不同细胞因子组合对体外培养人脐带血造血干细胞的扩增效果  

Influence of different cytokine groupings on the proliferation of hematopoietic stem cells derived from human umbilical cord blood in vitro

作　　者：刘云霞[1] 孟杰[2] 刘立新[1] 郭亚春[1] 王庆林[3] 徐大为[1] 

机构地区：[1]承德医学院基础部,河北省承德市067000 [2]承德医学院附属医院神经外科,河北省承德市067000 [3]湖南师范大学医学部免疫教研室,湖南省长沙市410006

出　　处：《中国组织工程研究与临床康复》2007年第3期401-404,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：目的:观察细胞因子对培养的人脐带血造血干细胞的扩增效果,寻求体外最佳细胞因子组合。方法:实验于2002-06/2004-04在承德医学院基础研究所及承德医学院附属医院中心实验室完成。①以足月顺产健康新生儿的脐带血(由承德医学院附属医院妇产科提供,新生儿家属均签署实验知情同意书)作为实验标本。无菌条件下平均采血量30～60mL,梯度离心分离脐带血单个核细胞。②细胞体外扩增DMEM培养体系(含体积分数0.2的胎牛血清)为0.25μg/L干细胞因子,0.25μg/L白细胞介素3,0.5μg/L白细胞介素6,0.25μg/L粒-巨噬细胞集落刺激因子,0.8μg/L粒细胞集落刺激因子,0.05μg/L血小板生成素,0.8μg/LFLt-3配基。此7种细胞因子及剂量按不同组合分为6组:干细胞因子+白细胞介素3+白细胞介素6组、干细胞因子+白细胞介素3+白细胞介素6+粒-巨噬细胞集落刺激因子组、干细胞因子+白细胞介素3+白细胞介素6+粒细胞集落刺激因子组、干细胞因子+白细胞介素3+白细胞介素6+粒-巨噬细胞集落刺激因子+粒细胞集落刺激因子组、干细胞因子+白细胞介素3+白细胞介素6+血小板生成素+FLt-3配基组、干细胞因子+血小板生成素+FLt-3配基组。各组单个核细胞终浓度为1×108个L-1。③培养第7,14,21天进行细胞形态学观察;培养第0,5,10,14,18,21天观察不同细胞因子组合对脐带血干细胞扩增的效果;应用流式细胞仪对培养前后细胞表面标志CD34+进行检测。结果:①脐带血干细胞形态学观察结果:体外培养第14天,细胞胞体小,胞浆量少,胞核体积大,多不规则,为早期造血干细胞。②细胞体外扩增培养情况:应用干细胞因子+白细胞介素3+白细胞介素6+血小板生成素+FLt-3配基这一细胞因子组合,脐带血干细胞可保持>98%的细胞存活率;培养第7天出现典型早期造血干细胞,第21天细胞总数达到高峰(F=60.228,P<0.01)。③细胞表面标志CD34+的动AIM： To observe the role of cytokine on the proliferation of the hematopoietic stem cell （HSC） derived from human umbilical cord blood （UCB）, search for the optimal cytokine grouping in vitro. METHODS： The experiment was conducted at the Central Laboratory, Affiliated Hospital and Basic Institute of Chengde Medical College from June 2002 to April 2004. （1）UCB from full-term normaldelivery healthy neonate （provided by Department of Gynaecology and Obstetrics, Affiliated Hospital of Chengde Medical College; Family members of neonates signed the consent） was used as samples. 30-60 mL blood was collected averagely under sterile condition. The mononuclear cells were separated from UCB by the density gradient separation method. （2）The cells were cultured in DMEM mediums with fetal calf serum （FCS） of 0.2 volume fraction, which contained 0.25 μg/L stem cell factor （SCF）, 0.25 μg/L intedeukin 3 （IL3）, 0.5 μg/L intedeukin 6 （IL6）, 0.25 μg/L granule-macrophage colony stimulating factor （GMCSF）, 0.8 μg/L granulocyte colony-stimulating factor （GCSF）, 0.05 μg/L thrombopoietin （TPO）, 0.8 μg/L FLt-3 ligandin. These 7 kinds of cytokines and dosage were divided into 6 groups： SCF＋IL3＋IL6 group, SCF＋IL3＋IL6＋GMCSF group, SCF＋IL3＋IL6＋GCSF group, SCF＋IL3＋IL6＋GMCSF＋GCSF group, SCF＋IL3＋IL6＋TPO＋FLt-3 ligandin group and SCF＋TPO＋FLt-3 ligandin group. Final concentration of mononuclear cell was 1×10^8 L^-1 in each group. （3）Cell morphological observation at days 7, 14 and 21： The effect of different cytokine groupings on the proliferation of the HSC was observed at days 0, 5, 10, 14, 18 and 21. CD34^＋ cellular marker was investigated before and after culture with flow cytometer. RESULTS： （1）Outcome of morphological observation of cord blood stem cell： At day 14, cells cultured in vitro were with small body, small amount of cytoplasma, big irregular nucleus, naming eady HSC. （2）Culture of cell amplification in vitrcr. In the

关 键 词：造血干细胞 胎血 细胞因子类 基因扩增 

分 类 号：R394.2[医药卫生—医学遗传学]