基于HepaRG活细胞的N-乙酰基转移酶1活性测定方法的建立与评价

Development and evaluation of the method for N-acetyltransferase activity assay by using living HepaRG cells

目的建立基于人类肝癌HepaRG活细胞的N-乙酰基转移酶1(NAT1)活性测定方法,并对其测定结果进行评价。方法 HepaRG细胞用含10%牛血清和青、链霉素培养液进行培养。用免疫印迹技术考察培养24,48和72 h的HepaRG细胞中NAT1表达情况;用噻唑蓝实验测定NAT1底物4-氨基水杨酸(4-ASA)孵育24和48 h对细胞活力的影响,分别用液-质联用技术及免疫印迹技术考察不同浓度4-ASA对HepaRG细胞中NAT1活性及蛋白表达的影响,比较基于HepaRG活细胞的NAT1活性测定方法与传统组织/细胞裂解法对NAT1活性测定结果的影响。结果与培养24 h的HepaRG细胞相比(0. 71±4. 00×10^(-3)),培养48和72 h的HepaRG细胞中NAT1的表达量显著增加,分别为(0. 80±7. 00×10^(-3))和(1. 04±0. 04),差异均有统计学意义(均P <0. 05)。与对照组相比,浓度为5～100μmol·L^(-1)的4-ASA孵育HepaRG细胞24和48 h,对其活力无明显影响(均P> 0. 05); 25～100μmol·L^(-1)的4-ASA孵育HepaRG细胞8 h,对NAT1蛋白表达无明显影响(均P> 0. 05),且50～75μmol·L^(-1)的4-ASA孵育HepaRG细胞,培养液中4-ASA代谢物的生成量与底物浓度成正比。NAT1活性测定结果显示,HepaRG活细胞法测得的结果略低于传统的组织/细胞裂解法所测得的结果(P <0. 05或P <0. 01),但两者的酶促反应趋势比较相似。结论以4-ASA为反应底物建立的基于HepaRG活细胞的NAT1活性测定方法具有操作简单、结果稳定、可动态检测等优势,有望用于NAT1活性的动态测定及NAT1特异性抑制的高通量筛选。

Objective To develop and evaluate the method for N-acetyltransferase 1(NAT1)activity assay by using living human hepatoma HepaRG cell line.Methods HepaRG cells were cultured by 10%bovine serum medium with penicillin-streptomycin.Protein expression of NAT1 in HepaRG cells was evaluated after 24,48 and 72 h cells culture by Western-blot technique;effect of NAT1 substrate 4-aminosalicylic acid(4-ASA)on cells viability was assessed by MTT assay at 24 and 48 h post 4-ASA;effect of 4-ASA concentration on NAT1 activity and protein expression were determined by using HPLC/MS and Western-bolt technique,respectively.Moreover,the determined NAT1 activity values were also compared between the methods using living HepaRG cells and the traditional method using tissue/cells lysate.Results Comparedto the cells cultured for 24 h[(0.71±4.00×10-3)],NAT1 expression in the cells cultured for 48 and 72 h were both significantly increased[(0.80±7.00×10-3)and(1.04±0.04)](all P<0.05).After 24 and 48 h exposure to 5-100μmol·L-14-ASA,the cells viability didn’t markedly changed when compared to that of control(all P>0.05);NAT1 expression also unchanged by 25-100μmol·L-14-ASA exposure for 8 h(all P>0.05);and when exposed in 50-75μmol·L-14-ASA,the produced 4-ASA metabolite in the medium was increased in a concentration-dependent manner.Results from NAT1 activity assay showed that the determined values from method using living HepaRG cells was lower than that from traditional method using tissue/cells lysate(P<0.05 or P<0.01),but the tendency of enzymatic reaction was consistent well between these two methods.Conclusion By using 4-ASA as the substrate,the developed NAT1 activity assay in living HepaRG cells could be used for dynamic NAT1 activity evaluation and high throughput screening of NAT1 inhibitors,for its simplicity in operation,stability in determination,and detectable in dynamic process.