猪骨髓间充质干细胞的分离培养与鉴定

Isolation and Culture of Porcine Bone Marrow Mesenchymal Stem Cells

目的:建立猪骨髓间充质干细胞(pMSCs)体外分离培养、纯化和鉴定的方法,为下一步实验研究奠定基础。方法:采用密度梯度离心法获得骨髓单核细胞,接种后形成单层贴壁的成纤维样细胞。免疫荧光及PCR检测细胞表面标志及多能性基因的表达,并鉴定分离细胞的多向诱导分化潜能。结果:体外培养的原代细胞10天达到融合,传代后仍具有成纤维样的形态;免疫荧光结果见波形蛋白(Vimention)和Oct4标记阳性,CD45阴性;PCR分子检测见多能性基因OCT-4,nanog的表达;细胞具有分化为成骨细胞和成脂细胞的能力。结论:采用密度梯度离心法获得的pMSCs体外增殖能力强,纯度高,具有间充质干细胞的特性,pMSCs分离培养体系的成功建立为下一步实验研究奠定基础。

Objective： To establish the methods of isolation, purification and detection of porcine bone marrow mesenchymal stem cells in vitro, and provide the experimental basis for the following research. Methods： By density gradient centrifugation method, the adherent fibroblast-like cells were obtain in single layer after plating. We detected the expression of surface marker and pluripotency gene through Immune fluorescence and PCR, also verified its capability of differentiation. Results： The primary cells were confluent for 10 days and had classic fibroblast-like shape. Cell markers Oct-4and vimentin were positive, but CD45 was negative, pluripotency gene Oct-4, nanog was expression highly. The cultured cells can differentiated into osteoblasts and adipocyte in vitro. Conclusion： The Proliferation ability and purity of pMSCs derived from density gradient centrifugation method was better, which showed the characteristics of Mesenchymal Stem Cell. The proposal method could be use for the following research.