鸡肝原代细胞药物代谢模型的建立与优化  

The establishment and optimization of the drug metabolic model of adult chicken primary hepatocytes

作　　者：刘丽[1] 季辉[1] 彭麟[1] 阮祥春[1] 吉利伟[1] 江善祥[1] 

机构地区：[1]南京农业大学动物医学院,江苏南京210095

出　　处：《南京农业大学学报》2015年第1期127-133,共7页Journal of Nanjing Agricultural University

摘　　要：[目的]建立成年鸡原代肝细胞的药物代谢模型并对其进行优化。[方法]以改进的二步灌流法分离8~12周龄的雄性黄羽鸡肝细胞,比较在不同细胞基础培养液条件下鸡肝细胞体外培养的情况,利用倒置显微镜观察肝细胞的形态,利用MTT检测法绘制生长曲线,测定细胞培养上清液中乳酸脱氢酶（LDH）活性,用q PCR和Western blot测定肝细胞中CYP450酶亚型CYP1A4、CYP1A5和CYP3A37 mRNA相对表达水平和3种酶蛋白的表达量。[结果]离体灌流操作简便,获得的细胞存活率高达90%以上;用含0.5 mg·L^-1牛胰岛素、10 g·L^-1双抗（100 U·m L^-1青霉素和链霉素）和体积分数为10%胎牛血清的DMEM基础培养液体外培养鸡肝细胞,贴壁培养时可见到肝细胞较为明显的分化过程,贴壁后呈上皮细胞样生长,多角形,胞浆内容物均匀丰富,细胞核清晰透亮,少数有双核,细胞生长状态良好。生长曲线和LDH活性测定结果显示：培养3~5 d时细胞状态稳定,并且在肝原代细胞培养的8 d内,CYP1A4、CYP1A5和CYP3A37 mRNA相对表达水平和蛋白的表达量没有显著差异。[结论]以改进的二步灌流法分离鸡肝细胞操作简便,分离的鸡肝原代细胞存活率高,用含0.5 mg·L^-1胰岛素、100 U·m L^-1青霉素、100μg·m L^-1链霉素和10%胎牛血清的DMEM基础培养液体外培养鸡肝原代细胞效果最好,培养3~5 d是进行体外药物代谢等试验的最佳时机。[Objectives]To establish the drug metabolic model in vitro,the primary hepatocytes culture methods and the drug metabolic model were optimized. [Methods]The chicken primary hepatocytes were isolated by modified two-step in situ collagenase perfusion from8-10 week-old yellow-feathered male broilers. Meanwhile,effects of different mediums on the morphology and growth of the cells were investigated. The culture condition was optimized by comparing the effects between DMEM medium,1640 medium,William＇s E medium and Leibovitz＇s L^-15（ L^-15） medium. The morphology of the cells was observed under the inverted microscope and the growth curve was drawn by MTT assay. The lactate dehydrogenase（ LDH） concentration in supernatant was determined with a LDH test kit. And the relative expression of mRNA about CYP1A4,CYP1A5 and CYP3A37 was quantitated by the q PCR method,while protein relative expression about CYP1A4,CYP1A5 and CYP3A37 was quantitated by Western blot. [Results]The isolated method of the chicken primary hepatocytes by modified two-step in situ collagenase perfusion in vitro is simple. The well-dispersed and pure hepatocytes with high viability（ 90%） were obtained. By comparing the effects of the four mediums,the adherence of the chicken primary hepatocytes were best by using William＇ s E medium,followed by DMEM medium and L- 15 medium,then 1640 medium slightly worse. But the chicken primary hepatocytes maintained a long stable period on the morphology and growth in the long process by using DMEM medium,long-term training 2-3 weeks,and compared with William＇ s E medium,DMEM medium is more economical,so the chicken primary hepatocytes were cultured by using DMEM medium supplemented with 0. 5 mg·L^-1insulin,1% streptomycin and 10% fetal bovine serum foundation. The cells attached on the surface of petri dishes with 4 h after inoculation（ 5×105m L^-1）. The morphology of chicken hepatocytes which was observed under the inverted microscope was effectively maintained in the culture medium. Under

关 键 词：成年鸡 离体灌流 鸡肝细胞 药物代谢模型 优化 

分 类 号：S859.1[农业科学—临床兽医学]