作者：寇晨光 曹成波 燕晓雯 罗兵 周岩冰
【摘要】 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?1、IL?6、TNF?α分泌功能，而单独培养组未检测到IL?1、IL?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。
【关键词】 肝细胞;枯否细胞;共同培养技术;清蛋白类;细胞因子类;大鼠
[ABSTRACT] 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 deve?loped 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,P<0.05), and that of ALT and AST, at the same time points as above, were higher (t=2.446-3.108,P<0.05). 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.
[KEY WORDS] hepatocyte; Kupffer cell; coculture technique;albumins; cytokines; rats
肝细胞分离、培养是研究肝脏和肝细胞的良好方法。尽管肝细胞在体内拥有相当强的增殖能力，但要肝细胞在体外保持分化状态且能继续增殖却非常困难。要明确影响肝细胞生长、分化和死亡的因素等，仍然面临认识水平和技术的挑战。实验已证实，肝实质细胞与非肝实质细胞或非肝细胞的共同培养可有效延长肝细胞的生存时间，促进肝细胞增殖;有助于保持细胞间特有的胆管结构，促进形成缝隙连接;可促进肝细胞的合成和分泌功能;有助于肝细胞维持其解毒功能[1?4]。目前进行的共同培养主要有肝细胞?非肝细胞培养或肝实质细胞?非实质细胞培养，而对肝细胞与Kupffer细胞的共同培养的报道还较少。本实验在体外将肝实质细胞与Kupffer细胞共同培养，观察体外联合培养体系中肝细胞形态、生长情况、功能的变化以及Kupffer细胞细胞因子分泌情况,旨在为肝细胞在病毒学、免疫学、药理学及肝细胞移植等方面的研究提供良好的平台。