一种新生大鼠肝细胞分离改良方法的建立

An improved approach for seperating hepatocytes from neonatal rats in vitro

目的尝试建立分离并获得稳定培养的新生大鼠原代肝细胞(HC)的简易方法。方法采用多步酶消化法和组织块培养贴壁法分离大鼠原代HC,经低速离心、选择性贴壁培养并纯化肝细胞。结果最初获得单细胞数量约1～2×10~6个/只,经0.4%台盼蓝染色细胞瞬时活率>72%,单细胞悬液和组织块两种培养方法均在3～5 d发现细胞可长满瓶底的70%～80%,经传代纯化以后细胞经糖原染色和抗CK-18免疫组化染色鉴定,发现HC纯度>95%,细胞生长状态良好,可供一般实验室使用。结论成功建立了获取原代培养HC的简易方法,细胞数量、纯度、活率较好,可供一般实验室实验研究。

Objective To establish a simple approach for isolating hepatocytes(HCs) from neonatal rats.Methods Multi-step enzymatic digestion and tissue adhering method were used to isolate primary HCs from neonatal rats,and after low-speed centrifugation and selective adherence culture,the HCs were identified by PAS and anti-CK-18 immunocytochemical staining. Results The numbers of separated HCs were about 1-2×10~6 cells per rat. After 0.4% trypan blue staining,the transient cell activity was >80%,and after cell suspension or tissue block were cultured for 3 to 5 days,the cells spread 70% to 80% of the bottom of each bottle. The purity of the HCs was greater than 95%,and the cell growth were in good condition. Conclusion The primary parenchymal cells of liver obtained were pure and in good activity,which might be used in laboratory for further experiments.