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目的：建立一种简便、经济、高产的同步分离培养肝细胞以及肝星状细胞的方法。方法：在参照国内外方法的基础上加以改良，首先采用肝脏原位胶原酶灌注消化的方法，获得总细胞悬液，经多次低速离心分离肝细胞；再用Nycodenz 作为分离介质，通过密度梯度离心法从非实质细胞中得到肝星状细胞。通过台盼蓝染色方法鉴定细胞的活力，用倒置相差显微镜、立体显微镜、 CK-18、白蛋白免疫荧光细胞化学染色对培养的肝细胞形态以及功能进行检测。使用Desmin、alpha-SMA 免疫荧光细胞化学对肝星状细胞进行鉴定。结果：成功的在体外同步分离、培养肝细胞及肝星状细胞，肝细胞产率为5-6× 107/ 只小鼠，两只小鼠肝星状细胞产率达1× 106个。细胞存活率及纯度均可达90%。肝细胞在培养24h 后呈不规则铺路石样形态，此为典型的肝细胞形态，其标志分子CK-18 以及白蛋白免疫荧光染色阳性。倒置相差显微镜下可见贴壁后的肝星状细胞呈典型的星形细胞形态，且其标志分子 Desmin、alpha-SMA 免疫荧光染色阳性。结论：改良的原位灌注以及分离方法可以同时分离并且培养具有高活性和功能的肝细胞和肝星状细胞。

英文摘要:

Objective: To establish a simple, economical and high-efficiency technique to isolate and culture the mouse hepatocytes and hepatic stellate cells coinstantaneous.Methods:In situ collagenase perfusion of liver was performed for the total cell suspension and then repeated low-speed centrifugation was used for the separation of hepatocytes. Hepatic stellate cells could be obtained from non-parenchymal cells by density gradient centrifugation using Nycodenz as a separation medium. Cell viability was identified by Typan staining. The inverted phase contrast microscope, stereoscopic microscope and immunofluorescence staining of CK-18 and albumin were used to determine the morphology and function of hepatocytes. Immunofluorescence staining of desmin and alpha-SMA were used to determined hepatic stellate cells.Results:Hepatocytes and hepatic stellate cells were separated synchronously and cultured . 5-6× 107 hepatocytes were harvested per mouse and the yield of hepatic stellate cells was 1× 106 or so per two mice. Both cell viability and purity could reach 90%. After incubation for 24h, hepatocytes presented typical irregular cobblestone morphology and positive staining of CK-18 and albumin. Hepatic stellate cells showed typical morphology of star-shapes and positive staining of Desmin and alpha-SMA.Conclusion:Improved situ perfusion method can be used for simultaneous isolation of hepatocytes and hepatic stellate cells with high viability and function.

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