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目的:探讨体外分离培养大鼠小肝细胞(SHC)的方法,并对其进行表型鉴定。方法:利用二乙基亚硝胺饲喂法,建立SD大鼠肝硬化模型。采用改进后两步胶原酶灌注消化法分离细胞。光镜下观察细胞形态。电镜下观察细胞形态及超微结构。采用计数法测定细胞生长曲线,流式细胞术(FCM)测定细胞周期。通过免疫细胞化学染色法进行表型鉴定。结果:原代培养的大鼠SHC,24h内即可见贴壁伸展,48h左右克隆形成。光镜下可观察SHC核仁小而清晰,胞质少,细胞体积较小;电镜下可见SHC表面少量短而小的微绒毛突起,细胞体积小,核浆比例高,呈现未分化细胞的形态。免疫细胞化学染色显示SHC胞质AFP、CK-7、CK-8及CK-19呈棕黄色阳性信号。结论:改进后的两步胶原酶灌注消化方法能较容易地获得大量纯度较高的SHC。
Objective: To investigate the method of isolation and culture of rat small hepatocytes (SHC) in vitro and to identify its phenotype. Methods: Diethylnitrosamine feeding was used to establish a model of SD rat cirrhosis. Cells were isolated by modified two-step collagenase perfusion digestion. Light microscope observation of cell morphology. Electron microscope observation of cell morphology and ultrastructure. Cell growth curve was determined by counting method, and cell cycle was determined by flow cytometry (FCM). Phenotypic identification by immunocytochemical staining. Results: Primary cultured rat SHC showed adherent spreading within 24 hours and formed around 48 hours. Light microscopic observation of SHC small and clear nucleoli, less cytoplasm, smaller cell size; electron microscopy can be seen a small number of short SHC surface microvilli protrusions, small cells, a high proportion of nuclear cytoplasm, showing undifferentiated cells morphology . Immunocytochemical staining showed positive cytoplasmic AFP, CK-7, CK-8 and CK-19 in SHC. Conclusion: The improved two-step collagenase perfusion digestion method can easily obtain a large number of high purity SHC.