【摘要】 背景:体外扩增是目前解决脐带血干细胞移植所面临的单份脐血造血干祖细胞数不足问题的主要手段。已有许多关于不同来源的间充质干细胞联合不同细胞因子支持脐血造血干祖细胞体外扩增的报道,而单独应用间充质干细胞对人脐血CD34+细胞体外扩增的报道仍很少。目的:观察应用脐带源间充质干细胞作基质层的体外培养体系对脐血CD34+细胞体外扩增的支持作用。设计、时间及地点:对比观察实验,于2006-03/2007-05在中国医学科学院中国协和医科大学血液学研究所、实验血液学国家重点实验室完成。材料:经产妇知情同意,采集健康足月分娩胎儿脐带9份。方法:应用贴壁培养的方法从正常足月出生儿脐带中分离培养间充质干细胞,通过细胞形态学、免疫表型、分化实验进行鉴定。利用免疫磁珠分离法从正常足月出生儿脐带血中分离CD34+细胞。应用3种不同培养体系进行脐血CD34+细胞的体外扩增:单独应用脐带源间充质干细胞作基质层、细胞因子培养体系和间充质干细胞联合细胞因子培养体系。主要观察指标:应用细胞计数法、集落培养计数法和流式细胞学检测法对扩增后细胞数量、集落形成能力和免疫表型进行分析比较。RT-PCR检测间充质干细胞中细胞因子的表达情况。结果:培养第14天后,间充质干细胞组的CD34+细胞比例明显高于细胞因子联合间充质干细胞组;CD34+细胞总数为培养前的(4.19±1.37)倍,明显高于细胞因子组。培养第7天和第14天,间充质干细胞组的CD34+CD38-细胞亚群比例均高于另两组。RT-PCR结果显示,脐带源间充质干细胞体外可表达干细胞因子和血小板生成素早期干细胞效应因子。结论:单独应用脐带源间充质干细胞可有效地对脐血CD34+细胞进行体外扩增,更有利于保持较早期干、祖细胞的扩增。更多还原

【Abstract】 BACKGROUND:Ex vivo expansion of hematopoietic stem cells was suggested as the best way of overcoming problems caused by limited hematopoietic cell number for cord blood transplantation.In many studies,mesenchymal stem cells(MSCs) of different origins as a cell feeder layer in the presence of recombinant cytokines have been used for support of umbilical cord blood(UCB)-derived committed hematopoietic progenitor cells(HPCs).However,studies on human UCB CD34+ cell in vitro amplification using MSCs are few.OBJECTIVE:To explore the effects of in vitro culture system on ex vivo expansion of UCB CD34+ cells using human UCB-derived MSCs as stromal layer.DESIGN,TIME AND SETTING:A controlled study was performed at the State Key Laboratory of Experimental Hematology,Institute of Hematology,Chinese Academy of Medical Science & Peking Union Medical College from March 2006 to May 2007.MATERIALS:Nine samples of UCB were obtained from healthy full-term delivery fetus after the consent of puerpera.METHODS:MSCs were isolated and cultured from human umbilical cord by adherence,and were identified by cellular morphology,immunophenotype and cell differentiation experiments.UCB CD34+ cells were isolated by immunomagnetic beads sorter from normal full-term newborn infants.UCB CD34+ cells were cultured in three various culture conditions:culture condition A,only supplemented with recombinant cytokines without a cell feeder layer;culture condition B,only supplemented with UCB-derived MSCs as a cell feeder layer without recombinant cytokines;culture condition C,supplemented with UCB-derived MSCs as a cell feeder layer and recombinant cytokines.MAIN OUTCOME MEASURES:Ex vivo expansion of total cells,colony culture forming capabilities and immunophenotypes were analyzed using cytometry,colony culture counting method and flow cytology.Expression of cytokines from UCB-derived MSCs was investigated by reverse transcription-polymerase chain reaction.RESULTS:After 14 days,the proportion of CD34+ cells in condition B was higher than supplemented with cytokines combined with MSCs(condition C),and the expansion of CD34+ cells in condition B increased up to(4.19±1.37) times superior to supplemented with recombinant cytokines(condition A).The culture cells in condition B showed highest proportion of CD34+CD38-cells of the three culture conditions at 7 and 14 days.The results of RT-PCR showed that human UCB-derived MSCs expressed stem cell factor and thrombopoietin,which were early-effect hematopoietic cytokines.CONCLUSION:UCB-MSCs can effectively amplify UCB-CD34+ cells in vitro,and can better maintain amplification of primitive hematopoietic progenitor cells.更多还原