基于Caco-2细胞裂解液的DPP-4抑制剂筛选方法的建立

Establishment of DPP-4 Inhibitors Screening Model Based on Caco-2 Cell Lysate

使用结直肠腺癌Caco-2细胞裂解液对西格列汀的DPP-4抑制率和抑制类型进行测定,建立体外筛选DPP-4抑制剂的模型。将成熟分化的Caco-2细胞于Tris-HCl缓冲液内超声破碎制成细胞裂解液,以甘氨酰-脯氨酸-7-氨基-4-甲基香豆素(Gly-Pro-AMC)为底物对裂解液中DPP-4的比活力进行测定。以Gly-Pro-AMC为底物、稀释后的Caco-2细胞裂解液为DPP-4酶溶液,使用一系列底物浓度测得西格列汀对DPP-4抑制的IC50,使用双倒数作图法在一系列底物以及抑制剂浓度下测得西格列汀的抑制类型。在实验条件下测得西格列汀的IC50为18.68 nmol/L(95%CI,15.44~21.90 nmol/L)(拟合曲线R^(2)>0.99),抑制类型为竞争性抑制(拟合曲线R^(2)>0.99)。测得模型条件下的酶反应动力学参数Km为(75.11±2.15)μmol/L,Vmax为(1.00±0.03)μmol/(L·min),以及西格列汀的Ki为6.07 nmol/L(95%CI,5.61~6.58 nmol/L)(拟合曲线R2>0.99)。使用该模型测得西格列汀的DPP-4抑制率和抑制类型与文献报道相符,证明该模型可以用于DPP-4抑制剂的筛选。

To establish a model for screening DPP-4 inhibitors in vitro to determine the DPP-4 inhibition rate and inhibition type by using human colorectal adenocarcinoma Caco-2 cell lysate,mature and diferentiated Caco-2 were broken into cell lysate ultrasonically in Tris-HCI buffer,and the specific activity of DPP-4 enzyme in lysate was determined with Cly-Pro-7-amido-4-methylcoumarin hydrobromide(Gly-Pro-AMC)as substrate.With Gly-Pro-AMC as substrate and diluted Caco-2 cell lysate as DPP-4 enzyme solution,the ICso of sitagliptin for DPP-4 inhibition was determined by a series of substrate concentrations and the inhibition type was determined by a double-reciprocal plot using a series of substrate and inhibitor concentrations.The ICso of sitagliptin for DPP-4 inhibition was 18.68 nmol/L(95%CI,15.44~21.90 nmol/L)under experimental conditions(fitted curve R^(2)>0.99),and the inhibition type was competitive inhibition(fited curve R^(2)>0.99).Under the model conditions,the enzymekinetics parameters K.and Vmax were(75.11±2.15)μmol/L and(1.00±0.03)μmol/(L·min)respectively.And the K,of sitagliptin was 6.07 nmol/L(95%CI,5.61~6.58 nmol/L)(fitted curve R?>0.99).The inhibition rate and inhibition type of sitagliptin determined by this model was consistent with literature reports,proving that this model is capable of screening DPP-4 inhibitors.