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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 a method for the isolation, purification and identification of porcine bone marrow mesenchymal stem cells (pMSCs) in vitro and lay the foundation for the further experimental study. Methods: Bone marrow mononuclear cells were obtained by density gradient centrifugation. After inoculation, monolayers formed fibroblast-like cells. Immunofluorescence and PCR were used to detect the cell surface markers and the expression of pluripotency genes, and to identify the multidirectional differentiation potential of the isolated cells. Results: Primary cultured cells in vitro reached confluence within 10 days and still had fibroblast-like morphology after passage. Vimention and Oct4 labeling were positive for immunofluorescence and negative for CD45. PCR detection of pluripotency gene OCT- 4, the expression of nanog; cells have the ability to differentiate into osteoblasts and adipocytes. CONCLUSION: The pMSCs obtained by density gradient centrifugation have strong proliferative ability and high purity in vitro and have the characteristics of mesenchymal stem cells. The successful establishment of pMSCs isolation and culture system lays the foundation for the further experimental research.