大鼠肝细胞与Kupffer细胞共同培养对两者功能和形态影响

THE EFFECT OF CO-CULTURE IN VITRO OF Kupffer CELLS WITH HEPATOCYTES ON MORPHOLOGY AND FUNCTION OF HEPATOCYTES IN RATS

目的观察大鼠肝实质细胞与Kupffer细胞体外共同培养时对两者生长、形态及功能的影响。方法采用原位二步Ⅳ型胶原灌注法、Percoll液密度梯度离心法分离Wistar大鼠肝实质细胞与Kupffer细胞;体外将肝实质细胞与Kupffer细胞按6∶1比例共同培养。观察不同情况下肝细胞生存时间和形态,每隔24 h检测培养上清液中清蛋白和谷丙转氨酶（ALT）、谷草转氨酶（AST）的水平,并在培养36 h用放免法检测上清液中白细胞介素1（IL-1）、白细胞介素6（IL-6）、肿瘤坏死因子α（TNF-α）含量。结果单独培养组肝细胞的生长、增殖迅速,并向正常肝细胞的形态演变,肝细胞可培养存活至15 d;共同培养组肝细胞生长增殖缓慢,细胞可培养存活至10 d。共同培养组上清液中清蛋白水平在24、36、48、60 h比单独培养组低（t=2.551~3.139,P〈0.05）;ALT、AST水平在24、36、48、60 h比单独培养组高（t=2.446~3.108,P〈0.05）;共同培养组Kupffer细胞保持IL-1I、L-6、TNF-α分泌功能,而单独培养组未检测到IL-1I、L-6、TNF-α。结论大鼠Kupffer细胞和肝实质细胞在适宜的培养条件下可进行共同培养,二者可以保持良好的分泌功能,可用于实验研究。

Objective To observe the growth,morphology,and function of hepatocytes when cultured in vitro with Kupffer cells in rats. Methods In-situ Ⅳcollagenase two-step perfusion method and low speed gradient centrifugtion by percoll fluid were used to isolate parenchymal hepatic cells（PHC） and Kupffer cells in Wistar rat,respectively.Cocultivation of PHC and Kupffer cells was done in vitro according to 6∶1 ratio.The live time and form of the cells under different circumstances were observed.ALT and AST levels in culture supernatant were detected at 24-hour-interval,and the concentration of IL-1,IL-6 and TNF-α measured by radioimmunoassay at 36 hours. Results In solitary-culture group,the hepatocytes grew quickly and developed to the form of normal liver cells,which could survive 15 days;for those in co-culture group,the cells grew and generated slowly,which survived 10 days.The levels of albumen in supernate of co-culture group,at 24 h,36 h,48 h and 60 h,were lower than that in solitary-culture group（t=2.551-3.139,P0.05）,and that of ALT and AST,at the same time points as above,were higher（t=2.446-3.108,P0.05）.In co-culture group,the secretory function of IL-1,IL-6,and TNF-α was retained,and in solitary-culture group,no IL-1,IL-6,and TNF-α were detected. Conclusion Hepatocytes and Kupffer cells of rat can be cultured together under a suitable condition,and the favourable secretory function of the cells be maintained for empirical study.