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目的:探索原代培养大鼠肝细胞的最佳分离方法。方法:对比观察不同的肝细胞分离技术的培养效果。培养前,以4％台盼蓝染色判定肝细胞活性,培养48h后,观察肝细胞贴壁及生长状况。结果:①灌流消化法优于剪切消化法;②在多种灌流消化法中,经门静脉灌流消化法优于经胆总管灌流消化法及经腹主动脉灌流消化法;③在门静脉灌流的基础上,0.1％胶原酶Ⅰ37℃热消化10～15分钟,效果最好,而0.25％胰蛋白酶消化10～12分钟次之,用含EDTA的D-Hank液直接灌流分离法效果极差。结论:原代培养肝细胞的得率及活性与不同分离法包括灌流途径、灌流液、消化时间等有关。
Objective: To explore the best method of primary culture of rat hepatocytes. Methods: The effects of different hepatocyte separation techniques were observed. Before culturing, the activity of hepatocytes was judged by trypan blue staining of 4%, and after 48 hours of culturing, the attachment and growth of hepatocytes were observed. Results: ① perfusion digestion method is better than shearing digestion; ② in a variety of perfusion digestion method, by portal vein digestion was better than by the common bile duct perfusion digestion and abdominal aorta digestion; ③ in the portal vein perfusion basal , 0.1% collagenase Ⅰ thermal digestion at 37 ℃ 10 to 15 minutes, the best, and 0.25% trypsin digestion 10 to 12 minutes followed by EDTA-containing D-Hank direct perfusion separation method very poor. Conclusion: The yield and activity of primary cultured hepatocytes are related to different isolation methods including perfusion pathway, perfusate, digestion time and so on.