【摘要】 目的 分离培养脐带血间充质干细胞并检测其生物学特性。方法 在无菌条件下用密度梯度离心的方法获得脐血单个核细胞，接种含10%胎牛血清的DMEM培养基中。单个核细胞行贴壁培养后，进行细胞形态学观察，绘制细胞生长曲线，分析细胞周期，检测细胞表面抗原。结果 采用Percoll(1.073 g/mL)分离的脐血间充质干细胞大小较为均匀，梭形或星形的成纤维细胞样细胞。细胞生长曲线测定表明接后第5天细胞进入指数增生期，至第9天后数量减少;流式细胞检测表明50%～70%细胞为CD29和CD45阳性。结论 体外分离培养脐血间充质干细胞生长稳定，可作为组织工程的种子细胞。
【关键词】 脐血;间充质干细胞;细胞周期;免疫细胞化学
Abstract: Objective Isolation and cultivation of mesenchymal stem cells (MSCs) in human umbilical cord in vitro, and determine their biological properties. Methods The mononuclear cells were isolated by density gradient centrifugation from human umbilical cord blood in sterile condition, and cultured in DMEM medium containing 10% fetal bovine serum. After the adherent mononuclear cells were obtained, the shape of cells were observed by microscope, then the cell growth curve, the cell cycle and the cell surface antigens were obtained by immunocytochemistry and flow cytometry methods. Results MSCs obtained by Percoll (1.073 g/mL) were similar in size, spindle-shaped or star-shaped fibroblasts-liked cells. Cell growth curve analysis indicated that MSCs were in the exponential stage after 5d and in the stationary stages after 9d. Flow cytometry analysis showed that the CD29 and CD44 positive cells were about 50%～70%. Conclusions The human umbilical cord derived mesenchymal stem cells were grown stably in vitro and can be used as the seed-cells in tissue engineering.
Key words:human umbilical cord blood; mesenchymal stem cells; cell cycle; immunocytochemistry
间充质干细胞(mesenchymal stem cells，MSCs)在一定条件下具有多向分化的潜能，是组织工程研究中重要的种子细胞来源。寻找来源丰富并不受伦理学制约的间充质干细胞成为近年来的研究热点[1]。脐血(umbilical cord blood, UCB)在胚胎娩出后，与胎盘一起存在的医疗废物。与骨髓相比，UCB来源更丰富，取材方便，具有肿瘤和微生物污染机会少等优点。有人认为脐血中也存在间充质干细胞(Umbilical cord blood-derived mesenchymal stem cells，UCB-MSCs)。如果从脐血中培养出MSCs，与胚胎干细胞相比，应用和研究则不受伦理的制约，蕴藏着巨大的临床应用价值[2,3]。本研究将探讨人UCB-MSCs体外培养的方法、细胞的生长曲线、增殖周期和细胞表面标志等方面，分析UCB-MSCs 作为间充质干细胞来源的可行性。
1 材料与方法
1.1 UCB-MSCs的分离与培养
9 份脐血标本均来源于辽宁医学院附属第一医院，均获得产妇同意。以等量的PBS缓冲液混匀，缓慢滴入到等量的淋巴细胞分离液(Fercoll，1.073 g/mL,美国Sigma公司)，650 g离心15 min。抽取白膜层细胞，以等量的PBS缓冲液洗涤离心，基础培养液(DMEM/F12，10%胎牛血清、100 U/mL青霉素和链霉素、2 mmol/L L-谷氨酰胺)重悬，接种至100 mm培养皿，置于37 ℃、饱和湿度、5%CO2的培养箱内培养。第5天首次全量换液，之后每周2次换液，2周后达60%～80%融合时，0.25%胰酶(含0.02%EDTA)常规消化传代。
1.2 UCB-MSCs细胞表面抗原分析
分别取第1、5、9代细胞，胰蛋白酶消化，制成单细胞悬液。含2%牛血清白蛋白的PBS缓冲液冲洗细胞，室温下分别与CD29-PE、CD34-PE、CD44-PE及CD45-PE单克隆抗体避光孵育15 min。PBS缓冲液冲洗细胞，离心，弃上清。加入含1%多聚甲醛的PBS缓冲液0.3 mL，使用同型对照单克隆抗体确定背景标记，流式细胞仪分析细胞表面抗原。
1.3 UCB-MSCs体外增殖能力检测