人脐带血间充质干细胞的分离培养及增殖能力  

Isolation,cultivation and reproductive activity of human umbilical cord blood mesenchymal stem cells

作　　者：闫曼[1] 杨依勇[2] 秦书俭[2] 郑德宇[2] 

机构地区：[1]辽宁医学院附属一院手外科,辽宁省锦州市121001 [2]辽宁医学院解剖学教研室,辽宁省锦州市121001

出　　处：《中国组织工程研究与临床康复》2010年第10期1739-1742,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：辽宁省教育厅青年基金(05L140);课题名称"降低诱导后脐血源性MSCs免疫排斥反应的研究"~~

摘　　要：背景:随年龄的增加,骨髓来源的间充质干细胞在数量和质量上均受到影响,并对供体损伤较大,寻找替代的间充质干细胞来源成为研究热点,而脐带血中是否含有间充质干细胞仍存在争议。目的:拟从人脐带血中分离培养间充质干细胞,并对其生物学特性进行检测。方法:无菌条件下,应用Percoll密度梯度离心法分离脐血标本,收获中间层细胞,加入含胎牛血清、青霉素和链霉素、L-谷氨酰胺的DMEM基础培养液,再经反复贴壁纯化,除去悬浮生长细胞,得到较多呈融合状态的贴壁细胞,待细胞达60%～80%融合时胰酶消化传代。分别取第1,5,9代细胞,观察细胞形态变化,流式细胞仪检测细胞表面抗原的表达,绘制细胞生长曲线,MTT法检测细胞活性。结果与结论:分离的脐血间充质干细胞大小均匀,呈梭形或星形的成纤维细胞样细胞。第1,5,9代脐带血间充质干细胞高表达CD29,CD105和CD166,低表达CD34和CD45;接种后5d细胞进入指数增生期,9d后数量减少,群体倍增时间为(53.5±8.32)h;细胞生长状态良好,在细胞活力方面1～7代基本相似(P>0.05),至第9代细胞增殖活力稍下降,但仍无明显差异(P>0.05)。结果证实可在体外成功从脐血中分离培养出间充质干细胞,第1～9代细胞均具有良好的增殖能力。BACKGROUND： With increased age, bone marrow mesenchymal stem cells （BMSCs） are influenced with regard to quantity and quality, which will induce great damages to the donors. Many studies have focused on seeking substitute MSC source. In contrast, it remains controversial whether umbilical cord blood contains MSCs. OBJECTIVE： To isolate MSCs from human umbilical cord blood, and to detect their biological properties. METHODS： Umbilical cord blood samples were sterilely isolated using Percoll density gradient centrifugation to harvest intermediate layer cells. DMEM medium containing fetal bovine serum, penicillin, streptomycin and L-glutamine was added. Following several adherences and purification, the floating cells were discarded. Thus, many adherent cells with a confluence were collected. When cells were 60%-80% confluent, cells were digested by trypsin for subculture. Cells at passages, 1, 5 and 9 were obtained and their morphological changes were observed. Cell surface antigens were measured using flow cytometry. Growth curves were drawn, and cell viability was determined utilizing MTT. RESULTS AND CONCLUSION： Isolated umbilical cord blood MSCs presented an even size, showing spindle or star-shape fibroblasts-like cells. Umbilical cord blood MSCs at 1, 5, 9 passages were greatly positive for CD29, CD105 and CD166, but weakly positive for CD34 and CD45. Following 5 days of incubation, cells entered logarithmic growth phase. The number was decreased at day 9. Population doubling time was （53.5±8.32） hours. Cells grew well. Cells at 1-7 passages showed similar viability （P 0.05）. Till passage 9, cell proliferation viability was decreased, but no significant difference was determined （P 0.05）. Results verified that MSCs can be successfully isolated from umbilical cord blood in vitro. Cells at passages 1-9 presented a good reproductive activity.

关 键 词：细胞周期 生长增殖 活性 脐血 间充质干细胞 

分 类 号：R394.2[医药卫生—医学遗传学]