不同培养条件下人脐血间充质干细胞的差异  

Difference of human umbilical cord blood derived mesenchymal stem cells in differential culture conditions

作　　者：孙海梅[1] 季凤清[1] 李荣平[1] 王屹[1] 曾晓蓓[1] 张立新[1] 

机构地区：[1]首都医科大学组织学与胚胎学教研室,北京市100069

出　　处：《中国临床康复》2006年第45期51-53,I0006,共4页Chinese Journal of Clinical Rehabilitation

基　　金：北京市优秀人才培养基金项目(20042D0501805);北京市教育委员会科技发展计划面上项目(KM200610025005)~~

摘　　要：目的:观察不同条件对人脐血间充质干细胞培养的影响。方法:实验于2004-10/2005-10在首都医科大学组织学胚胎学教研室实验室完成。①用复方枸橼酸血液保存液和肝素钠两种不同的抗凝血剂保存的人脐带血,经密度梯度离心法分离新鲜脐带血,获取的单个核细胞,经椎虫蓝染色进行活细胞计数,比较所获得的单个核细胞数。②用不同密度1×109L-1,2×109L-1,5×109L-1接种单个核细胞,倒置显微镜下观察细胞形态。用流式细胞仪检测所培养的细胞免疫表型。③分别用高糖和低糖DMEM培养液(含体积分数0.1的胎牛血清)培养单个核细胞,其他条件相同,从72h后对所形成的细胞克隆进行计数,对两种培养基形成的细胞克隆和生长状况进行比较。结果:①不同抗凝血剂抗凝的人脐带血所获得的单个核细胞数及细胞生长状态:用肝素钠抗凝血剂保存的人脐带血所获得的单个核细胞数和细胞生长状态明显优于用复方枸橼酸血液保存液保存的人脐带血(P<0.05)。②不同接种密度人脐血间充质干细胞的贴壁生长状态及人脐血间充质干细胞的免疫表型:较低密度接种细胞并不能形成大量贴壁细胞,而以5×109L-1密度接种,72h后单个核细胞开始贴壁并开始形成细胞克隆,在10d后出现梭形细胞,培养过程中细胞形态逐渐形成均一梭形。用流式细胞仪检测所培养的细胞为非造血间充质干细胞。③两种培养基形成的细胞克隆数和生长状况:在10d以后,用低糖DMEM组培养的单个核细胞形成的细胞克隆数与高糖DMEM组相比较差异有显著性意义(P<0.05)。结论:①肝素钠作为人脐带血保存液能获得大量的单个核细胞,有利于人脐血间充质干细胞的培养。②5×109L-1细胞接种密度有利于人脐血间充质干细胞贴壁生长。③用低糖DMEM作为培养人脐血间充质干细胞的培养基有利于细胞克隆的形成和维持,对保持人脐血间充质�AIM： To observe the influence of differential conditions on human umbilical cord blood derived mesenchymalstem cells. METHODS： The experiment was conducted at the Department of Histology and Embryology, Capital Medical University from October 2004 to October 2005. ①Human umbilical cord blood was anticoagulated by sodium citrate and heparin sodium. Mononuclear cells isolated from human umbilical cord blood by density gradient centrifugation were counted and stained with trypan blue. Number of the mononuclear cells was compared. ② Mononuclear cells were cultured with three different density 1 ×10^9 L^-1, 2×10^9 L^-1 and 5×10^9 L^-1. Morphology characteristics of human umbilical cord blood derived mesenchymal stem cells were observed by inverted microscope. Flow cytometry was used to detect immune phenotype of this kind of cell. ③ Mononuclear cells were cultured in high glucose DMEM and low glucose DMEM （containing 0.1 volume fraction fetal calf serum）, and others were the same, the number of cell clone was counted 72 hours later. The number of cell clone and growth state were compared in different media. RESULTS： ① Number of mononuclear cells of human umbilical cord blood that anticoagulated by different anticoagulants and the cell growth： Number of Mononuclear cells that anticoagulated by heparin sodium was more and the cell growth was significantly better than those anticoagulated by sodium citrate （P 〈 0.05）. ②Adherence growth state of human umbilical cord blood derived mesenchymalstem cells in different density and the immunophenotype： A mass of adhered cells were observed in 5× 10^9 L^-1 cultured but not in low cells density culture. Seventy-two hours later mononuclear cells began to adhere to the wall and form cell clone. Ten days later fusiform cells appeared, and during culture cells formed fusiform gradually. This type of cells were confirmed that it is non- hematopoietic mesenchymal stem cells by flow cytometry. ③Number of cell clone formed in two culture medium and the

关 键 词：胎血 干细胞 细胞培养技术 

分 类 号：R394.2[医药卫生—医学遗传学]