人脐带间充质干细胞体外长期培养及向肝样细胞的分化(英文)  

Long-term in vitro culture and hepatocytic differentiation of human umbilical cord-derived mesenchymal stem cells

作　　者：朱争艳[1] 阎俊卿[1] 韩涛[1] 杜智[1] 骆莹[1] 王鹏[1] 高英堂[1] 刘彤[1] 

机构地区：[1]天津市第三中心医院卫生部人工细胞工程技术研究中心,天津市300170

出　　处：《中国组织工程研究与临床康复》2009年第49期9792-9796,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：目的:观察人脐带间充质干细胞体外长期培养的生物学特性,探讨其向肝样细胞分化的可能性。方法:脐带来源于天津市第三中心医院住院的健康足月产妇,采用酶消化法分离培养人脐带间充质干细胞,待细胞生长至80%～90%融合时传代。取传至第9代细胞接种于12孔板内,调整细胞浓度为5×1010L-1,加入含肝细胞生长因子、成纤维生长因子4、抑瘤素的DMEM培养基,诱导28d。MTT法测定细胞生长活性,流式细胞仪检测细胞周期,免疫细胞化学及流式细胞仪鉴定细胞表型;免疫细胞化学染色鉴定诱导后肝细胞特异表面标志物的表达,以及糖原染色结果。结果:从人脐带中分离得到的间充质干细胞贴壁生长,形态类似于成纤维细胞,80%以上处于G0/G1期,具有良好的生长活性,可在体外稳定传代20次以上;CD29,CD90,CD105,Vimentin呈阳性表达,基本不表达造血细胞标志CD34和内皮细胞标志CD31。随诱导时间的延长,圆形细胞逐渐增多,形态似肝细胞;诱导1周时细胞开始表达肝细胞表面标志物甲胎蛋白、细胞胶蛋白18、细胞胶蛋白19;诱导3周时开始表达成熟肝细胞标志白蛋白;诱导4周时白蛋白表达增强,且诱导细胞糖原染色呈阳性。结论:从人脐带中可成功分离出间充质干细胞,实现体外长期培养,并可诱导分化为肝样细胞。OBJECTIVE：To investigate biological characteristics of human umbilical cord-derived mesenchymal stem cells,and to explore the possibility of hepatocyte-like cells differentiation.METHODS：The umbilical cord was provided by healthy term birth woman in Tianjin Third Central Hospital.Mesenchymal stem cells were isolated from human umbilical cord by enzyme digestion method.Cells were passaged at 80%-90% confluent.The ninth passage of cells at a density of 5×10^10/L were seeded in 12-well culture plate and incubated with DMEM containing hepatocyte growth factor,fibroblast growth factor-4 and oncostatin for 28 days.Cell growth activity was detected by MTT method;cell cycle was detected by flow cytometry;surface immunological marker in MSC was detected by immunocytochemical stain and flow cytometry;specific surface phenotype of hepatocyte was detected by immunocytochemical staining.Function characteristic of hepatocyte was determined by staining for glycogen.RESULTS：MSCs were isolated from human umbilical cord and presented with fibroblastic morphology.80% of cells were at G0/G1 phase with good growth activity and stably passaged over 20 times.These cells were positive for CD29,CD105,and Vimentin,but negative for CD34 and CD31.MSCs were induced to hepatocyte-1ike cells that were positive for alpha fetoprotein,CK18,CK19 at 1 week and albumin at 3 weeks.At 4 weeks,induced cells were positive for glycogen staining.CONCLUSION：MSCs isolated from human umbilical cord can be cultured in a long periods time in vitro and are able to differentiate into functional hepatocyte-like cells.

关 键 词：脐带间充质干细胞 肝样细胞 分化 

分 类 号：R394.2[医药卫生—医学遗传学]