大鼠肝干细胞的分离培养  

THE ISOLATION AND CULTURE OF RAT HEPATIC STEM CELLS

作　　者：邹清雁[1] 张宜俊[1] 孔祥平[1] 张淑莲[1] 郑曲波[1] 肖荣 罗峰 

机构地区：[1]解放军第458医院解放军传染病防治中心,广州市510602

出　　处：《实用肝脏病杂志》2000年第4期195-198,共4页Journal of Practical Hepatology

摘　　要：目的 分离大鼠胎肝细胞,观察人肝细胞生长肽(HGP)和小鼠白血病抑制因子(mLIF)对其生长和肝干细胞集落形成的影响。方法 从大鼠胚胎(12日龄)肝中分离肝细胞,采用MTT比色法、~3H-TdR掺入法和碱性磷酸酶(AKP)检测法。结果 人HGP能剂量依赖性地促进胎肝细胞的DNA合成和增殖,500～2000u/ml的mLIF能明显促进肝干细胞集落的生长和维持未分化状态。肝干细胞培养基的组成为:5％FBS(国产或进口),10％国产NCS,0.14mM的β-ME,40ng/ml伴白蛋白,10ng/ml的人bFGF,0.5μg～1.0μg/ml的HGP,1000u/ml mLIF,1％非必需氨基酸的高糖DMEM培养基。AKP染色阳性细胞在上述培养基中传至6代仍生长良好和保持未分化状态。结论 从胎鼠肝中能分离到肝干细胞,人HGP能促进鼠胎肝细胞的生长,而mLIF对促进肝干细胞生长和维持未分化是必要的。Objective Aim To dissociate the embryonic hepatocytes and define the growth requirement for proliferation of hepatic stem cells. Methods The liver of murine embryos about 12-14 days were digested by collagenase to dissociate the embryonic hepatocytes. The effects of HGP and mLIF on the hepatocytes were selected by the methods of testing the hepatocytes' dedydroxygenase activity,3H-TdR incorparation and alkaline-phosphatase(AKP)stain. Results The human hepatocyte growth peptide(HGP)can apparently promote the DNA systhesis and proliferation of the embryonic hepatocytes in a dose dependent manner,and 500-1000u/ ml mLIF can apparently promote the growth of the hepatocyte stem cells and maintain them undifferenti-ated. The hepatocyte stem cell optimal culturing condition which composed of DMEM(high glucose)containing 4. 5g/L glucose, 10% newborn calf serum (NBS) , 5%FBS, 10fig/L human bFGF, 0. 5 - 1. 0μg/ml HGP, 1×106u/L mLIF,40μg/L Conalbumin,and 0. 14mmolβ-Mercaptoethanol(β-ME)were established. The alkaline - phosphatase -positive cells can be maintained growth well and undifferentiation until 6 passages. Conclusion hepatocyte stem cells can be abtained from the murine embryonic liver,HGP can promote the growth of embryonic liver cells,and mLIF can promote the growth of hepatocyte stem cells and is essential to keep them undif-ferentiated.

关 键 词：肝 肝前体细胞 肝干细胞 大鼠 胚胎 碱性磷酸酶染色 

分 类 号：R329-33[医药卫生—人体解剖和组织胚胎学]