摘要
背景:间充质干细胞主要来源于骨髓,但骨髓源性间充质干细胞存在病毒污染的可能,且随年龄增长其细胞数量和扩增分化能力会出现明显下降,因此需要寻找新的间充质干细胞来源。目的:拟采用组织块培养法从人脐带中分离间充质干细胞,并观察其生物学特性。设计、时间及地点:观察性实验,于2007-03/2008-05在吉林大学白求恩医学院组胚教研室完成。材料:脐带来自正常足月产健康婴儿,产妇及家属均知情同意。方法:采用组织块法分离培养和扩增间充质干细胞:将脐带剪碎至1mm×1mm×1mm大小组织块,置于培养瓶中,并在组织块表面覆盖载玻片防止其漂浮,加入含体积分数为0.10胎牛血清的DMEM/F12培养液培养。细胞贴壁后,每隔三四天更换培养基,待细胞出现集落生长达60%以上汇合后,弃组织块,消化后,按1:2～1:3的比例传代。取第7代细胞在含地塞米松、抗坏血酸、β-磷酸甘油、含体积分数为0.10胎牛血清的DMEM/F12培养液与含体积分数为0.10胎牛血清、地塞米松、胰岛素、吲哚美辛、1-甲基-3-异丁基-黄嘌呤、抗坏血酸、青霉素和链霉素的DMEM/F12培养液中诱导其成骨及成脂分化。主要观察指标:光、电镜观察获得的人脐带源性间充质干细胞形态,MTT法检测细胞增殖率,流式细胞术检测细胞周期、免疫表型,碱性磷酸酶染色及油红O染色鉴定细胞分化能力。结果:组织块贴壁后1周可见多数呈长梭形或扁平形的成纤维样细胞,2周后,细胞形态变为均一的纺锤形。透射电镜观察第5代和第7代细胞均为幼稚形态的细胞,细胞膜表面不光滑,核大,不规则,核仁明显,常染色质多,异染色质少,胞浆少,胞浆内可见较多游离核糖体,此外还可见粗面内质网和线粒体。流式细胞术分析细胞周期显示,第3,5,7代70%以上的细胞处于G0/G1期;第3,5,7代细胞均强烈表达CD105、CD90、CD44,不表达CD34、CD31、CD45。第7代细胞在体外可定向诱导分化为成骨细胞和脂肪细胞。结论:采用组织块培养法可从人脐带中分离出具有间充质干细胞特性的细胞。
BACKGROUND： Although the main source of mesenchymal stem cells （MSCs） is bone marrow, bone marrow may be detrimental due to the virus contaminative, which may induce the decline in MSCs number and differentiation potential with increasing age. Therefore, we should find a new source of MSCs. OBJECTIVE： To isolate mesenchymal stem cells （MSCs） from human umbilical cord, and to investigate biological characteristics. DESIGN, TIME AND SETTING： An observational experiment was performed at the Department of Histology and Embryology, Norman Bethune Medical College in Jilin University from March 2007 to May 2008. MATERIALS： Human umbilical cords were obtained from healthy full-term natural delivered newborns. Informed consents were obtained from women and their family numbers. METHODS： Human umbilical cord mesenchymal stem cells （hUCMSCs） were separated by culture of tissue adherence. The umbilical cord was sliced into small fragments of 1 mm^3 in diameter, and plated in culture flasks, and the glass slides were covered to prevent them to float, then cultured in growth medium consisting of Dulbecco＇s Medified Essential/F12 Media （DMEM/F12）, 0.10 volume fraction of fetal bovine serum. After the cells adhered to the flasks, the medium was changed every 3-4 days. When the cells reached 60% convergence, they were detached with trypsin and were passaged at 1： 2-1：3. At the seventh passage 7, MSCs were detected osteogenic and adipegenic differentiation respectively by treated with osteogenic induction DMEM/F12 medium consisting of supplemented with 0.10 volume fraction of fetal bovine serum, dexamethasone, antiscorbic acid, β-glycerophosphate, as well as the adipogenic induction medium consisting of DMEM/F12 supplemented with 0.10 volume fraction of fetal bovine serum, dexamethasone, insulin, indomethacin, isobutylrnethylxanthine （IBMX）, antiscorbic acid, penicillin and streptomycin. MAIN OUTCOME MEASURES： Morpbologic appearance of UCMSCs was observed by an optical microscope and electronic microscope. The proliferation rate was measured by MTT assay. Cell cycle and surface antigens were measured by Flow cytometry. The differentiation potential was demonstrated by alkaline phosphatase and oil red O stains respectively. RESULTS： After one week of incubation, long spindle or flat fibroblast-like cells were presented. After two weeks, the cells presented a homogeneous population of spindle fibroblast-like cells. The hUCMSCs at passage 5, 7 under a transmission electron microscope was infantile morphologic appearance; the surface of cell membrane was not smooth; the nuclus was irregular and large, with evidence nucleoli, more euchromadn, less heterochromatin. The cytoplasm was less, with more free ribosome and rough endoplasmic reticulun, mitochondria. Flow cytometry analysis revealed that CD44, CD90, CD 105 were highly expressed on the surface of passages 3, 5, 7 cells, but there was negative for CD34, CD31, CD45. The cell cycle at the passages 3, 5 and 7 showed the percentage of G0/G1 was more than 70% respectively. The assays in vitro demonstrated the cells of passage 7 exhibited multipotential differentiation into osteogenic and adipogenic cells. CONCLUSION： Cells from the human umbilical cords have been isolated by tissue culture method, which have the biological characteristics of MSCs.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2008年第47期9221-9225,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
