【摘要】 探讨建立兔骨髓间质干细胞(BMSCs)的体外分离纯化、扩增和鉴定的方法,为BMSCs的进一步诱导分化和应用奠定基础。首先抽取兔髂骨骨髓,采用Percoll密度梯度离心法得到骨髓单个核细胞,接种后形成单层贴壁细胞,经胰蛋白酶消化后传代培养扩增,倒置相差显微镜观察细胞生长状态,细胞免疫组化检测CD73、CD34。结果表明,成功建立了兔BMSCs体外分离及培养扩增的方法,发现BMSCs表现贴壁生长,P0代时呈集落生长,细胞呈梭形,传代后细胞增殖速度加快,形态开始多样化;细胞免疫组化显示BMSCs表达CD73,未表达CD34,反复传代后CD73表达效率增高。上述研究表明,Percoll密度梯度离心联合骨髓贴壁法,能有效分离、纯化和扩增BMSCs,提取的细胞具有BMSCs的生长特性和抗原表型,其中P3~P6代细胞增殖能力强,可用于进一步的研究工作。更多还原

【Abstract】 This study established the method for isolation, amplification and identification of the rabbit bone marrow mesenchymal stem cells(BMSCs) in vitro, which provided an experimental foundation for the further differentiation and the clinical application of BMSCs. Bone marrow was first aspirated from rabbit iliac and the BMSCs were isolated and cultivated by Percoll density gradient centrifugation which formed monolayer adherent cells after inoculation. Subculture amplification was conducted after trypsin digestion, and the growth characteristics of primary and passage cells were observed by inverted phase contrast microscopes, and CD73 and CD34 were detected by immunohistochemistry. The results showed that the rabbit BMSCs were successfully isolated and cultured in vitro, which grew with adherence, and the primary and passage cells were spindle-shaped and grew in colonies. After subculture, the proliferation rate was accelerated and forms began to diversify. Cell immunohistochemistry indicated that CD73 was expressed in BMSCs while CD34 was not, and the expression efficiency was increased after many generations. The above research showed that Percoll density gradient centrifugation combined with bone marrow adherent method was a simple and reliable method to isolate, purify and amplify rabbit BMSCs. The extracted cells had the growth characteristics and antigenic phenotype of BMSCs, of which the cells in P3~P6 generation had strong cell reproductive capacity and could be used for further research work.更多还原