用组织贴壁法从整根脐带分离培养间充质干细胞及其生物学特性的检测  

Isolation and characterization of mesenchymal stem cells derived from whole human umbilical cord by applying a direct explant technique

作　　者：张颢[1,2] 张斌[2] 程梅[1] 陶艳玲[1] 扈江伟[2] 徐曼[2] 陈虎[2] 

机构地区：[1]哈尔滨市第一医院哈尔滨血液病肿瘤研究所,黑龙江哈尔滨150010 [2]军事医学科学院附属医院造血干细胞移植科,北京100071

出　　处：《基础医学与临床》2012年第1期71-76,共6页Basic and Clinical Medicine

基　　金：国家高技术研究发展计划项目(863项目)(2011AA020114);军队临床高新技术重大项目(2010gxjs100);黑龙江省卫生厅科研课题(2011-518);首都临床特色应用研究(Z111107058811107);哈尔滨市科技创新人才研究专项基金(2009RFQQS027)

摘　　要：目的建立从整根脐带分离培养间充质干细胞(UC-MSCs)的技术,并对其生物学特性进行检测。方法用组织贴壁法分离UC-MSCs,并通过传代进行纯化和扩增培养,绘制生长曲线,用流式细胞仪检测UC-MSCs表面抗原及细胞周期;在特定诱导体系中,检测UC-MSCs向脂肪、成骨及软骨分化的潜能;用RT-PCR检测多能干细胞标志多能干细胞标志Oct-4,Sox-2,Nanog mRNA水平。结果成功建立了UC-MSCs分离培养的方法;贴壁细胞均表达CD73、CD90及CD105,不表达造血细胞表型CD34、CD45和HLA-DR;细胞倍增时间为(24.04±0.49)h,G0～G1期和S+G2+M期所占比例分别为81.56%和18.44%;UC-MSCs能够向脂肪、成骨和软骨分化;表达Oct-4,Sox-2,Nanog基因。结论组织贴壁法是一种较好的分离培养UC-MSCs的方法,培养的细胞为更原始的间充质干细胞,增殖能力强。Objective To establish a technique for isolating mesenchymal stem cells from umbilical cord and their biological characteristics. Methods Mesenchymal stem cells （MSCs） were isolated from umbilical cord （UC） by applying a explant technique and explanding cultured in vitro. Growth curves were drawn, and The phenotypes and cell cycle were evaluated by flow cytometry. UC-MSCs were induced to differentiate into adipocytes, osteocytes and chondrocytes in special differentiation condition. Mutidifferentiation related genes and stem cell-related transcription factors, Nanog, Oct-4 Sox-2 were detected by TR-PCR. Results UC-MSCs were isolated using a explant technique, and characteristic of plastic adherence and fibroblast-like morphology, the adherent cells displayed an abundant presence of CD73, CD90, CD105 and absence of CD34, CD45, HLA-DR. Cell cycle showed that there were percentages of stem cells as G0/G1 81.56 % and S ＋ G2 ＋ M 18.44% respectively. When cultured in differentiation media, they differentiated into adipocytes, osteocytes and chondrocytes. RT-PCR reactions confirmed that their mutidifferentiation related genes were positive. Moreover, stem cell-related transcription factors, Nanog, Oct-4 Sox- 2 were positively expressed in UC-MSCs. Conclusions The explant technique is a feasible method to obtain MSCs from whole human umbilical cord, and UC can be considered as a novel and convenient source of adult MSCs displaying primitive pluripotent sten cells and high expansion potential.

关 键 词：脐带 间充质干细胞 分化 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]