人脐带间充质干细胞在3种不同培养体系中的生长状况及腺病毒感染效率  

Growth state and adenovirus infection efficiency of human umbilical cord-derived mesenchymal stem cells in 3 different culture systems

作　　者：洪敬欣[1] 张茜真[2,3] 韩俊领[1,4] 刘辉[2] 刘剑[1] 邱录贵[1,4] 

机构地区：[1]协和干细胞基因工程有限公司,天津市脐带血造血干细胞库,天津市300384 [2]解放军第二军医大学东方肝胆外科医院病毒基因治疗实验室,上海市200438 [3]浙江理工大学生命科学学院新元医学与生物技术研究所,浙江省杭州市310018 [4]中国医学科学院,北京协和医学院,血液学研究所,血液病医院,天津市300020

出　　处：《中国组织工程研究与临床康复》2010年第1期42-47,共6页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：天津市科技创新专项资金(08FDZDSH03000)~~

摘　　要：背景：目前所报道的脐带间充质干细胞体外培养条件及培养效率不尽相同,尚缺乏统一标准。而且由于不同来源的间充质干细胞生物学特征尚有一定差异,因此建立脐带间充质干细胞简便、高效的培养体系十分必要。目的：观察人脐带来源的间充质干细胞在体外不同培养体系中的生长状态,以及不同腺病毒感染的效率。方法：采用胶原酶消化法从正常足月新生儿脐带中分离出间充质干细胞,贴壁法纯化培养,细胞贴壁后利用低糖DMEM,MesenPRO RSTM Medium和STEMPRO MSC SFM这3种培养体系进行体外扩增。取对数生长期的第3~5脐带间充质干细胞,应用腺病毒Ad5-EGFP,Ad5/11-EGFP,Ad5/35-EGFP分别以感染复数=1,10,100进行感染,分别于感染后24,56,72h倒置荧光显微镜观察病毒感染及绿色荧光表达情况。结果与结论：使用低糖DMEM培养的细胞初期融合时间长,STEMPRO MSC SFM培养的细胞虽然连接紧密,但消化传代后不易贴壁,而MesenPRO RSTM Medium培养的细胞在相同时间内能达到较高的细胞密度,更适于脐带间充质干细胞的体外扩增。Ad5/35-EGFP感染脐带间充质干细胞的效率明显高于其他两种腺病毒,但可导致细胞凋亡;腺病毒Ad5/11-EGFP对脐带间充质干细胞的感染效率较佳,随着感染复数的升高,所表达的荧光强度也逐渐增大。BACKGROUND： In vitro culture condition and culture efficiency are different in reported umbilical cord-derived mesenchymal stem cells, and lacked of unified standards. Different derived mesenchymal stem cells have different biological properties. Therefore, it is very necessary to establish a simple and high-performance culture system for umbilical cord-derived mesenchymal stem cells. OBJECTIVE： To observe the growth state of human umbilical cord-derived mesenchymal stem cells in different culture systems in vitro and adenovirus infection efficiency. METHODS： Mesenchymal stem cells were separated from healthy full-termed delivery fetus using collagenase digestion method and purified by adherent culture. These cells were cultured and amplified in DMEM （low glucose）, MesenPRO RS？ Medium and STEMPRO？ MSC SFM in vitro. The 3-5 passage mesenchymal stem cells were infected by the Ad5-EGFP, Ad5/11-EGFP, Ad5/35-EGFP as multiplicity of infection （MOI）=1, 10, 100. Viral infection and green fluorescence expression were observed at post-infection 24, 56 and 72 hours using inverted fluorescence microscope. RESULTS AND CONCLUSION： The cell morphology in STEMPR？ MSC SFM was different from other two culture system and these cells were not easy to adherent after trypsin digestion. Cell doubling time in the MesenPRO RS？ Medium was shorter than other two groups. Mesenchymal stem cells were infected by Ad5/35-EGFP with higher efficiency than other two kinds of adenovirus, but part of cells appeared apoptosis. The infection efficiency of Ad5/11-EGFP was highest. The fluorescence intensity was gradually increased with increased MOI.

关 键 词：培养体系 腺病毒 感染效率 脐带间充质干细胞 干细胞 

分 类 号：R394.2[医药卫生—医学遗传学]