双峰驼CYP2D6酶体外孵育体系的建立与优化

为了研究双峰驼CYP2D6酶体外活性,建立双峰驼肝微粒体孵育体系并对孵育体系中探针底物浓度、肝微粒体蛋白浓度和孵育时间等进行优化研究。首先采用改良差速离心法制备双峰驼肝微粒体、BCA法测定双峰驼肝微粒体蛋白浓度、CO还原差示光谱法检测CYP总酶含量,然后采用HPLC法跟踪检测孵育体系中CYP2D6酶特异性底物的主要代谢产物去甲右美沙芬含量进而优化孵育条件。结果表明,双峰驼肝微粒体蛋白浓度为5.565 0mg/mL±0.519 7mg/mL,CYP总酶含量为0.177 7nmol/mg±0.050 3nmol/mg;肝微粒体孵育体系的最适底物浓度为250μg/mL,肝微粒体蛋白浓度为5.565 0mg/mL,最适孵育时间为40min。所制备的双峰驼肝微粒体各项指标和优化后的肝微粒体孵育条件均能满足后续对双峰驼CYP2D6酶体外活性研究的基本要求。

Establishment and Optimization of Incubation System of Bactrian Camel Studying CYP2D6 Enzyme in Vitro

In order to study the in vitro activities of Bactrian Camel CYP2D6 enzyme,the liver microsome in-cubation system was established and optimized by studying the concentration of probe substrate,protein content of liver microsome and incubation time,etc.Firstly,liver microsomes of bactrian camel was pre-pared by modified differential centrifugation method,the protein content of bactrian camel liver microsomes was detected by using BCA method and the total content of CYP enzyme was determined by using CO re-duction method.Secondly,the incubation system was optimized by detecting and tracking the concentration of dextrophan,an active essential metabolite of dextromethorphan,in incubation system by using HPLC method.The results showed that the liver microsomal protein content of bactrian camel was 5.565 0 mg/mL±0.519 7 mg/mL,total CYP enzyme content was 0.177 7 nmol/mg±0.050 3 nmol/mg and the optimum substrate concentration in the liver microsome incubation system was 250 μg/mL,the op-timum concentration of protein in liver microsomes was 5.565 0 mg/mL and the optimal incubation time was 40 min,respectively.Therefore,the prepared bactrian camel liver microsome and optimized incubation system can meet the basic requirements of further research on bactrian camel CYP2D6 enzyme activities in vitro.