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

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间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：无菌条件下取新生儿脐带血，以密度梯度离心法分离单个核细胞，以含体积分数为10％胎生血清的α-MEM培养基进行体外培养，原代培养5-7d后半量换液，后每隔三四天全量换液一次。待细胞贴壁后，按处理方法不周分为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，逍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细胞诱导分化能力。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等间充质干细胞表面标志，而CD34、CD45和CD106等早阴性表达。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：脐带血中存在间充质干细胞，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 mesenchymal 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 UCB-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 total of 28 UCB samples were obtained from full-term normal delivery, Department of Maternity, Shanghai Red House Hospital. The written informed consent was obtained from the puerpera and their families. METHODS： Neonatal umbilical cord blood was collected under sterile condition. Mononuclear cells （MNCs） were Separated from using lymphocyte isolation medium by centrifugation and suspended in a-minimum essential medium （a-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 fusiferm fibroblast-like cells were fell off, the cell suspension was transferred to new culture dish; in group 2, when the round megakaryocytes dominated the majority, 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 of 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. CONCLUSION： MSCs exist in UCB, which have multi-differentiation capacity, and passage steadily. The modified in vitro culture method improves culture success rate of UCB-MSCs.