脐带间充质干细胞的体外分离及生物学特性观察

背景:脐带组织依赖于母体免疫系统的保护,且胚胎自身免疫系统相对不发育、MHC表达低下,故来源于脐带的间充质干细胞的生物学特性已成为目前研究热点.目的:拟在体外分离培养人脐带组织间充质干细胞,并观察其多向分化能力.设计、时间及地点:细胞学体外观察,于2006-10/2007-05在四川大学组织工程重建实验室完成.材料:脐带来源于胎龄37～40周的健康新生儿,由四川大学华西第二医院产房提供.方法:取脐带沿血管长轴切开,去掉血管,再将脐带重新缝合形成环状,灌入胶原酶悬液,6～8 h后灌洗离心,获取细胞后贴壁法分离培养、扩增,细胞呈集落生长后传代.取传至第5代细胞,分别加入成骨、成脂诱导分化培养基.主要观察指标:脐带间充质干细胞的形态、生长状况、表面抗原分子的表达、多向分化潜能.结果:去除血管后获取脐带组织细胞的方法可获得贴壁生长的细胞,呈短棒状或梭形样细胞,易扩增和形成集落;高表达基质细胞抗原CD29,CD51,CD71,而不表达CD34,CD45及HLA-DR等造血干细胞抗原分子;成骨诱导后茜素红染色胞浆中有大量的钙沉积,碱性磷酸酶钙钴法染色胞质呈灰黑色,阳性细胞率＞85%;成脂诱导后油红O染色示胞浆充满油滴空泡.结论:脐带组织存在具有分化能力的间充质干细胞,并可在体外进行培养扩增形成集落细胞传代,传代细胞表达基质细胞表面抗原,能够向成骨细胞、成脂肪细胞方向分化.

Isolation and biological characteristics of umbilical cord mesenchymal stem cells

BACKGROUND:Cord tissues,which immune system is relatively non-development with low expression of MHC,rely on the protection of the mother's immune system.Accordingly,studies underlying biological characteristics of umbilical cord mesenchymal stem cells (MSCs) arouse more and more attention.OBJECTIVE:To isolate,separate,and culture MSCs from the tissues of umbilical cord,and to explore the ability of multi-directional differentiation.DESIGN,TIME AND SETTING:The in vitro cytology experiment was performed at the Laboratory of Tissue Engineering of Sichuan University from October 2006 to May 2007.MATERIALS:The umbilical cord of healthy neonate with 37-40 weeks gestational age was obtained from delivery room of the Second West China Hospital,Sichuan University.METHODS:Pieces of umbilical cord were dissected along its long axis,vessel was pulled away,then cord was tied together with a suture creating "loops",then it was placed into collagenase solution,after 6-8 hours,suspended cells obtained from the loops suspension were centrifuged and cultured with adherent method,pass,aged when single cell-derived colonies were appeared.Finally,the 5th generation of cells was induced differentiation towards osteoblasts and lipoblasts.MAIN OUTCOME MEASURES:Morphology,growth,surface antigen expression,as well as the multiple differentiation potential of umbilical cord MSCs.RESULTS:The adherent cells obtained from umbilical cord after removal vessels showed short rod-like or fusiform shape,which can be proliferated and form single cell-derived colonies.Cells isolated from the umbilical cord high expressed integrin markers (CD29,CD51,CD71) but not hematopoietic lineage markers (CD34,CD45 and HLA-DR).These colonies can differentiate into osteoblasts that produce mineralized matrices,stained by alizarin red and alkaline phosphatase,and differentiate adipocytes that accumulate lipid vacuoles and are demonstrated by morphology,stained by oil red.CONCLUSION:Umbilical cord tissue contains a high number of MSC-like elements forming colonies that may be successfully expanded in culture.The proliferation colonies present matrix ceil immunophenotypes,and can differentiate into osteobiasts and adipocytes.