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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。
To establish a method for the isolation, purification and identification of rabbit bone marrow mesenchymal stem cells (BMSCs) in vitro, and to lay the foundation for the further differentiation and application of BMSCs. Bone marrow mononuclear cells were obtained by Percoll density gradient centrifugation. Monolayer adherent cells were obtained after inoculation. The cells were passaged and subcultured by trypsinization. The cells were observed under inverted phase contrast microscope for cell growth status, cell immunohistochemistry Detection of CD73, CD34. The results showed that BMSCs were successfully established in vitro and cultured. The results showed that BMSCs grew in an adherent manner, with colonies growing on P0 generation and spindle-shaped cells. After passage, the proliferation rate of cells increased and the morphology began to diversify. Cellular immunity Histochemistry showed that CD73 was expressed in BMSCs, CD34 was not expressed, CD73 expression was increased after repeated passages. The above studies show that Percoll density gradient centrifugation combined with bone marrow adherent method can effectively separate, purify and amplify BMSCs. The extracted cells have the growth characteristics and antigenic phenotype of BMSCs. The P3 to P6 generation cells have strong proliferative capacity and can be used for further Research work.