郝世凯　苏相相　洪敬欣

【摘 要】 目的：比较胶原酶的不同消化时间，观察所获得的间充质干细胞的数量，确定最为理想的消化时间。方法：分别采用3h、5h、7h的消化时间分离脐带间充质干细胞；通过传代进行纯化和扩增培养，绘制生长曲线；用流式细胞仪检测其表面标志。结果：分离出的间充质干细胞贴壁后为梭形，呈平行排列生长或漩涡状生长，并且消化时间为5h时收获到的细胞数量最多；流式细胞仪检测结果显示，获得的间充质干细胞均表达CD73、CD105、HLA-ABC，不表达CD34、CD45、HLA-DR。结论：胶原酶消化法从人脐带中分离培养的细胞具有间充质干细胞的生物学特性，并且消化5h收获得的间充质干细胞数量最多，生长状况最好。

【关键词】 胶原酶；脐带；间充质干细胞；分离；消化时间

【中图分类号】R3292 【文献标志码】 A 【文章编号】1007-8517（2014）24-0036-05

The impact of different collagenase digestion time on UCMSCs separation

HAO Shi-kai，SU Xiang-xiang，HONG Jing-xin

UNION STEMCELL&GENE ENGINEERING CO.LTD，Tianjin 300384，China

Abstract：Objective Comparing the quantity of MSCs acquired from umbilical cord that digested by collagenase II in different hours， ascertain an ideal digesting time and culture and appraise the cells acquired.Methods MSCs were isolated from human umbilical cord through collagenase digestion respectively in 3h， 5h， 7h， then passaged to purify and culture， drew the growth curve.Their surface markers and cells cycle were detected by flow cytometry.Results MSCs isolated from umbilical cord adhered with a spindle-shape， they parallel along their longitudinal axis or grew in whirl manner. After 5hours digestion， we got the biggest quantity with best growth status， and successfully built the method for MSCs culture and increasing. Detected by flow cytometry， all of adherent cells express CD73、CD105、HLA-ABC，none of them express CD34、CD45、HLA-DR.Conclusion Cells isolated from umbilical cord by digestion have the biological characteristics of MSCs. And 5 hours is the best time for digestion to acquire the most and best MSCs.

Keywords：Collagenase； Umbilical cord； Mesenchymal Stem Cells； Isolate； Digestion time

间充质干细胞（mesenchymal stem cells，MSCs）是一种多潜能干细胞，具有自我更新、高度增殖和多向分化的潜能，在人体生长、发育过程中存在于多种组织中。目前，MSCs主要来源于人骨髓，但成人骨髓中MSCs容易被病毒感染，且强的免疫原性等因素限制了其临床应用[1]。此外骨髓来源的间充质干细胞还存在以下问题：随着年龄的老化，干细胞数目显著降低，增殖分化能力大幅度衰退；制备过程不容易质控；移植给异体可能引起免疫反应；取材时对患者有损伤，患者有骨髓疾病时不能采集，即使是健康供者，亦不能抽取太多的骨髓。这都限制了骨髓间充质干细胞临床应用，使得寻找骨髓以外其他可替代的间充质干细胞来源成为一个重要的问题[2]。……