骨髓间充质干细胞向成骨细胞诱导分化的超微结构改变  

Ultrastructure characteristics of osteoblasts differentiated from bone marrow mesenchymal stem cells

作　　者：王志顺 王江泳 王保芝[2] 王琳[2] 刘长安[3] 王丽 刘贵生 

机构地区：[1]鹿泉市人民医院骨科,河北省鹿泉市050200 [2]河北医科大学人体解剖学教研室,河北省石家庄050017 [3]白求恩国际和平医院骨科,河北省石家庄050010 [4]河市北医科大学电镜实验中心,河北省石家庄市050017

出　　处：《中国组织工程研究与临床康复》2009年第1期11-16,共6页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：河北省科技攻关项目资助;项目编号2007000823~~

摘　　要：背景:目前尚缺乏骨髓间充质干细胞及其诱导分化后成骨细胞超微结构特点的观察。目的:采用全骨髓培养-贴壁筛选法分离培养骨髓间充质干细胞,诱导其向成骨细胞方向分化,利用光镜和电镜观察诱导和未诱导分化细胞的细微和超微结构特点。设计、时间及地点:观察实验,于2007-03/2008-04在河北医科大学人体解剖教研室和白求恩国际和平医院骨科完成。材料:成年新西兰大白兔由白求恩国际和平医院实验动物中心提供。HitachiS-3500N型扫描电镜和Hitachi-7500型透射电镜。方法:从新西兰大白兔髂后上嵴处抽取骨髓。经原代及传代培养后,取部分第3代细胞加入诱导剂,诱导剂为10mmol/Lβ-甘油磷酸钠,10-8mmol/L地塞米松,50mg/L维生素C。培养2周后,诱导和未加诱导的骨髓间充质干细胞进行碱性磷酸酶、骨形态发生蛋白和钙化结节检测。主要观察指标:光镜观察骨髓间充质干细胞及其诱导分化后成骨细胞的一般形态学特征;电镜观察骨髓间充质干细胞和成骨细胞的超微结构特点。结果:培养的第3代骨髓间充质干细胞,其碱性磷酸酶染色呈强阳性反应;钙化结节染色和骨形态发生蛋白免疫组织化学检测,均呈现阴性结果。经向成骨细胞诱导分化后,其碱性磷酸酶染色,钙化结节染色和骨形态发生蛋白免疫组织化学检测,均呈现阳性反应。光镜和扫描电镜观察显示,骨髓间充质干细胞大部分呈长梭形,少量呈星形;经向成骨细胞诱导分化后,细胞体积增大,更加饱满,似鱼群样排列。透射电镜观察显示,骨髓间充质干细胞的细胞器较丰富,向成骨细胞诱导分化后,其线粒体明显增多,出现较多的基质小泡、髓样体和空泡状结构。结论:骨髓间充质干细胞成骨诱导分化后细胞的超微结构表现为胞浆中出现增多的基质小泡、髓样体和空泡状结构。BACKGROUND： There is lack of study about bone marrow mesenchymal stem cells （BMSCs） and ultrastructure characteristics of osteoblasts after differentiation. OBJECTIVE： To isolate and culture BMSCs by the whole bone marrow culture and adherence method, to induce the differentiation of BMSCs into osteoblasts, and to investigate the tiny and ultrastructure characteristics of induced and non-induced cells with an optical microscope and electron microscope. DESIGN, TIME AND SETTING： This experiment was performed at the Department of Human Anatomy, Hebei Medical University and Department of Orthopaedics, Bethune International Peace Hospital from March 2007 to April 2008. MATERIALS： Adult New Zealand rabbits were obtained from Animal Experiment Center of Bethune International Peace Hospital. Scanning electron microscope （SEM）, Hitachi S-3500N and transmission electron microscope （TEM）, Hitachi-7500 were used in this study. METHODS： Bone marrow was collected from posterior superior iliac spine of New Zealand rabbits. After primary culture and passage, some the third passage of cells were incubated in 10^-8mmol/L dexamethasone, 50 mg/L ascorbate and 10 mmol/L -glycerol phosphate. BMSCs were identified by alkaline phosphatase （ALP）, bone morphogenetic protein and calcified node staining. MAIN OUTCOME MEASURES： General morphological characteristics of BMSCs and differentiated osteoblasts were observed by the optical microscope and their ultrastructural characteristics by the electron microscope. RESULTS： At the third passage, BMSCs were positive for ALP staining and negative for bone morphgenetic protein （BMP） by immunohistochemical staining and calcified node staining, but induced BMSCs were positive for ALP staining, BMP immunohistochemistry and calcified node stainings. The BMSCs showed long fusiform shape mostly and a few showed astero-form by the optical microscope and scanning electron microscope. After induced, cell volume increased, the abundant organellas in BMSCs were observed by

关 键 词：骨髓间充质干细胞 细胞培养 诱导分化 骨形态发生蛋白 超微结构 电镜 

分 类 号：R394.2[医药卫生—医学遗传学]