兔骨髓间充质干细胞的分离、培养与鉴定

Experimental Study on Isolation,Culture and Identify of Rabbit Bone Marrow-derived Mesenchymal Stem Cells in Vitro

目的建立一种兔骨髓间充质干细胞(Mesenchymal stem cell,MSC)体外分离培养、扩增及鉴定的方法。方法骨髓穿刺法抽取新西兰白兔髂骨骨髓5ml,应用密度梯度离心法分离纯化MSC并进行体外扩增,倒置显微镜下观察原代及传代细胞的形态、生长情况,计数细胞数目,绘制细胞生长曲线。HE染色光镜观察细胞形态,采用CD44及CD34抗体进行间接免疫荧光标记鉴定培养的干细胞。结果成功建立了兔MSC体外分离及培养扩增的方法。生长动力学分析发现,传代细胞随传代次数的增加其增殖能力逐渐下降,第3～5代细胞增殖能力强,生长旺盛。所分离培养的细胞均表达CD44,不表达CD34。结论在体外采用密度梯度离心及贴壁培养法可获得高纯度的兔骨髓间充质干细胞,第3～5代细胞增殖能力强,可用于进一步的研究工作。

Objective To explore the approaches of isolation, culture and identify the rabbit bone marrow-derived mesenchymal stem cells in vitro. Methods MSCs were extracted from rabbit bone marrow （5.0 ml） and purified via Density gradient centrifugation, then proliferated in vitro. The cell growth was observed under Phase-contrast microscope and the growth curve of cell proliferation was drawn accordingly. Light microscopy were used to study the morphologic features and indirect immunofluorescence was used to examine the expression of cell surface antibody of CD44 and CD34. Results MSCs of rabbit were isolated, cultured in vitro. The proliferation of MSCs decreased while the cell proliferated more generations. The MSCs of 3rd and 5th generation proliferated more rapidly than those of 7^th and 9^th generation. These cultured cells showed immunoreactivity to CD44 but not CD34. Conclusion Purified MSCs of rabbit could be obtained via Density gradient centrifugation. Cells of 3-5 generation with high proliferation were effective to the further experiment.