绿色荧光蛋白转基因小鼠胎肝干细胞在体内外定向分化的潜能  

Protential of directed differentiation of fetal liver stem cells in green fluorescent protein transgenic mice invivo and in vitro

作　　者：黄启科[1] 党立力[1] 阮柏[1] 张卓超[1] 刘杰[1] 王兴[1] 郑志刚[1] 陶开山[1] 

机构地区：[1]第四军医大学西京医院肝胆胰脾外科,西安710032

出　　处：《中华消化外科杂志》2014年第5期358-363,共6页Chinese Journal of Digestive Surgery

基　　金：国家自然科学基金（81170419、81272648）;陕西省科技创新工程重大科技专项（2012KTCL03-01）

摘　　要：目的探讨绿色荧光蛋白转基因小鼠胎肝干细胞（FLSCs）在体内及体外定向分化的潜能。方法取孕13d绿色荧光蛋白（GFP）转基因C57BL／6J小鼠胚胎肝脏，经机械消化和胶原酶消化获得FLSCs并扩大培养；成熟肝细胞来自于同品系小鼠成体肝脏，通过两步灌注胶原酶消化法提取，流式细胞仪鉴定其干细胞标志物的表达及自我增殖能力，RT—PCR及Westernblot测定AFP表达；分别将FLSCs经肝细胞生长因子（HGF）及TGF-β诱导分化后，Real—timePCR检测Alb、角蛋白CK一7、8、19mRNA以及Westernblot检测Alb、角蛋白CK-7、8、19蛋白在诱导前后的表达变化，鉴定其向肝细胞和胆管细胞双向分化的能力；经尾静脉注射FLSCs到野生型C57BL／6J肝切除小鼠体内，观察其在肝内重构肝组织的能力。计量资料采用t检验，各时相点比较采用重复测量的方差分析，两两比较采用LSD—t检验。结果成功克隆FLSCs细胞。流式细胞仪检测分离的FLSCs表达上皮细胞黏附分子（EpCAM）、CDl33、CD49f干细胞标志物，其阳性表达率分别为78．6％±3．3％、31．7％±2．5％、47．6％±1．8％。AFPmRNA在FLCs和成熟肝细胞中的相对表达量分别为0．640±0．115和0．053±0．012，蛋白相对表达量分别为1．3834±0．265和0．243±0．227，两者比较，差异有统计学意义（t=8．772，5．354，P〈0．05）。超低黏附培养皿培养FLPCs和成熟肝细胞，其形成克隆球数目分别为（234．04±57．0）个和（5．04±2．0）个，两者比较，差异有统计学意义（t=12．711，P〈O．05）。体外HGF诱导FLSCs分化，Alb、CK-8mRNA表达分别在8、10d到达峰值，分别为0．851±0．030和0．771±0．031；两者蛋白水平于第10天达峰值，分别为4．573±0．015和1．562±0．029。体外TGF—B诱导FISCs分化，CK-7、CK-19mRNA在第12天达峰值，分别为15．454±2．152和10．675±1．822；两者蛋白水平于第14天达峰�Objective To investigate the potential of directed differentiation of fetal liver stem cells （FLSCs） in mice with sponataneous green fluorescence in ,vitro and in vivo. Methods FLSCs were taken from green fluorescent protein transgenic mice at embryonic day 13. After mechanical dissection and enzyme digestion with collagenase, FLSCs were further cultured. The expression of stem cell related markers and the self-renewal ability were identified by flow cytometl3,. The expression of alpha fetoprotein （AFP） was detected by RT-PCR and Western-blot. FLSCs were induced to differentiation by hepatic growth factor （HGF） and transforming growth factor [3 （TGF-[3）, respectively, and the ability of dual-direction （hepatocyte and cholangiocyte） differentiation was meas- ured by exploring the expression alteration of Albumin （Alb）, cytokeratin （CK）-7, 8, 19 by using RT-PCR and the expression alteration of CK-7, 8, 19 and Alb by using Western blot. Suspended FLSCs were injected to wild type C57BL/6J mouce hepatectomy nlodel via caudal vein, and the ability of hepatic tissue reconstruction was observed. All data were analyzed using the analysis of variance or LSD-t test. Results FLSCs cells were successfully cloned. Stem cell related markers including epithelial cell adhesion molecule （EpCAM） , CD133 and CD49f were detected by flow cytometry. The positive expressions of EpCAM, CD133 and CD49f were 78.6% ± 3.3% , 31.7% ±2.5%and 47.6% ± 1.8% , respectively. The relative mRNA and protein expression levels of AFP in FLSCs and mature hepatocytes were 0. 640 ±0. 115, 0. 053 ± O. 012 and 1. 383 ± 0. 265, 0. 243 ± 0. 227, there were significant differences in AFP expression between FLSCs and mature hepatocytes （ t = 8. 772, 5. 354, P 〈 0. 05 ）. FLSCs and mature hepatocytes were both cultured in ultra-low attachment dishes for 2 weeks. There was a significant difference in the clone formation rate beteween the FLSCs and mature hepatocytes, which were 234.0 ±57.0 and 5.0 ± 2. O, respectively

关 键 词：绿色荧光蛋白转基因小鼠 胎肝干细胞 分离 鉴定 示踪 

分 类 号：R575[医药卫生—消化系统]