【摘要】 背景:脊柱和创伤骨科的重建修复对骨的需求量极大,但现实的供需缺点和矛盾限制了植骨运用,急需找寻一种替代途径。目的:探索骨髓间充质干细胞体外分离培养的最佳实验条件,并观察其生物学特征。设计、时间及地点:细胞水平的体外组织工程实验,于2007-05/2008-05在苏州大学附属儿童医院骨科实验室完成。材料:选择4周龄雄性新西兰大耳白兔,由苏州大学动物中心提供。方法:自股骨大转子及髓腔抽取新西兰大耳白兔骨髓,采用密度梯度离心法获取离心层骨髓单个核细胞,体外接种连续培养观察,原代接种后48h首次换液,此后每二三天全量换液;待细胞长至90%汇合时,用胰酶和EDTA混合液消化传代。主要观察指标:采用倒置显微镜下进行细胞生长形态观察,四唑盐比色法测定细胞活性并描绘细胞生长曲线;另选择P3代细胞进行成骨条件培养液培养4周,应用Gomori钙钴染色法鉴定成骨分化的标志物——碱性磷酸酶阳性细胞,应用Von-Kossa改良染色法测定钙节结。结果:分离培养的骨髓间充质干细胞生长形态和增殖活性良好,接种后的细胞由短梭形向长梭形、三角形乃至多角性发展,各代细胞有明显的生长潜伏期、对数增长期和平台期,细胞可以连续培养至P9代。经成骨诱导分化的细胞碱性磷酸酶染色阳性率为93%,矿化结节染色阳性。结论:骨髓间充质干细胞体外分离、扩增简单可行,可以定向诱导为成骨细胞。更多还原

【Abstract】 BACKGROUND:Spinal reconstruction and orthopedic trauma demand a great amount of bones,but the imbalance between supply and demand restricts the usage of implant bone,so to find a new way is urgent.OBJECTIVE:To explore the best experimental conditions for isolation and cultivation of bone marrow mesenchymal cells(BMSCs) and to observe its biological features in vitro.DESIGN,TIME AND SETTING:The experiment was performed in the orthopedics laboratory of The Children’s Hospital of Soochow University from May 2007 to May 2008.MATERIALS:Four-week-old big ear male New Zealand white rabbits were provided by the Animal Center of Soochow University.METHODS:BMSCs were separated by density gradient centrifugation method from bone marrow of New Zealand white rabbits,then were cultivated and sub-cultivated in vitro.The cells were removed into new culture medium 48 hours after primary inoculation,and then the total medium exchange was made every two or three days.Mixture of trypsin and EDTA was used for digestion and passage when cells reached 90% confluence.MAIN OUTCOME MEASURES:Using inverted microscope,cell morphology was observed,cell viability was measured with the MTT method,according to which cell growth curve was drawn.Following P3 generation of cells in osteogenesis conditioned medium were chosen to culture for 4 weeks,alkaline phosphatase positive cells,marking osteogenic differentiation,were identified using Gomori staining method,and calcium nodules were determined with the modified Von-Kossa staining.RESULTS:Following isolation and culture,BMSCs grew with normal appearance and vigor.After inoculation,cells presented from short fusiform to long fusiform,triangular and even polygon.Each generation of cells had remarkable latency,increased logarithmic phase and plateau phase,and cells could be cultured to P9 generation continuously.The rate of alkaline phosphatase positive cells was 93%.Staining of mineralization nodules was positive CONCLUSION:In vitro isolation and cultivation of BMSCs is relatively simple and feasible,and BMSCs can be differentiated into osteoblasts directionally.更多还原