脐血间充质干细胞的体外扩增及向类肝细胞分化的实验研究  

Experimental study on the amplification and differentiation of human umbilical cord blood mesenchymal stem cells into functional hepatocypte-like cells in vitro

作　　者：韩翠萍[1] 刘吉勇[1] 高蕾[2] 裴庆山[1] 孙欣欣[1] 文婷玉[1] 

机构地区：[1]山东大学附属省立医院,济南250021 [2]深圳市宝安区人民医院

出　　处：《山东医药》2010年第7期3-5,共3页Shandong Medical Journal

基　　金：山东省自然科学基金资助项目(Y2002C10)

摘　　要：目的探讨人脐带血间充质干细胞(UBC-MSCs)向类肝细胞定向分化的可能性。方法无菌条件下采集新生儿脐带血75份,采用密度梯度离心法与直接贴壁法相结合分离UBC-MSCs,体外培养传代,获得第4代集落生长细胞作流式细胞仪表面抗原测定,并应用a-FGF、b-FGF、HGF、OSM等多种成分,分阶段向肝细胞定向诱导分化。倒置相差显微镜观察细胞形态变化,取诱导后4周和6周的细胞分别采用RT-PCR法检测ALB、AFP和CK-19基因mRNA水平,分析类肝细胞诱导表达情况。结果按108/ml接种,用含15%FBS的DMEM/F12培养基培养可成功获取UBC-MSCs。细胞呈长梭形,细胞表面抗原测定显示强烈表达CD29、CD44,而不表达造血细胞的标志物CD34、CD45。分阶段诱导6周后,细胞形态呈多角形,并检测到肝细胞表面标志物白蛋白、AFP和CK-19。结论新生儿脐血中可分离出MSCs,并可在体外进行培养扩增,分阶段诱导可分化为类肝细胞。Objective To investigate the feasibility of human umbilical cord blood mesenchymalstem cells （UCB- MSCs） induced into functional hepatocypte-like cells in vitro. Methods Full-term UCB-MSCs samples were obtained in sterile condition and isolated by density gradient centrifugation and directly adherence growth, then were cultured in vitro. After the fourth passage of UCB-MSCs, surface antigens were detected with flow cytometry, and then were induced into func- tional hepatocypte-like cells by stages with tile application of many ingredients such as a-FGF,b-FGF, HGF,OSM and so on. The changes of cell shape were observed by inverted phase contrast microscope. The levels of albumin（ALB） , al pha-fetopmtein （AFP） , cytokeration （CK-19） mRNA of the induced cells were detected by RT-PCR 4 and 6 weeks after the indue- tion, and the expression of induced functional hepatocypte-like cells were analyzed. Results UBC-MSCs were obtained sue- cessfully under the conditions of inoculum density at 10S/ml and DMEM/F12 including 15% FBS. HUCB- MSCs displayed long spindle shape and expressed CD44 and CD29 strongly,but CD34 and CD45 were expressed negatively. After 6 weeks of the induction by stages,the cells showed polygon ,and the cell surface markers such as AFPmRNA（ ＋ ） ,ALBmRNA（ ＋ ） and CK-19mRNA（ ＋ ） were detected. Conclusions MSCs can be obtained from human umbilical cord blood,and are capable of amplifying and differentiating into functional hepatocypte-like cells with several different eytokines step by step in vitro.

关 键 词：脐血 间充质干细胞 类肝细胞 分化 

分 类 号：Q813.1[生物学—生物工程]