三维共培养免髓核细胞诱导骨髓间充质干细胞分化

背景:在椎间盘组织工程研究领域,种子细胞主要有髓核细胞与骨髓间充质干细胞.髓核细胞来源有限,取材不便,增殖能力差,体外培养困难.目的:利用海藻酸盐微球模拟的三维环境下,将兔髓核细胞与骨髓间充质干细胞共培养,观察骨髓间充质干细胞的分化情况.设计、时间及地点:细胞观察实验,于2007-12/2008-10在华中科技大学同济医学院附属同济医院骨科实验室进行.材料;4月龄健康新西兰大耳白兔6只,购自华中科技大学同济医学院动物实验中心.方法:应用密度梯度离心法及贴壁法分离纯化兔骨髓间充质干细胞,应用Ⅱ型胶原酶消化法及贴壁法分离培养兔椎间盘髓核细胞.取传至第3代骨髓间充质干细胞,用脂质体介导法将绿色荧光蛋白质粒转入,经G418筛选后,分为2组:单独培养组将转染后的骨髓间充质干细胞单独培养,共培养组将其与髓核细胞按1:1比例于海藻酸盐凝胶微球中进行共培养.共培养14 d后,溶解海藻酸盐凝胶珠,通过流式分选收集骨髓间充质干细胞.主要观察指标:利用RT-PCR法和Westernblot技术检测骨髓间充质干细胞中Ⅱ型胶原、聚集蛋白聚糖的表达.结果:共培养14 d后,共培养组有Ⅱ型胶原、聚集蛋白聚糖mRNA及蛋白的表达,而单独培养组均呈阴性表达.结论:通过三维共培养,兔椎间盘髓核细胞能够在体外诱导骨髓间充质干细胞向髓核样细胞方向分化.

Rabbit nucleus pulposus cells induce the differentiation of mesenchymal stem cells after co-culture in a three-dimensional environment

BACKGROUND: In the field of intervertebral disc tissue engineering, seed cells primarily consist of nucleus pulposus cells (NPCs) and mesenchymal stem cells (MSCs). NPCs have been known to have several shortcomings: limited source, inconvenient collection, poor proliferative capacity, and difficult in vitro culture.OBJECTIVE: To investigate the proliferation of MSCs after co-culture with NPCs in alginate beads-simulated three-dimensional environment.DESIGN, TIME AND SETTING: A cell observation was performed at the Laboratory of Orthopedics, Tongji Hospital, Tongji Medical College of Huazhong University of Science and Technology between December 2007 and October 2008.MATERIALS: Six healthy New Zealand rabbits of 4 months old were provided by the Laboratory Animal Center, Tongji Medical College of Huazhong University of Science and Technology and were used for the present study.METHODS: Rabbit MSCs were isolated and purified using density gradient centrifugation and adherence methods. Rabbit NPCs were isolated and purified using collagenase Ⅱ digestion and adherence methods. Following liposome-mediated green fluorescent protein tranfection and G418 screening, MSCs of passage 3 were cultured either alone (control group) or with NPCs at a ratio of 1:1 (co-culture group) in alginate beads. After 14 days of culture, alginate beads were dissolved and MSCs were collected by flow cytometry sorting.MAIN OUTCOME MEASURES: The expression of collagen Ⅱ and aggrecan in MSCs was detected by RT-PCR and Western blot techniques.RESULTS: mRNA and protein expression of collagen Ⅱ and aggrecan was observed in the co-culture group, but not in the control group, after 14 days of culture.CONCLUSION: MSC differentiation towards nucleus pulposus-like cells can be induced by co-culture with NPCs in a three-dimensional environment.