改良法体外培养脐带血间充质干细胞及其生物学特性分析析☆

背景:目前报道的脐带血间充质干细胞分离成功率较低,且缺乏较为统一的鉴定方法.目的:对传统的体外分离培养脐带血问充质干细胞方法加以改良,以提高细胞培养成功率,并进行生物学特性观察.设计、时间及地点:细胞学体外观察,于2006-04/2007-01在上海交通大学医学院附属第九人民医院完成.材料:取自足月健康顺产新生儿的脐带血标本28份,由上海市红房子医院产科提供,经产妇和家属同意.方法:无菌条件下取新牛儿脐带血,以密度梯度离心法分离单个核细胞,以含体积分数为10%胎牛血清的α-MEM培养基进行体外培养,原代培养5～7 d后半量换液,后每隔三四天全量换液一次.待细胞贴壁后,按处理方法不同分为2组:改良1组当皿底圆形巨核细胞融合、梭形成纤维样细胞脱落时将细胞悬液移入新的皿中培养:改良2组待皿底圆形巨核细胞渐渐占据优势时,将培养基换为含体积分数为15%小牛血清的α-MEM培养液,当圆形巨核细胞大部脱落后换回含体积分数为10%胎牛血清的α-MEM培养基.取第5代脐带血间充质干细胞,进行体外成骨及成脂诱导.主要观察指标:显微镜下观察脐带血间充质干细胞的形态,流式细胞仪测定细胞免疫表型,碱性磷酸酶染色及油红染色检测绌胞诱导分化能力.结果:28份脐带血中20份培养出贴壁细胞(改良1组6份/10份,改良2组14份/18份),其中13份培养出能融合且可稳定传代的成纤维样细胞(改良1组4份,改良2组9份),成功率为46.4%,可传至22代且形态无变化,强烈表达CD105、CD29等问充质干细胞表面标志,而CD34、CD45和CD106等呈阴性表达.在特定诱导条件下,脐带血间充质干细胞可分化为成骨细胞和脂肪细胞.结论:脐带血中存在问充质干细胞,具有多向分化潜能,且易于体外扩增、传代稳定,体外培养方法经改良后可提高脐带血间充质干细胞的培养成功率.

In vitro improved culture and biological Characteristics of umbilical cord blood mesenchymaI stem cells

BACKGROUND:Studies have demonstrated that the success rate of isolation of umbilical cord blood mesenchymal stem cells (UCB-MSCs)is low,which also lacks of unified identification method.OBJECTIVE:To modify the traditional in vitro isolation and culture method of UC8-MSCs to obtain a higher success rate and to observe its biological characteristics.DESIGN,TIME AND SETTING:In vitro observation of cytology.The study was performed at the Ninth People's Hospital of Shanghai Jiao Tong University Medical College between April 2006 and January 2007.MATERIALS:A totaI of 28 UCB samples were obtained from full-term normal delivery.Department of Matemity,Shanghai Red House Hospital.The written informed consent was obtained from the puerpera and their families.METHODS:NeonataI umbilical cord blood was collected under sterile condition.Mononuclear cells (MNCs) were separated from using lymphocyte isolation medium by centrifugation and suspended in α-minimum essential medium(α-MEM)containing 10% fetal bovine serum.The medium was half exchanged after 5-7 days of primary culture and then was totally exchanged every 3-4 days.The confluent cells were divided into 2 groups.In group 1.when the round megakaryocytes were confluent and fusiform fibroblast-like cells were fell off.the cell suspension was transferred to new culture dish;in group 2.when the round megakaryocytes dominated the majodty,the culture medium was replaced by α-MEM containing 15% calf serum,followed by culture in α-MEM containing 10% fetal bovine serum when the round megakaryocytes fell off.The fifth passage of UCB-MSCs was harvested for subsequent osteoinduction in vitro.MAIN OUTCOME MEASURES:The cell morphology was observed by microscopy;Flow cytometry was used to examine the surface antigen phenotype;alkaline phosphatase and oil red staining was performed to detect cell differentiation capacity.RESULTS:Of 28 samples of UCB,attaching cells were obtained from 20 samples(6/10 in group 1,14/18 in group 2),fibroblast-like cells that could passage steadily(4 samples in group 1,9 in group 2)were cultured from 13 0f 20 samples with success rate of 46.4%,among which MSCs were passaged steadily up to P22.UCB-MSCs were all positive for MSC-related antigens such as CD29 and CD105,but negative for CD34,CD45 and CD106.Incubation of UCB-MSCs under special condition resulted in a differentiation of osteoblast and adipocyte.CONCLUSIONMSCs exist in UCB,which have multi-differentiation capacity,and passage steadily.The modified in vitro culture method improves culture success rale of UCB-MSCs.