原代猪肝细胞及主动脉内皮细胞的提取和培养  

Extraction and culture of primary porcine hepatocytes and aortic endothelial cells

作　　者：王权成 陶开山 李霄 张玄 白鸽 王博 窦科峰 Wang Quancheng;Tao Kaishan;Li Xiao;Zhang Xuan;Bai Ge;Wang Bo;Dou Kefeng(Department of Hepatobiliary Surgery,Xijing Hospital,Air Force Military Medical University,Institute of Organ Transplantation,Xi＇an 710032,Shaanxi,China.)

机构地区：[1]空军军医大学西京医院肝胆外科全军器官移植研究所,陕西西安710032

出　　处：《实用器官移植电子杂志》2018年第5期363-367,共5页Practical Journal of Organ Transplantation(Electronic Version)

基　　金：国家"973"计划基金资助项目(2015CB554100);国家重点研发计划资助项目(2017YFC1103703);国家"863"计划基金资助项目(2012AA021005);国家自然科学基金资助项目(81300361;81270549;81670593;81470873;81671838);陕西省自然科学基础研究计划资助项目(2017JM8014);陕西省科技统筹创新工程计划资助项目(2012KTCL03-01;2011KTCL03-15);西京医院学科助推计划基金资助项目(XJZT12M09;XJZT13Z01;XJZT14Z04)

摘　　要：目的建立有效高活力的原代猪肝细胞和主动脉内皮细胞提取和培养的方法。为异种移植排斥反应的靶细胞——血管内皮细胞和肝细胞的研究提供基础。方法从野生巴马猪分离肝脏和主动脉血管,通过蠕动泵灌注Ⅱ型胶原酶消化的方法提取猪肝细胞,低速离心和差速贴壁法纯化肝细胞,过碘酸雪夫(PAS)染色、白蛋白与肝细胞核因子4α免疫荧光染色对原代肝细胞进行鉴定;利用Ⅰ型胶原酶血管管腔灌注消化法提取猪主动脉内皮细胞,并通过检测因子Ⅷ相关抗原vWF及内皮细胞吞噬乙酰化低密度脂蛋白的方法进行鉴定。结果通过本文方法提取到了大量高活力的原代猪肝细胞和主动脉内皮细胞,猪肝细胞可以连续培养一周,内皮细胞可进行传代培养和冻存。两种细胞分别表达肝细胞和内皮细胞标志性蛋白。结论本研究提供的原代猪内皮细胞和肝细胞提取方法是制备大量高活力的原代内皮细胞及肝细胞的可靠方案。Objective To establish an effective method for the extraction and culture of vigorous primary porcine hepatocytes and aortic endothelial cells, and provide the basis for the research of porcine vascular endothelial cells and hepatocytes which are the target cells in xenograft rejection. Methods Liver and aortic blood vessel were isolated from wild Bama pigs, followed by primary porcine hepatocytes extraction using peristaltic pump perfusion and type II collagenase for digestion. Then porcine hepatocytes were purified by low speed centrifugation and differential adherence methods. Primary hepatocytes were identified by PAS staining, immunofluorescence staining for albumin and hepatocyte nuclear factor 4α. Endothelial cells of porcine aorta were extracted by type I collagenase digestion, and authenticated by testing factor VIII associated antigen vWF and endocytosis of acetylated DiI-Ac-LDL. Results A large number of highly viable primary porcine hepatocytes and aortic endothelial cells were extracted, and they can express hepatocyte and endothelial cell marker proteins respectively. Conclusion The primary pig endothelial cells and hepatocyte extraction methods provided in this study are reliable methods for preparing a large number of highly viable primary endothelial cells and hepatocytes.

关 键 词：猪 原代肝细胞 主动脉内皮细胞 异种移植排斥 

分 类 号：R657.3[医药卫生—外科学]