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.

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。