构建兔髓核细胞诱导人骨髓间充质干细胞向类髓核细胞分化的体外模型

背景:目前尚缺乏诱导骨髓间充质干细胞向髓核细胞转化的体外模型.目的:建立兔髓核细胞诱导人骨髓间充质干细胞分化为类髓核细胞的体外模型,为髓核细胞体内移植培养种子细胞提供实验依据.设计、时间及地点:对比观察,于2008-08/2009-03在青岛大学医学院附属医院骨科研究所和中心实验室完成.材料:8周龄新西兰大白兔2只用于兔髓核细胞的分离培养:人椎间盘髓核组织为术中取自16岁先天性脊柱侧弯患者T_(12)L_1、L_(1.2)椎间盘髓核组织,患者家属知情同意.人骨髓间充质干细胞株为广州赛业生物科技有限公司产品.方法:将人骨髓间充质干细胞和兔髓核细胞新式分层共培养(应用去掉挂臂、膜孔为0.4 μm的小室,膜底与贴壁细胞相接触,膜上下两种细胞比例为50%:50%)7 d,同时设立传统的分层培养(带有挂臂的小室)组、单独人骨髓间充质干细胞培养组和髓核细胞培养组为对照.主要观察指标:反转录-聚合酶链反应和Western blot检测蛋白聚糖和Ⅱ型胶原的表达.结果:传统分层培养组及单独人骨髓间充质干细胞培养组不表达蛋白聚糖和Ⅱ型胶原.新式分层培养组、单独髓核细胞培养组Ⅱ型胶原和蛋白聚糖mRNA表达均高于传统分层培养组、单独人骨髓间充质干细胞培养组(P＜0.01).新式分层培养组与单独髓核细胞培养组比较及传统分层培养组与单独人骨髓间充质干细胞培养组比较,差异无显著性意义(P＞0.05).结论:新式分层培养人骨髓间充质干细胞能较高的表达蛋白聚糖和Ⅱ型胶原,接近椎间盘未退变人的髓核细胞表达水平,表明成功建立兔髓核细胞诱导人骨髓间充质干细胞向类髓核细胞分化的体外模型,为椎间盘退变的细胞治疗奠定了基础.

Construction of in vitro models of human bone marrow-derived mesenchymal stem cells differentiated into nucleus pulposus-like cells in rabbits

BACKGROUND:There is notan effective method for inducing bone marrow-derived mesenchymal stem cells (BMSCs) differentiatied in to intervertebral disc cells.OBJECTIVE:To establish a model of human BMSCs (hBMSCs) differentiated in to nucleus pulposus-like cells in vitro.DESIGN,TIME AND SETTING:A single sample contrast.The experiment was performed at the Orthopaedics Institute and Central Laboratory,the Affiliated Hospital of Qingdao University Medical College,from August 2008 to March 2009.MATERIALS:Two New Zealand white rabbits with 8-week-old were used to culture nucleus pulposus cells.Human nucleus pulposus was obtained from a aged 16 years patient with T_(12)L_1,L_(1.2) congenital scoliosis.The informed consent was obtained from his patients,hBMSCs were purchased from Salye Biological Science and Technology Co.,Ltd.(Guangzhou,China).METHODS:hBMSCs and nucleus pulposus cells of rabbit were co-cultured in new-style culture model (in 6-well plates and inserts with track-etched membrane having 0.4 μm pores without arms) for 7 days,conventionally laminar culture,solitary hBMSCs and nucleus pulpesus cells culture were set up as controls at same time.MAIN OUTCOME MEASURES:The expressions of collagen Ⅱ and aggrecan mRNA were detected by RT-PCR and Western blot.RESULTS:hBMSCs in the conventionally laminar and single hBMSCs groups were negative to collagen Ⅱ and aggrecan.The collagen Ⅱ and aggrecan expression of hBMSCs were upregulated evidently in new-style culture group,which was similar to nucleus pulposus cells that from degenerate intervertebral disc of human,but these expression did not appear in conventionally laminar culture and solitary hBMSCs culture (P ＜ 0.01).The difference among new-style culture group and other groups had no significance (P ＞ 0.05).CONCLUSION:A new-style model of hBMSCs differentiation to nucleus pulposus-like cells is constructed in vitro,which can highly express collagen Ⅱ and aggrecan and provide a foundation for intervertebral disc degeneration cellular treatment.