摘要
目的:建立细胞色素P450酶3A4(CYP3A4)、1A2(CYP1A2)及2E1(CYP2E1)的快速免疫印迹(Western blot)检测方法。方法:培养HepG2细胞并提取总蛋白,饲养大鼠并制备肝组织匀浆液、S9以及微粒体,以4种标本为对象分别以不同的封闭时间、抗体孵育时间和分离胶类型等常规Western blot条件设置A、B、C、D、E及F组用以考察CYP3A4的最佳检测条件;使用Western blot快速孵育仪,分别以不同的Western blot封闭和抗体孵育时间条件设置A、B、C和D、E、F及G、H、I组分别用以考察CYP3A4、CYP1A2及CYP2E1的最佳检测条件。结果:常规Western blot不同条件下,对CYP3A4的检测中C组检测灵敏度在HepG2、肝微粒体样品检测中高于A、B、D、E及F组(P<0.05),在组织匀浆、S9样品检测中C组检测灵敏度高于A、B、E组(P<0.05);在使用Western blot快速孵育仪孵育条件下,对CYP3A4的检测C组灵敏度在HepG2、肝组织匀浆及S9样品检测中高于A、B组(P<0.05),在肝微粒体样品检测中高于B组(P<0.05);对CYP1A2的各组样品检测中,F组的检测背景干净、条带的均一性均优于D、E组;对CYP2E1检测显示,在肝组织匀浆、S9及肝微粒体样品中I组的检测灵敏度高于G、H组(P<0.05)。结论:与常规检测条件相比,改进后的Western blot封闭孵育方式能够有效地对CYP3A4、CYP1A2及CYP2E1进行快速检测,缩短了封闭孵育的时间。
Objective:To establish a rapid Western blot detection method for cytochrome P450 enzymes 3A4(CYP3A4),1A2(CYP1A2),and 2E1(CYP2E1).Methods:HepG2 cells were cultured and total protein was extracted,rats were raised for preparing liver tissue homogenates,S9 and microsomes were prepared as the objects of this study.According to the conventional Western blot conditions,four samples were grouped as A,B,C,D,E and F with different including blocking time,antibody incubation time,separation gel concentration.The groups of were designed to investigate the optimal conditions of observing CYP3A4.Adopting Western blot fast incubator,the A,B,C and D,E,F together with G,H,I groups were set with the conditions of different Western blot blocking and incubation time to develop rapid detection methods of CYP3A4,CYP1A2,and CYP2E1.Results:Under different conventional Western blot conditions:the test sensitivity of group C was higher than that of group A,B,D,E and F in HepG2 and liver microsome samples(P<0.05)for the detection of CYP3A4;in tissue homogenates and S9 samples,the detection sensitivity of group C was higher than group A,B,and E(P<0.05);under the conditions of incubation with Western blot rapid incubator,the detection sensitivity of CYP3A4 in group C was higher than group A and B in HepG2,tissue homogenate,and S9 samples(P<0.05),and the detection sensitivity of samples in liver microsomes was higher than group B(P<0.05).in detecting each group of samples of CYP1A2,the detection background of group F is cleaner and its uniformity of bands is better than group D and E.The detection of CYP2E1 revealed that,the detection sensitivity of group I was higher than the group G and H in tissue homogenate,S9,and liver microsomes(P<0.05).Conclusion:Compared with normal testing conditions,Western blot blocking incubation method improves fast detection on CYP3A4,CYP1A2 and CYP2E1 quickly and efficiently,and shortens the blocking incubation time.
作者
何俊奇
杨畅
陆定艳
李靖
刘欢
王永林
刘亭
HE Junqi;YANG Chang;LU Dingyan;LI Jing;LIU Huan;WANG Yonglin;LIU Ting(Guizhou Provincial Key Laboratory of Pharmaceutics&State Key Laboratory of Functions and Applications of Medicinal Plants,Guizhou Medical University,Guiyang 550004,Guizhou,China;School of Pharmacy,Guizhou Medical University,Guiyang 550025,Guizhou,China)
出处
《贵州医科大学学报》
CAS
2020年第10期1117-1122,共6页
Journal of Guizhou Medical University
基金
国家自然科学基金(81603189,U1812403)
贵州省科技计划项目[黔科合基础(2019)1280]
贵州省科学技术厅人才团队项目[黔科合平台人才(2016)5613\5677]
中央引导地方科技专项项目[黔科中引地(2018)4006]。
