen1-OATP1A2 using a Liposome transfection reagent LipofectamineTM 2000. After geneticin (G-418; 600 mg/ml) treatment, single colonies were selected and characterized. Western blot analysis and real-time PCR were used to verify the success of transfection. Different concentrations of probe drug fexofenadine was used to verify HEK293-OATP1A2 transport function. The cells were incubated with different concentrations of fentanyl. HEK293-OATP1A2 uptake experiment was performed with inhibitor naringenin. Results: Fexofenadine and fentanyl showed a significantly increased uptake (P < 0.05) into the HEK293-OATP1A2 compared with the HEK293-VC cells. The uptake value of Fexofenadine at 100 nM was 2.8-fold to that of control. The OATP1A2 inhibitor naringenin at a concentration of 100 μg/mL significantly reduced the cellular fexofenadine and fentanyl net uptake to 66.8±0.6% and 86.5±0.5% of control (fexofenadine uptake without inhibitor), respectively (P <0.05). Conclusion: HEK293 cell line with overexpression of OATP1A2 was successfully constructed, and OATP1A2 mediating fentanyl transport in vitro has been confirmed.%目的:建立稳定表达有机阴离子转运多肽1A2(organic anion-transporting polypeptide 1A2,OATP1A2)的HEK293细胞株,体外研究OATP1A2是否转运芬太尼(Fentanyl).方法:用脂质体转染法将pIRES2-ZsGreen1-OATP1A2质粒转染至HEK293细胞中,G418(600 mg/ml)筛选单克隆阳性细胞,采用Western blot、RT-PCR证实HEK293-OATP1A2构建成功;用不同浓度的探针药物非索非那定(Fexofenadine,FEX)验证HEK293-OATP1A2的转运功能;运用不同浓度的芬太尼孵育细胞,另取两组用相同浓度的芬太尼孵育的细胞,分别加或不加抑制剂柚皮素(Naringenin,100μg/mL)进行HEK293-OATP1A2转运实验.结果:FEX浓度为1、10、100 nM时HEK293-OATP1A2实验组与HEK293-VC对照组,FEX的吸收值均有显著的统计学差异(P<0.05),且FEX浓度为100 nM时,实验组对FEX的吸收值是对照组的2.8倍;加入抑制剂柚皮素组FEX吸收值较不加抑制剂时减少了66.8±0.6%.芬太尼转运结果显示,芬太尼浓度为1、10、100、1000 nM时实验组与对照组比较,芬太尼的吸收值均有显著的统计学差异(P<0.05),且芬太尼浓度为1000 nM时,实验组对芬太尼的吸收值是对照组的2.2倍;加入抑制剂组芬太尼的吸收值较未加抑制剂组减少了86.5±0.5%(P<0.05).结论:成功建立了稳定表达OATP1A2的HEK293细胞株,体外研究发现OATP1A2能介导芬太尼的转运.
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