摘要 目的 探讨一种改良的简易灌注分离小鼠肝细胞的方法。 方法 采用改良的简易两步灌注法分离小鼠肝细胞，即使用连有头皮针的注射器进行肝门静脉插管并灌流冲洗，再用Ⅳ型胶原酶灌流消化；分离肝细胞后采用室温静止法进行纯化；利用锥虫蓝染色法鉴定细胞存活率；培养板预先用鼠尾胶原铺备，利用倒置显微镜观察细胞形态；细胞贴壁后1～10 d检测培养液上清液中的白蛋白的分泌情况，判断肝细胞的生物学活性和功能。 结果 成功建立了一种简单易行的分离与培养小鼠原代肝细胞的方法。培养的小鼠肝细胞在接种后4 h基本完成贴壁，细胞可稳定贴壁至少1周，成活率可达85%以上且功能良好，可用于后续实验。 结论 改良的简易两步灌注法是一种简单易行、经济高效的分离纯化小鼠肝细胞的方法，适合小鼠肝细胞的分离。 Abstract：Objective To investigate a modified simple perfusion method for the isolation of mouse hepatocytes. Methods Mouse hepatocytes were isolated by the modified two-step simple perfusion method. A syringe with scalp needle was used for hepatic portal vein intubation and perfusion, and then type Ⅳ collagenase was used for digestion. The hepatocytes were purified by room-temperature immobilization after isolation. Trypan blue staining was used to measure cell viability; the culture plate was preliminarily prepared with rat tail collagen, and then an inverted microscope was used to observe the morphology of the cells; the secretion of albumin in the supernatant of culture medium was measured at 1-10 days after cell attachment to determine the biological activity and function of hepatocytes. Results A simple and easy method for the isolation and culture of primary mouse hepatocytes was successfully established. The cultured mouse hepatocytes basically adhered to the wall at 4 h after inoculation, with stable attachment for at least 1 week. Cell viability reached more than 85% and the cells had good function and could be used for subsequent experiments. Conclusion The modified two-step simple perfusion method is a simple and cost-effective method for the isolation and purification of mouse hepatocytes and is suitable for the isolation of mouse hepatocytes
