HBV转基因小鼠肝原代细胞分离纯化及其在肝损伤研究中的应用  

Isolation and purification of primary hepatocytes from HBV transgenic mice and their application in the study of liver injury

作　　者：贺云 黄敏健 陈艺生 HE Yun;HUANG Minjian;CHEN Yisheng(Key Laboratory of Gastrointestinal Cancer,Ministry of Education,Fujian Key Laboratory of Tumor Microbiology,School of Basic Medical Sciences,Fujian Medical University,Fuzhou 350122,China;Center for Experimental Research in Clinical Medicine,Fujian Provincial Hospital,Fujian Medical University,Fuzhou 350001)

机构地区：[1]福建医科大学基础医学院,消化道恶性肿瘤教育部重点实验室,福建省肿瘤微生物学重点实验室,福州350122 [2]福建省立医院临床医学实验研究中心,福建医科大学,福州350001

出　　处：《中国实验动物学报》2022年第4期526-532,共7页Acta Laboratorium Animalis Scientia Sinica

基　　金：福建医科大学启航基金项目(2018QH1006,2020QH1150)。

摘　　要：目的分离鉴定乙型肝炎病毒(HBV)转基因小鼠肝原代细胞,为HBV体外相关研究提供有利模型。方法HBV转基因C57 BL/6小鼠深度麻醉后,通过二步灌注法,首先用不含钙灌注液将肝中的血液冲洗干净,然后用含钙的IV型胶原缓冲液将肝细胞消化下,通过3次低速低温离心法纯化去除杂质,则成功分离提取肝原代细胞。进而通过糖原染色法对提取的细胞进行鉴定,并检测7 d内肝细胞的增殖活力,以及第1天和第3天细胞凋亡情况。最后,将提取的肝细胞应用于过氧化氢诱导肝细胞损伤表型检测,包括细胞内活性氧、线粒体膜电位、多个氧化应激因子转录水平检测和LDH泄露情况。结果(1)分离的HBV小鼠肝原代细胞为典型性肝细胞形态,并呈现糖原阳性染色结果;(2)肝细胞活性检测发现肝细胞贴壁后,活性增强,第5~6天时活性最高,然后逐渐下降;(3)对比肝细胞提取后第1天和第3天的细胞死亡情况,未发生明显自发性死亡;(4)细胞通过过氧化氢诱导后,发现胞内活性氧积聚,线粒体膜电位改变,氧化应激因子转录水平上调,以及乳酸脱氢酶泄露增加。结论在本实验条件下,成功分离鉴定HBV小鼠肝原代细胞,并应用于肝损伤相关实验。Objective To isolate and identify primary hepatocytes from hepatitis B virus(HBV)transgenic mice,and provide a useful model for the study of HBV in vitro.Methods HBV transgenic C57 BL/6 mice were anesthetized and primary mouse hepatocytes(PMHs)were isolated and purified via a two-step infusion method.First,the liver was flushed with a calcium-free infusion,then digested with calcium-containing type IV collagen buffer.Hepatocytes were purified by centrifugation at low-speed and low-temperature three times,and the purified PMHs were confirmed by glycogen staining.In addition,the viability of PMHs within 7 days and the apoptotic ratio on days 1 and 3 were detected.Furthermore,PMHs were used to detect the effects of liver injury induced by hydrogen peroxide,including intracellular reactive oxygen species,mitochondrial membrane potential,oxidative stress factor level detection,and lactate dehydrogenase(LDH)leakage.Results(1)The PMHs isolated from HBV transgenic mice showed typical hepatocyte morphology and positive glycogen staining.(2)The viability of hepatocytes increased after being adhered to a plate,and peaked at days 5 to 6,and then decreased gradually.(3)There was no difference between the apoptosis ratio on day 1 and day 3,with no spontaneous death.(4)Hydrogen peroxide had serious effects,including reactive oxygen accumulation,a loss in mitochondrial membrane potential,upregulation of oxidative stress factors,and LDH leakage.Conclusions Taken together,these data indicate that the PMHs of HBV can be successfully isolated and applied to liver injury research.

关 键 词：原代细胞 二步灌注法 模型 肝损伤 

分 类 号：Q95-33[生物学—动物学]