小鼠胚胎干细胞分化为肝前体细胞及其脾内移植的实验研究  

Engrafted hepatic precursor cells induced from mouse embryonic stem cells differentiate into functional hepatocytes in rat spleen

作　　者：江海洪[1] 王宇明[1] 刘国栋[1] 陈耀凯[1] 李俊刚[1] 赵文利[1] 

机构地区：[1]第三军医大学西南医院全军感染病研究所,重庆400038

出　　处：《第三军医大学学报》2006年第6期503-506,共4页Journal of Third Military Medical University

基　　金：国家高技术研究发展计划("863")资助项目(2001AA216161)~~

摘　　要：目的将体外小鼠胚胎干细胞(ESC)诱导为肝前体细胞(HPCs)并移植到预处理的C57/BL6小鼠脾脏,检测HPCs在小鼠脾脏内分化和移植细胞的功能及其在脾脏组织中的整合情况,探讨肝细胞合适的替换治疗细胞来源。方法体外培养ESC并制备拟胚体(EBs),体外诱导EBs 18 d后得到HPCs,然后用Hochest 3342荧光标记HPCs并制备成6.0×107/m l细胞悬液。移植受体小鼠用2乙-酰氨基芴(2-AAF)处理,移植前受体鼠经过7 Gy60Coγ全身辐照和50%肝脏切除,造成小鼠体内对肝细胞的极大需求;然后将0.1 m l含6.0×106HPCs细胞悬液经尾静脉注射移植入脾脏,细胞移植4周后,检测移植细胞在小鼠脾脏整合分化情况。结果体外培养ESC并制备出EBs,EBs经体外诱导为HPCs,并将HPCs移植到小鼠脾脏,4周后观察到移植细胞能够嵌合到受体脾脏组织,在脾脏组织中未形成畸胎瘤,并且移植细胞能表达肝细胞较特异标志物白蛋白、CK19和糖原PAS染色呈阳性反应。结论本实验建立一种将体外ESC诱导的HPCs移植并整合到脾脏的细胞移植技术,为研究肝细胞分化的分子机制和肝病的细胞替换治疗提供基础。Objective To investigate differentiation of mouse embryonic stem cells （ESC） into hepatic precursor cells （HPCs） in vitro and transplant the induced HPCs into the rat spleen, then to explore ESC as a graft source of hepatocytes in the spleen. Methods Mouse ESC were cultured on the feeder layer of mouse embryonic fibroblasts （MEF） in the medium containing leukemia inhibitory factor （LIF）, and embryonic bodies （EBs） were prepared. HPCs were selectively differentiated at day 18 in the medium including acid-fibroblast growth factor （acid-FGF）, HGF, recombinant human oncostatin M （OSM） and ITS et al, added at different time point according to developmental biology of liver. In order to test the induced HPCs, albumin （ALB） was detected with immunohistochemistry by fluorescent microscope, CK19 was examined by immunohis- tochemical method and stained with DAB staining, glycogen of differentiated HPCs was observed with periodic acid Schiff＇s test （PAS）. Transplanted cells were labeled by fluorescence dye Hochest 3342. Mice were injected with 0.4 mg 2-acetylaminofluorene （2-AAF） once a week. Fourteen days after injection, partial hepatectomy （PHx） （50%） followed radiation （RT,7 Gy） was performed in mice. HPCs （6. 0 × 10^6） were transplanted into the spleen intravenously through the caudal vein. Four weeks after cell transplantation, the spleen spec- imen was obtained for examination. Results ALB and CK19 were positive in induced HPCs. Induced HPCs/ hepatocyte-like cells engrafted and did not formed teratomas 4 weeks after cell transplantation in the spleen. By tracing the HPCs labeled with Hochest3342 in the spleen, we found these cells were incorporated into the spleen tissue and could generate ALB. Conclusion HPCs/hepatocyte-like cells derived from mouse ESC engrafted without forming teratomas and differentiated into functional hepatocytes in the spleen.

关 键 词：胚胎干细胞 脾脏 肝前体细胞 细胞移植 2-乙酰氨基芴 

分 类 号：R322.21[医药卫生—人体解剖和组织胚胎学] R322.47[医药卫生—基础医学]