密度梯度离心与贴壁法分离培养兔骨髓间充质干细胞及其生物学特性观察

Isolation and culture of rabbit bone marrow mesenchymal stem cells by density gradient centrifugation combined with adherent method and their biological characteristics observation

背景：获取更大量、更高纯度、有活性的骨髓间充质干细胞是干细胞移植及组织工程研究发展的基础。目的：拟建立一套体外分离培养及大量扩增兔骨髓间充质干细胞的方法，并对细胞生物学特性进行观察。设计、时间及地点：细胞学体外观察，于2006—12／2007—07在山东省烟台市莱阳中心医院骨科试验室完成。材料：清洁级1月龄雌性新西兰大白兔1只。方法：采用密度梯度离心法和贴壁培养法相结合的方式，分离兔骨髓间充质干细胞，应用细胞刮收集呈长梭形的细胞，形成克隆后用胰蛋白酶＋EDTA消化，按1：3传代进行体外扩增培养。主要观察指标：倒置相差显微镜下观察细胞形态特征，免疫细胞化学SABC法鉴定CD34、CD44抗原的表达，观察细胞生长特性，测定细胞活力。结果：原代分离培养的贴壁细胞呈长梭形，漩涡状排列，约14d达90％以上融合；传代后增殖迅速，细胞为单一的梭形，排列更加有序。所培养的细胞CD44呈阳性表达，而CD34呈阴性。第1～5代细胞培养3—5d为对数生长期，第7代以后对数增长期延长。第1～5代细胞成活率均〉94％，其中第3，4代成活率高达97％；至第7代细胞活力呈明显下降趋势，细胞成活率仅为78％。结论：应用密度梯度离心结合贴壁法，可成功分离并扩增兔骨髓间充质干细胞，且细胞生长稳定，增殖力强．可作为细胞移植的种子细胞。

BACKGROUND： To collect bone marrow mesenchymal stem cells （BMSCs） with high quantity, high purity and activity is the foundation of stem cell transplantation and tissue engineering research. OBJECTIVE： To establish a method for in vitro isolation and cultivation of BMSCs from rabbits and to study their biological characteristics. DESIGN, TIME AND SETTING： The cytology in vitro experiment was performed at the Laboratory of Department of Orthopedics, Laiyang Central Hospital from December 2006 to July 2007. MATERIALS： One clean female New Zealand rabbits aged I month was used in this study. METHODS： BMSCs were separated from rabbits by the density gradient centrifugation combined with adherent method. Spindle cells were collected, and then digested in trypsin＋ethylenediamine tetraacetic acid （EDTA） after clone. Subsequently, these cells were cultured in vitro according to 1 ： 3 passage. MAIN OUTCOME MEASURES： Cell morphology was observed with an inverted phase contrast microscope. CD34 and CD44 antigen expression was identified by immunocytochemistry to detect the cell growth characteristics and activity. RESULTS： Primary culture of adhered cells was spindle and whirlpool-shaped. About 14 days later, 90% cells were confluent. After passage, cells were spindle and distributed in order. Cultured cells were positive for CD44, but negative for CD34. First to fifty passages of cells entered logarithmic phase after 3-5 days of culture. From the seventh passage, logarithmic phase was longer. The survival rate of the first to fifth passages of cells was 〉94%, and the third and fourth passages of cells was 97%. However, the activity of the seventh passage of cells was significantly decreased, and the survival rate was only 78%. CONCLUSION： BMSCs can be obtained from bone marrow through density gradient centrifugation combined with adherent method. The cultured cells with stable growth rate and active reproductive activity can be used as seeds cell of cell transplantation.