人脐血间充质干细胞在体外向肝样细胞的诱导分化  

Differentiation of mesenchymal stem cells from umbilical cord blood into hepatocyte-like cells in the invitro culture

作　　者：迟作华[1] 张洹[1] 陆琰[1] 

机构地区：[1]暨南大学医学院血液病研究所,广州510632

出　　处：《中华器官移植杂志》2008年第3期159-163,共5页Chinese Journal of Organ Transplantation

基　　金：国务院侨办重点学科基金（2005）;国家自然科学基金（3067（）902）;广东省自然科学基金（06300546）

摘　　要：目的 探讨脐血间充质干细胞（MSCs）在体外能否分化成肝细胞。方法 分离人脐血MSCs，培养传代，流式细胞仪进行细胞表面标志检测。取培养至第三代的细胞，接种于六孔板内，分两个阶段进行细胞分化的诱导，第一阶段采用含地塞米松（终浓度为0．5μmol／L，下同）、肝细胞生长因子（HGF，10ng／m1）、表皮生长因子（10ng／m1）及1×ITS（胰岛素-转铁蛋白-硒）的F12培养基诱导2周，第二阶段用含地塞米松（0．5μmol／L）、HGF（10ng／m1）、抑瘤素M（10ng／m1）及1×ITS的F12培养基继续诱导2周。诱导期间于倒置显微镜下观察细胞的形态变化；采用逆转录聚合酶链反应检测分化细胞的甲胎蛋白（AFP）、白蛋白、人细胞角蛋白18（CK-18）及酪氨酸氨基转移酶（TAT）基因的表达，以免疫荧光染色法检测分化细胞胞浆中AFP、白蛋白、CK-18的表达；用电子显微镜观察分化细胞的超微结构。结果 培养的脐血MSCs不表达CDl4、CD34及CD45，也不表达CD49f、CD54及HLA-DR；部分表达CD106；强表达CDl3、CD29及CD44。未分化的脐血MSCs不表达AFP、TAT及白蛋白基因，弱表达CK-18基因；诱导分化1周后可检测到AFP基因的表达，诱导分化4周后，不仅表达AFP、CK-18和白蛋白基因，还表达TAT基因。免疫荧光染色显示，未分化的MSCs胞浆中无AFP、白蛋白及CK-18的表达；分化细胞则可以检测到上述物质的表达。诱导至第4周的分化细胞，可见核仁大且明显，细胞核周围有板层状的内质网，胞浆中可见脂滴及簇状糖原，线粒体丰富，细胞表面有微绒毛。结论 脐血MSCs在合适的诱导条件下可以分化为肝样细胞。Objective To discuss the possibility of differentiation of mesenchymal stem cells （MSCs） from umbilical cord blood （UCB） into hepatocyte-like cells in the in vitro culture. Methods MSCs were isolated from UCB, cultured and passaged. The surface markers were examined by flow cytometry. When cells were cultured to the third passage, they were inoculated into a 6-well plate. A two-stage induction method was used： MSCs in the first phase were cultured in DMEM/F12 medium supplemented with dexamethasone （final concentration of 0. 5 μmol/L, the same below）, hepatocyte growth factor （HGF, 10 ng/ml）, epidermal growth factor （10 ng/ml） and 1 ×insulin-transferrin-Se （ITS） for two weeks, then in DMEM/F12 supplemented with 0. 5 μmol/L dexamethasone, 10 ng/ml HGF, 1 ×ITS, 10 ng/ml Oncostatin M for another two weeks. Morphological changes were observed under a microscope. The gene expression correlated with hepatocytes was detected by using RT-PCR. Immunofluorescence staining was used to identify the expression of specific protein related to hepatocytes （AFP, Albumin, CK-18）. Ultrastructure was detected under an electron microscope. Results In the cultured MSCs from UCB, CD34/CD45/CD14, CD54, CD49f and HLA-DR were not detected, there was low expression of CD106 and strong expression of CD29, CD44 and CD13. The gene expression of AFP, albumin, CK-18 and TAT was discovered and three kinds of protein AFP, albumin and CK-18 were positively showed in cytoplasm after 4 weeks＇ induction. The hepatin granules and fatty drops in cytoplasm of cells induced for 4 weeks were found under an electron microscope. Conclusion The MSCs fromUCB can differentiate into hepatocyte-like cells in the in vitro culture under some conditions.

关 键 词：胎血 间质干细胞 细胞分化 肝细胞 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学] R692[医药卫生—基础医学]