人羊水来源干细胞的体外培养及其生物学性状分析  

In Vitro Culture of Human Amniotic Fluid-Derived Stem Cells and Analysis of Their Biological Properties

作　　者：李保伟[1] 刘慧[1] 刘大庆[1] 吕洋[1] 陈琳[1] 岳文[1] 李艳华[1] 裴雪涛[1] 

机构地区：[1]军事医学科学院野战输血研究所,北京100850

出　　处：《生物技术通讯》2010年第5期655-659,665,共6页Letters in Biotechnology

基　　金：国家高技术研究发展计划重大项目(2006AA02A107);北京市科委科技计划研发攻关类重大项目(D07050701350702)

摘　　要：目的:体外分离培养人羊水来源干细胞(hAFSC),观察分析其基本的生物学性状。方法:取孕中期产前诊断所抽取的羊水,离心收集细胞,然后贴壁培养获得hAFSC。在细胞培养的基础上,观察hAFSC的形态;研究其增殖能力;用流式细胞术测定细胞周期及不同代次细胞表面阶段特异性胚胎抗原4(SSEA-4)表达率的变化;利用RT-PCR方法检测细胞干性基因的表达;利用诱导培养基诱导,检测其向肝样细胞与成骨细胞分化的潜能。结果:在体外培养条件下,hAFSC表现为成纤维细胞形态;具有良好的增殖能力,群体倍增时间约为36 h;细胞周期检测G0/G1期细胞约占86.7%,S+G2/M期细胞约占13.3%;流式细胞术检测结果表明,hAFSC表达SSEA-4,且SSEA-4阳性率随传代次数呈现先上升后下降的趋势;hAFSC在mRNA水平上表达Nanog、Oct4和Rex1等胚胎干细胞标志性基因;经诱导培养基诱导后,hAFSC可以向肝样细胞与成骨细胞分化。结论:hAFSC是一群呈成纤维细胞样,有良好增殖能力,且具有多向分化潜能的细胞,加之其来源方便,伦理学限制较少,因此在细胞治疗及组织工程等方面有着广泛的应用前景。Objective： To isolate and culture human amniotic fluid-derived stem cells（hAFSC） in vitro,and to analyse biological properties of hAFSC for future study and clinical applications.Methods： Amniotic fluid was collected from the second trimester prenatal diagnosis,then centrifugated to collect cells and culture them,which named hAFSC.On the basis of cell culture,we observed their morphology and tested their ability of proliferation.The cell cycle and the stage-specific embryonic antigen 4（SSEA-4） expression in different cell passages were analyzed by flow cytometry.RT-PCR was used to detect the expression of multipotent genes in these cells.Multipotency of hAFSC was detected by hepatic medium and osteogenic medium.Results： In vitro culture conditions,hAFSC showed a homogeneous fibroblast morphology and good proliferation capacity with a doubling time of about 36 h.Flow cytometry results showed that hAFSC were positive for SSEA-4,and SSEA-4-positive cells were in-creased at first and then decreased during the passages of hAFSC.Cell cycle showed that 86.7% of hAFSC was in G0 / G1 phase,and 13.3% was in S＋G2 / M phase.Embryonic stem cell marker genes,such as Nanog,Oct4 and Rex1,were expressed in hAFSC in mRNA levels,and these cells could be induced to hepatocyte-like cells and osteogenic cells.Conclusion： The hAFSC were fibroblast-like stem cells with good proliferative capacity,and multiple differentiation potential,together with their accessibility,low ethical constraints,hAFSC show itself a wide range of application in cell therapy and tissue engineering.

关 键 词：人羊水来源干细胞 细胞培养 多潜能性 

分 类 号：Q813[生物学—生物工程]