大鼠骨髓间充质干细胞分离培养及其生物学特性观察  

Isolation,culture and biological characteristics of bone marrow mesenchymal stem cells in rats

作　　者：买霞[1] 陈莉[1] 扎拉嘎胡[1] 战丽艳[1] 徐瑞成[1] 

机构地区：[1]武装警察部队医学院细胞生物学教研室,天津市300162

出　　处：《中国组织工程研究与临床康复》2007年第3期463-466,I0002,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：武警医学院资助科研项目(WY2004-6)~~

摘　　要：目的:分离和培养大鼠骨髓间充质干细胞并观察其生物学特性。方法:实验于2004-05/2006-04在武装警察部队医学院细胞生物学实验室进行。在无菌条件下分离大鼠胫骨、股骨,用预冷磷酸盐缓冲液冲出骨髓,经Percoll梯度分离方法获得骨髓单个核细胞,接种含体积分数为0.10胎牛血清的IMDM培养基中。对单个核细胞行贴壁培养后,通过倒置光显微镜和透射电子显微镜进行细胞形态学观察,流式细胞术进行细胞周期分析,细胞计数法测定细胞生长曲线,免疫细胞化学染色显示c-kit表达。结果:①倒置光显微镜下观察采用Percoll(1.073g/mL)分离的骨髓间充质干细胞大小较为均匀,基本上呈梭形或星形的上皮样细胞,传代培养后的细胞体积增大,成纤维样细胞逐渐增多。②透射电子显微镜下观察骨髓间充质干细胞的核质比例较大,核仁大而明显,细胞表面有微绒毛,胞浆内可见核糖体和线粒体,其他细胞器较少,表现出未分化细胞的特征。③细胞生长曲线测定表明接种后第4天细胞进入指数增生期,至第7天进入平台期。④流式细胞术证明G2+S期细胞为11.8%。⑤体积较大的多角形细胞为c-kit阳性,体积较小的成纤维样细胞为阴性细胞,阳性颗粒主要分布在细胞膜和细胞质,细胞核未见表达,阳性细胞比率为55.3%。结论:成功地建立了一种分离培养大鼠骨髓间充质干细胞的方法,获得的细胞生长稳定,增殖活跃,可作为组织工程的种子细胞。AIM： To isolate and culture mesenchymal stem cells （MSCs） from bone marrow of adult rats and observe their biological properties. METHODS： The experiment was completed in the Laboratory of Cell Biology, Medical College of Chinese People＇s Armed Police Forces from May 2004 to April 2006. After the tibias and femurs were dissected from Pats, the marrow was flushed out with ice-cold phosphate buffered solution under aseptic condition. The mononuclear cells were obtained after PercoU gradient centrifugation and then were inoculated into IMDM medium containing 10% fetal bovine serum. After the adherent mononuclear cells were cultured, the morphologic changes of MSCs were detected by inverted light microscope and transmission electron microscope. Cell cycle was analyzed by flow cytometry, growth curve was measured by cell counting and immunocytochemistry was performed to identify c-kit specific antigens on the MSCs. RESULTS： （1）The results of inverted light microscope showed that the cultured MSCs collected by 1.073 g/mL Percoll appeared no difference in their bodies, generally were spindle-shaped or star-shaped epidermal-liked cells. The cellular bodies were swelled and fibroblast-like cells were increased after transplanted culture.（2）The results of transmission electron microscope showed that the ratio of nucleus/cytoplasm was larger. The nucleoli were larger and more obvious. There were more microvilli on cellular surface. Ribosome and mitochondria could be seen in cytoplasm. The ultrastructures of MSCs demonstrated the features of infantile cells. （3）The measurement of growth curve demonstrated that the time for cell duplication was the fourth day and the platform phase was the seventh day. （4）The cell cycle analysis showed that 11.8% of MSCs was in G2＋S phase.（5）The c-kit positive cells were about 55.3% polygon cells in cell membrane and cytoplasm, whereas fibroblast-like cells with smaller bodies were negative for c,kit. CONCLUSION： A successful method is established for the i

关 键 词：骨髓 间质干细胞 大鼠 细胞培养 免疫细胞化学 

分 类 号：R394.2[医药卫生—医学遗传学]