酶标仪法5α-还原酶抑制剂体外筛选模型的建立

Establishment of an in vitro screening model for steroid 5 alpha-reductase inhibitors with the microplate reader

目的:建立酶标仪法5α-还原酶抑制剂体外筛选模型。方法:取6只雌性SD大鼠肝脏制备5α-还原酶,检测酶活性后,利用96孔板、酶标仪及酶标仪法分析软件建立5α-还原酶抑制剂体外筛选模型,并通过已知5α-还原酶抑制剂爱普列特及非那甾胺验证模型的可靠性。实验分别设置0、30、60 nmol/L爱普列特组及0、30、60 nmol/L非那甾胺组,96孔板依次加入终浓度0～40μmol/L系列睾酮溶液、22μmol/L的NADPH、终浓度0～60 nmol/L爱普列特或0～60 nmol/L非那甾胺及20μl 5α-还原酶,Tris-HCl缓冲液将每孔溶液体积调至200μl。将96孔板放置于酶标仪中,分别测定0、10 min的A340 nm,并对读取数据进行分析。结果:5α-还原酶的Km值为3.794μmol/L,Vmax为0.271μmol/(L.min);爱普列特的酶抑制常数(Ki)为148.2 nmol/L,半数抑制浓度(IC50)为31.5 nmol/L,曲线图分析为反竞争性抑制剂;非那甾胺的Ki为158.8 nmol/L,IC50为13.6 nmol/L,曲线图分析为竞争性抑制剂;二者结果均同文献报道相同。结论:建立的体外快速筛选模型能够有效地筛选5α-还原酶抑制剂。

Objective： To establish an in vitro screening model for steroid 5 alpha-reductase inhibitors using the microplate reader. Methods ： Steroid 5 alpha-reductase was obtained from the liver of female rats, an in vitro screening model for steroid 5 alphareductase inhibitors established using the 96-well plate and microplate reader after determination of the enzymatic activity, and the reliability of the model verified with the known 5 alpha-reductase inhibitors epristeride and finasteride. Added to the 96-well plate were the final concentrations of testosterone （0-40 μmol/L）, NADPH （22 μmol/L）, epristeride （0-60 nmol/L） or finasteride （0-60 nmol/ L） and steroid 5 alpha-reductase （20 μl）, the total volume of each well adjusted to 200 μl with Tris-Hcl buffer. The 96-well plate was placed in the microplate reader, mixed and incubated at 37 ℃, followed by detection of the A34onm value at 0 and 10 min and analysis of the data. Results ： The Km value of steroid 5 alpha-reductase was 3. 794 μmol/L, with a Vm= of 0. 271 p.mol/（ L. min）. The Ki of epristeride was 148.2 nmol/L, with an IC50 of 31.5 nmol/L, and the enzymatic reaction kinetic curve suggested that epristeride was an uncompetitive enzyme inhibitor. The Ki of finasteride was 158.8 nmol/L, with an IC50 of 13.6 nmol/L. The enzymatic reaction kinetic curve showed that both epristeride and finasteride were competitive enzyme inhibitors, similar to those reported in the published literature. Conclusion ： A screening model was successfully established, which could rapidly and effectively screen steroid 5 alpha-reductase inhibitors in vitro.