小鼠成体肝脏干细胞体外培养模型的建立  

Establishment of adult hepatic stem cell in vitro culture model in normal adult mice

作　　者：王洪山[1] 宋陆军[1] 沈坤堂[1] 刘隽[1] 高晓东[1] 沈睿[1] 牛伟新[1] 秦新裕[1] 

机构地区：[1]复旦大学附属中山医院普外科,上海200032

出　　处：《中华实验外科杂志》2008年第3期386-388,F0004,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（30371388）;上海市科学技术委员会重大资助项目（04DZ19505）

摘　　要：目的观察正常成体小鼠肝细胞的增殖能力和分化潜能，分离成体小鼠肝脏内可能存在的干细胞或祖细胞，建立细胞培养模型。方法应用改良的Seglen二步法灌注和离心分离肝脏细胞，用含血清的改良DMEM培养基进行培养，持续观察超过60d。应用免疫荧光技术对肝细胞及其形成的克隆进行Albumin、AFP和CK19染色。结果部分肝脏细胞培养第2～3天后活化，迅速增殖并形成细胞克隆，培养30d后克隆内出现类似成熟的肝细胞，细胞克隆持续扩增超过60d。该类细胞培养第1天强阳性表达肝细胞标记物Albumin，培养第5天细胞克隆开始表达肝脏干细胞标记物AFP，第55天表达胆管细胞标记物CK19。结论在成体小鼠未损伤肝脏内存在一种成体肝脏祖细胞（adult hepatic progenitor cells，AHPCs），该细胞体外培养具有较强的增殖能力，可分化为肝细胞和胆管细胞，并成功建立了AHPCs的体外培养模型。Objective To isolate and culture candidate cells of adult hepatic stem cells or progenitor cells from uninjured adult mouse liver, investigate their ability of proliferation and potentials to differentiate in vitro and establish the cell culture model. Methods Hepatocytes, isolated by two-step collagenase perfusion and mechanical centrifugation, were cultured with modified DMEM, and observed under the phase contrast microscopy for over 60 days. The albumin, AFP and CK19 staining was peformed on hepatocytes and their colonies by immunotluorescence. Results A subpopulation of hepatocytes began to proliferate significantly and to form colonies while being activated after 2 -3 days in culture. Those colonies could expand continually more than 60 days, with some larger cells morphologically like mature hepatocytes appearing around day 30. Albumin was definitely expressed in all hepatocytes at 24 h after attachment. Within hepatocyte colonies, AFP was expressed at day 5, and CK19 was positive at day 55. Condusion Adult hepatic progenitor cells exist in uninjured adult mouse liver, which can proliferate significantly after being cultured in vitro, and hold bipotential to differentiate into hepatocytes and duct cells.

关 键 词：肝细胞移植 肝脏干细胞 细胞培养 

分 类 号：R-332[医药卫生]