人脐带间充质干细胞不同培养方案的比较与优化  

Comparison and optimization of different culture schemes of human umbilical cord mesenchymal stem cells

作　　者：屈玟 宋磊[1] 赵瑶[2] 胡振波[2] 

机构地区：[1]山东省潍坊医学院 [2]山东省潍坊医学院附属医院干细胞与再生医学实验室,山东潍坊261042

出　　处：《海南医学院学报》2017年第8期1009-1013,共5页Journal of Hainan Medical University

基　　金：国家自然科学基金(81570157);山东省医药卫生科技发展计划项目(2013ws0291)~~

摘　　要：目的:目前人脐带间充质干细胞已广泛的应用于各种基础研究以及治疗疾病的临床实验,但目前各个实验室所选取的培养方案各异,培养效果并不稳定。本实验旨在选择一种稳定可靠的人脐带间充质干细胞培养方案。方法:从脐带中分离间充质干细胞,采用组织贴壁法将其培养于8组不同配方的培养基中,通过比较间充质干细胞从组织中游出的时间,生长状态,细胞数量,表面抗原的表达等,选出较优的人脐带间充质干细胞培养方案。结果:从细胞生长状态比较,达优MSC基础培养基-血清替代物组(F组)、X-VIVO^(TM)15-胎牛血清组(B组)、X-VIVOT M15-胎牛血清+血清替代物组(C组)、迈键医药的干细胞无血清基础培养基-无血清替代物组(G组)所培养的间充质干细胞从脐带游出时间较早且细胞融合率达到80%~90%时间较其他组短;达优MSC基础培养基-血清替代物组人脐带间充质干细胞生长增殖最快(P<0.05)。选取生长较快的B、C、F、G组进行免疫表型分析,发现达优MSC基础培养基-血清替代物组(F组)所培养的间充质干细胞表面抗原CD73,CD90表达均在90%以上,而其他组CD90和CD73的表达都在90%以下(P<0.05),各组造血细胞表面抗原CD34,CD45表达均呈低表达。结论:达优MSC基础培养基-血清替代物组为较优的人脐带间充质干细胞培养方案。Objective.. At present, human umbilical cord mesenchymal stem cells （hucMSC） have been widely used in basic research and clinical trials in treatment of many diseases, but how to obtain a large number and high quality of stable and reliable mesenchymal stem cells for clinical application is still a scientific research needs to be resolved. Since each laboratory used different culture methods, the productivity of the different cultures may vary. Therefore, the purpose of this study is to optimize a stable and reliable hucMSC culture procedure. Methods： Mesenchymal stem cells derived from human umbilical cord were isolated and cultured in different culture medium. The immunophenotypes of the cells were detected by comparing the morphology, passage, proliferation and using flow cytometry and optimized cultural method was selected. Results： By comparison of the time of cells migrating out from the tissue, cell growth status, cell number and expression of CD90 andCD73 of the hucMSCs, we found that X-VIVO15＋ 10% fetal bovine serum group, X-VIVO15＋2 % fetal bovine serum and 2 %UltroserG group, and MSC basal medium＋UltraGRO-Advanced group had an earlier migration time from the umbilical cord and shorter confluence time to cover 80%-90% of the cuturing flask. The Huc-MSCs cultured with MSC basal medium＋ 2% UltraGRO- Advanced group revealed the fastest proliferation （P〈0.05）. The expression of CD90 and CD73 in Huc-MSCs cultured with MSC basal medium＋ 2% UltraGRO-Advanced group were above 90%, while the expression of CD90 and CD73 in other groups were below 90% （P〈0.05）. The expression of CD34 and CD45 surface antigens were low in hue-MSCs from all the groups. Conclusions： In summary, MSC basal medium-UltraGRO-Advanced group was the optimal culture medium for human umbilical cord mesenchymal stem cells.

关 键 词：间质干细胞 细胞培养技术 培养基 免疫表型 流式细胞术 

分 类 号：R730.54[医药卫生—肿瘤]