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目的探讨体外分离、混合培养纯化人脐血间充质干细胞的适宜体系,观察该人脐血间充质干细胞生物学特性。方法收集孕足月新生儿脐血,每3份脐血[(70—100)ml/份]混合;采用1.077 g/ml的淋巴细胞分离液(Ficoll)以1 500 r/min密度梯度离心法分离脐血中的单个核细胞,在Mesencult培养基中贴壁培养筛选人脐血间充质干细胞,显微镜下观察细胞生长形态变化,细胞计数并绘制细胞生长曲线,流式细胞仪检测细胞抗原表达,分析细胞周期;体外诱导脐血间充质干细胞成脂、成骨分化。结果密度梯度离心法所获得的单个核细胞,在培养基中培养约3—5 h开始出现贴壁生长,24 h后贴壁细胞增多,呈明显纺锤状,10 d后开始出现细胞克隆,3周后呈漩涡状生长。原代培养时间为15 d,P1代倍增时间为26 h。流式细胞仪鉴定:贴壁生长的细胞表达CD29、CD44和CD105,不表达CD34、CD45。分化潜能鉴定体外培养细胞可以成脂、成骨分化。结论所贴壁培养出的细胞的生长形态、流式表形、分化潜能具有人脐血间充质干细胞生物学特征,混合培养可以提高间充质干细胞培养成功率,实验中采用的培养体系适宜人脐血间充质干细胞的分离培养。
Objective To investigate the suitable system for the isolation and culture of human umbilical cord blood mesenchymal stem cells in vitro and to observe the biological characteristics of human umbilical cord blood mesenchymal stem cells. Methods Umbilical cord blood of pregnant neonatal rats was collected and mixed every 3 umbilical cord blood [(70-100) ml / serving]. Fetal lymphocytes were separated by Ficoll at 1.077 g / ml and centrifuged at 1 500 r / Umbilical cord blood mononuclear cells were isolated, cultured in Mesencult medium adherent screening of human umbilical cord blood mesenchymal stem cells were observed under a microscope, the growth of cell morphology, cell counting and mapping of cell growth curve, flow cytometry to detect cell antigen expression , Analysis of cell cycle; induced mesenchymal stem cells into adipogenic, osteogenic differentiation in vitro. Results The mononuclear cells obtained by density gradient centrifugation began to appear adherent growth in about 3-5 h culture medium. After 24 h, the number of adherent cells increased and became spindle-like, and cell clones began to appear after 10 d. 3 After a whirlpool-like growth. Primary culture time was 15 d, P1 generation doubling time was 26 h. Flow Cytometry Identification: Adherently growing cells express CD29, CD44 and CD105, but do not express CD34 and CD45. Differentiation Potential Identification of cultured cells can be adipogenic, osteogenic differentiation. Conclusion The growth morphology, flow-form and differentiation potential of adherent cells have the biological characteristics of human umbilical cord blood mesenchymal stem cells. Mixed culture can improve the success rate of mesenchymal stem cell culture. The culture system used in the experiment is suitable Isolation and culture of human umbilical cord blood mesenchymal stem cells.