全骨髓贴壁法培养人骨髓间充质干细胞的改良研究

Modified Method for Whole Bone Marrow Adherent Culture of Human Bone Marrow Mesenchymal Stem Cells

本研究旨在利用红细胞自然沉降性原理,以传统的全骨髓贴壁法为基础,探讨一种改良后简便而高效的人骨髓间充质干细胞培养方法。采用6孔板培养细胞,将骨髓标本用MSC完全培养液培养48 h,吸取上层无红细胞的上清层,置于新的培养孔以分离获取MSC。用倒置显微镜观察细胞形态及贴壁情况,记录细胞首次贴壁时间、首次传代时间,并以传统全骨髓贴壁法为对照;应用流式细胞术检测细胞周期、细胞表面标记;用油红O及碱性磷酸酶试剂盒分别对MSC成脂、成骨能力进行鉴定;应用计数板计数法描绘第1代,第3代,第5代MSC增殖曲线。结果表明,用改良法培养的MSC高表达CD90、CD105、CD13、CD44,低表达CD14、CD45、CD34;细胞周期中G0/G1期88.76%,G2/M期3.04%,S期8.2%,符合干细胞周期特征;增殖曲线呈典型"S"型;油红O与碱性磷酸酶染色均为阳性。同时与传统方法相比,改良方法的细胞贴壁率显著高于传统方法(P=0.0004),首次贴壁时间短(P<0.0001)、首次传代时间短(P=0.001)。结论:骨髓标本培养48 h后的上清层中含大量贴壁活性的悬浮MSC,改良方法是一种比传统贴壁法更具有便捷、高效等优点的培养方法。

This study was aimed to investigate a more convenient and efficient method to cultivate the human bone marrow mesenchymal stem cells by means of natural erythrocyte sedimentation principle, based on the whole bone marrow adherent method. The bone marrow was cultured with a six-well plate instead of the flasks. Firsly, the bone marrow specimen was cultivated with the MSC complete medium for 48 h,then the upper RBC-free supernatant layer was drawn and placed into the new wells to isolate MSC. Inverted microscope was used to observe the cell morphology and to record the adherent time of first cell passage, first passaging time. The traditional whole bone marrow adherent method was used as the control. The cell cycle and cell surface markers were detected by flow cytometry, and the differentiative capacity of MSC into osteocyte and adipocyte was identified by alkaline phosphatase kit and oil red O, respectively. Besides ,the proliferative curve of P1 ,P3 ,P5 of BMSC was depicted by counting method. The results showed that MSC cultured by the modified method highly expressed CD90, CD105, CD13, CD44 and lowly expressed CD14, CD45, CD34. Concerning the cell cycle feature, it was found that most of the cells were in G0/G1 phase （88.76%） , followed by G2/M phase （3.04%） and S phase （8.2%）, which was in accordance with stem cell cycle characteristics. The proliferative curve showed a typical ＂ S＂ type, and both the oil red O and alkaline phosphatase staining of MSC were positive. Compared with the traditional method, the modified method had the advantage of high adherence rate （ P = 0. 0001 ） and shorter passaging time for the first passage （ P = 0. 001 ）, with the statistically significant difference. It is concluded that there is a large number of adherent, active and suspended MSC in the RBC-free supernatant layer after the culture of bone marrow for 48 h. Isolating MSC by the modified method is more convenient and efficient than the traditional whole bone marrow adherent method.