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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14d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Western blot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14d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。
BACKGROUND: In the field of disc tissue engineering, seed cells mainly include nucleus pulposus cells and bone marrow mesenchymal stem cells. The sources of nucleus pulposus cells are limited, inconvenient to harvest, poor proliferation, and difficult to culture in vitro. OBJECTIVE: To observe the differentiation of bone marrow mesenchymal stem cells by co-culture rabbit nucleus pulposus cells with bone marrow mesenchymal stem cells in a three-dimensional environment simulated by alginate microspheres. DESIGN, TIME AND SETTING: The Cell Observation Experiment was performed at the Department of Orthopedics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology from December 2007 to October 2008. MATERIALS: Six healthy New Zealand white rabbits aged 4 months were purchased from Animal Experimental Center, Tongji Medical College, Huazhong University of Science and Technology. Methods: Rabbit bone marrow mesenchymal stem cells were isolated and purified by density gradient centrifugation and adherent method. The nucleus pulposus cells of rabbit intervertebral disc were isolated and cultured by type Ⅱ collagenase digestion and adhesion method. The third passage of bone marrow mesenchymal stem cells were collected and transfected with green fluorescent protein by liposome-mediated method. After screening by G418, the cells were divided into two groups: BMSCs were transfected Cultures were cultured alone and co-cultured with nucleus pulposus cells in alginate gel microspheres in a 1: 1 ratio. After co-cultivation for 14 days, alginate gel beads were dissolved and bone marrow mesenchymal stem cells were collected by flow cytometry. MAIN OUTCOME MEASURES: Expression of type Ⅱ collagen and aggrecan in bone marrow mesenchymal stem cells were detected by RT-PCR and Western blot. Results: After co-culture for 14 days, the expression of type Ⅱ collagen and aggrecan mRNA and protein in co-culture group was negative, but negative in culture group. Conclusion: By three-dimensional co-culture, nucleus pulposus cells of rabbit intervertebral disc can induce the differentiation of BMSCs into nucleus pulposus-like cells in vitro.