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该文建立了一种简便、高效的Wistar大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)分离方法,为研究其生物学特性奠定了基础。采用全骨髓贴壁培养与密度梯度离心结合筛选法无菌分离Wistar大鼠BMSCs,常规传代和成骨、成脂诱导后,经显微观察、细胞计数、免疫印迹、q PCR和流式细胞术检测,分析细胞形态、增殖与分化特性。结果表明,分离细胞传至3代后细胞呈漩涡状生长、形态为长梭形,细胞生长曲线呈“S”形,表面分子CD29、CD34、CD44、CD45和CD90的阳性率分别为98.21%、0.78%、91.62%、0.93%和99.27%。成脂和成骨诱导后,过氧化物酶体增殖物激活受体-γ2(PPAR-γ2)、脂蛋白脂肪酶(LPL)、骨髓基质细胞核心结合因子α1(Runx2)和骨形态发生蛋白-2(BMP-2)表达水平显著升高,细胞内分别出现了大量的红色脂滴和钙化结节。该研究运用全骨髓贴壁培养与密度梯度离心结合筛选法成功分离了BMSCs,分离细胞具有向成骨和成脂细胞分化的潜能与骨髓间充质干细胞的生物学特性。
This article established a simple and efficient method for the isolation of bone marrow mesenchymal stem cells (BMSCs) in Wistar rats, which laid the foundation for the study of its biological characteristics. BMSCs were isolated by whole bone marrow adherent culture and density gradient centrifugation, and were routinely passaged and osteogenesis. After adipogenic induction, the cells were stained by microscopy, cell counting, western blot, qPCR and flow cytometry Detection, analysis of cell morphology, proliferation and differentiation characteristics. The results showed that the cells grew in a swirling pattern with a long spindle shape and the cell growth curve was “S” shape after passage 3 passages. The positive rates of surface molecules CD29, CD34, CD44, CD45 and CD90 were 98.21 %, 0.78%, 91.62%, 0.93% and 99.27%. After induction of adipogenesis and osteogenesis, peroxisome proliferator-activated receptor-γ2 (PPAR-γ2), lipoprotein lipase (LPL), bone marrow stromal cell core binding factor α1 (Runx2) and bone morphogenetic protein- 2 (BMP-2) expression levels were significantly increased, a large number of intracellular red lipid droplets and calcified nodules. In this study, BMSCs were successfully isolated using whole bone marrow adherent culture and density gradient centrifugation combined with screening, and the biological characteristics of bone marrow mesenchymal stem cells and their potential to differentiate into osteogenic and adipogenic cells were observed.