LDH抑制剂筛选方法的优化及西藏药材中的应用

Optimization of LDH Inhibitor Screening Method and Its Application in Tibetan Medicinal Materials

高度表达的乳酸脱氢酶(LDH)与大多数肿瘤细胞的增殖和不良预后具有密切关系,通过建立LDH抑制剂高通量体外筛选模型,为筛选LDH小分子抑制剂提供快捷方法,为抗肿瘤天然产物的筛选提供一种富有潜力的策略。以丙酮酸钠和还原型烟酰胺腺嘌呤二核甘酸(NADH)为底物,加入适量乳酸脱氢酶,通过检测340 nm波长处吸光度的变化,确定底物与LDH的最适浓度及最适孵育时间,用棉酚作为阳性对照,验证筛选模型的科学性,并计算模型的Z’因子。结果表明,酶反应体系的最佳条件为:以0.422 mmol/L的NADH和0.067 mol/L的丙酮酸钠为底物,1 mg/mL乳酸脱氢酶,孵育25 min;测得该模型的Z’因子为0.88~0.90。试验建立的简单、快速、低成本的乳酸脱氢酶抑制剂筛选方法,为抗肿瘤药物筛选、药食同源食品开发利用提供一定研究基础。

The high expression of lactate dehydrogenase is closely related to the proliferation and poor prognosis of most tumor cells,by establishing a high-throughput in vitro screening model for LDH inhibitors,and a rapid method for screening LDH inhibitors and a potential strategy for the screening of natural antitumor products are provided.The optimum concentration and incubation time of the substrate and LDH were determined by detecting the change of the optical density at 340 nm,using NADH and sodium pyruvate as substrates and adding proper amount of lactate dehydrogenase,using gossypol as the positive control,the scientificity of the screening model was verified,and the Z’factor of the model was calculated.The results showed that the optimum condition of the enzyme reaction system was 0.422 mmol/L NADH and 0.067 mmol/L sodium pyruvate as substrate,1 mg/mL lactate dehydrogenase,incubated for 25 min.The Z’factor of the model was determined to be 0.88-0.9.A simple,rapid and low-cost screening method for lactate dehydrogenase inhibitors was established,to provide a certain research basis for the screening of antitumor drugs and the development and utilization of medicinal and edible homologous foods.