体外诱导脐血间充质干细胞分化为成骨细胞

背景:脐血间充质干细胞在特定的诱导条件下可以分化成为多种类型的细胞,易于体外培养扩增,但脐血中间充质干细胞的建立时间长、频率低是其主要缺点.目的:体外分离培养脐血间充质干细胞,并诱导其向成骨细胞方向分化.设计、时间及地点:细胞学体外观察,于2008 06/2009 01在青岛大学医学院试验室完成.材料:脐血取自足月正常分娩的产妇,由青岛市立医院妇产科提供.方法:采用percoll密度梯度法体外分离培养人脐血间充质干细胞,当细胞汇合90%单层细胞后胰蛋白酶消化传代.取第3代脐血间充质干细胞,以1×106密度接种,当细胞达50%～60%融合时,加入含0.1 μmol/L地塞米松、10mmol/L β-甘油磷酸钠、50 μmol/L维生素C的DMEM成骨细胞诱导液;对照组加入普通低糖DMEM培养液培养.主要观察指标:倒置显微镜下观察细胞生长及增殖情况,流式细胞仪检测细胞表面标志物的表达,细胞生长曲线分析,透射电镜观察细胞超微结构,von Kossa染色和碱性磷酸酶染色检测向成骨细胞诱导分化情况.结果;原代培养的脐血间充质干细胞形态与骨髓间充质干细胞相似,传代后细胞形态均一,呈梭形旋涡状排列.第3代脐血间充质干细胞高表达CD29,CD44,CD13,不表达CD34.生长潜伏期为两三天,第三四天进入对数增殖期,1个月后进入平台期.胞核呈类圆形或不规则形,核膜清晰,有一两个核仁,染色质较粗,胞质中细胞器丰富,细胞表面有微绒毛.第3代脐血间充质干细胞成骨诱导7 d后逐渐汇合呈铺路石状,14 d后von Kossa染色出现钙化结节,碱性磷酸酶染色呈阳性;对照组未见钙化结节,碱性磷酸酶染色呈阴性.结论:采用percoll密度梯度法可成功从人脐血中分离出间充质干细胞,并在体外诱导分化为成骨细胞.

Differentiation of umbilical cord blood mesenchymal stem cells into osteoblasts in vitro

BACKGROUND: Umbilical cord blood mesenchymal stem cells (UCB-MSCs) can differentiate into various types of cells under certain condisions, and easily proliferate in vitro. However, UCB-MSCs have long establishment time and low frequency.OBJECTIVE: To in vitro isolate and culture UCB-MSCs, and induce its differentiation into osteoblasts.DESIGN, TIM E AND SETTING: The in vitro cytological study was performed at the Laboratory of the Medical College of Qingdao University from June 2008 to January 2009.MATERIALS: UCB was obtained from term normal delivery women at the Department of Gynaecology and Obstetrics, Qingdao Municipal Hospital.METHODS: Human UCB-MSCs were isolated and cultured in vitro by Percoll density gradient. When reached 90% confluency,UCB-MSCs were digested by trypsin for subculture. At the third passage, UCB-MSCs at 1×106 were incubated. When reached 50% 60% cenfluency, UCB-MSCs were treated with DMEM supplemented with 0.1 μmol/L dexamathasone, 10 mmol/Lβ-sodium glycerophosphate and 50 μmol/L vitamin C. UCB-MSCs in the control group were incubated in low glucose DMEM.MAIN OUTCOME MEASURES: Growth and proliferation of MSCs were observed under the inverted microscope. Cell surface marker expression and cell growth curve were measured by flow cytometry. Cell ultrastructure was observed under the transmission electron microscope. Differentiation of UCB-MSCs into osteoblasts was determined by Won Kossa staining and alkaline phosphatase staining.RESULTS: Primary cultured UCB-MSCs had similar morphology to bone marrow mesenchymal stem cells. After passage, cell morphology was even, presenting spindle shape. UCB-MSCs at passage 3 highly expressed CD29, CD44, CD13, but did not express CD34. Growth latency was 2-3 days. Cells entered logarithm proliferation phase at days 3-4, and platform phase 1 month later. Nuclei presented round or irregular, with clear nuclear membrane, 1-2 nucleoli, rough chromatin, abundant organelles and microvilli. UCB-MSCs at passage 3 were gradually confluent following 3 days of osteogenic induction, with the presence of pavement-stone shape. 14 days later, calcified nodules by Von Kossa staining, and cells were positive for alkaline phosphatase staining. In the control group, no calcified nodules were found, and cells were negative for alkaline phosphatase staining.CONCLUSION: UCB-MSCs can be successfully isolated by Percoil density gradient, and induced to differentiate into osteoblasts in vitro.