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目的:建立一种能稳定获得高活力和高纯度原代小鼠肝脏细胞的分离、纯化及培养方法。方法:应用改良的Seglen二步法原位灌注和机械离心分离肝脏细胞,并用改良的高糖DMEM培养基进行培养。台盼蓝拒染法检测接种时肝脏细胞的存活率,倒置显微镜动态观察肝脏细胞形态变化,应用免疫荧光技术对肝脏细胞进行Albumin染色。结果:每只小鼠可获取肝脏细胞的总产量平均为1.35×106/g体重,肝脏细胞存活率>90%。倒置显微镜下观察贴壁前肝细胞直径为35.14μm±4.35μm,肝脏细胞在接种后3 h基本完成贴壁;肝脏细胞接种后24h,所有肝脏细胞均强阳性表达成熟肝脏细胞标志物Albumin,肝细胞纯度>95%。结论:改良的分离纯化及培养方法能稳定获得高产量、高活率及高纯度的小鼠肝脏细胞。
OBJECTIVE: To establish a method for the isolation, purification and culture of primary liver cells with high viability and purity. METHODS: Liver cells were isolated by in situ perfusion and mechanical centrifugation using a modified Seglen two-step method and cultured in modified high glucose DMEM medium. Trypan blue exclusion method was used to detect the survival rate of liver cells inoculated. The morphological changes of liver cells were observed under inverted microscope. Albumin staining of liver cells was performed by immunofluorescence. RESULTS: The average total liver cell yield per mouse was 1.35 × 10 6 / g body weight and liver cell survival was> 90%. The diameter of the pre-adherent hepatocytes was 35.14μm ± 4.35μm under inverted microscope. The hepatocytes were almost completely adhered 3 h after inoculation. The hepatocytes were strongly positive for mature liver cell marker Albumin after 24h of inoculation, Cell purity> 95%. Conclusion: The improved method of isolation, purification and culture can stably obtain mouse liver cells with high yield, high viability and high purity.