论文部分内容阅读
背景:目前对骨髓间充质干细胞常用的分离方法有密度梯度离心法、贴壁筛选分离法。目的:联合应用密度梯度离心法和贴壁筛选分离法体外分离培养、扩增兔骨髓间充质干细胞,并对其进行鉴定。设计、时间及地点:对比观察的细胞学实验,于2007-10/2008-03在上海市第六人民医院中心实验室完成。材料:2月龄新西兰纯种大耳白兔6只用于骨髓间充质干细胞取材与原代培养,1.073kg/L的Percoll分离液。方法:实验采用Percol分离液利用密度梯度离心法及结合贴壁分离筛选法来分离、纯化骨髓间充质干细胞,在采用密度梯度离心法得到骨髓间充质干细胞后,经贴壁培养及反复换液纯化骨髓间充质干细胞。分别取第3,5,7,9代骨髓间充质干细胞,行细胞计数,绘制细胞生长曲线。主要观察指标:倒置显微镜下观察原代及传代细胞的形态、生长情况。采用CD44及CD34抗体进行间接免疫荧光标记鉴定培养的干细胞。CD44染色呈阳性,CD34染色呈阴性,说明所提取、纯化的细胞是骨髓间充质干细胞。结果:增殖传代的骨髓间充质干细胞呈长梭形均匀分布生长,形态比原代培养的细胞更均匀,细胞生长旺盛、增殖迅速,胞核明显,核仁清晰,核浆比例大,细胞形态均匀,平行排列呈螺旋状或漩涡状,传代至第5代时无明显变化。随传代次数的增加,细胞增殖能力逐渐下降,第3~5代细胞增殖能力强。所分离培养的细胞均表达CD44,不表达CD34。结论:在体外采用密度梯度离心及贴壁培养法可获得高纯度的兔骨髓间充质干细胞。
BACKGROUND: Currently, the most commonly used separation methods for bone marrow mesenchymal stem cells are density gradient centrifugation and adherence screening. OBJECTIVE: To isolate and culture rabbit bone marrow mesenchymal stem cells (MSCs) in vitro by density gradient centrifugation and adherence screening. DESIGN, TIME AND SETTING: Comparative cytology experiments were performed at the Central Laboratory of the Sixth People’s Hospital of Shanghai from October 2007 to March 2008. MATERIALS: Six-month-old New Zealand purebred rabbits were used to extract bone marrow-derived mesenchymal stem cells and primary culture, 1.073kg / L Percoll separation liquid. Methods: Bone marrow mesenchymal stem cells were isolated and purified by density gradient centrifugation combined with adherent separation and screening using Percol separation liquid. After the bone marrow mesenchymal stem cells were obtained by density gradient centrifugation, Purification of bone marrow mesenchymal stem cells. The 3rd, 5th, 7th and 9th generations of bone marrow mesenchymal stem cells were taken, and the cell counts were counted and the cell growth curve was drawn. MAIN OUTCOME MEASURES: Morphology and growth of primary and subculture cells were observed under an inverted microscope. Indirect immunofluorescent labeling of CD44 and CD34 antibodies was used to identify cultured stem cells. CD44 staining was positive, CD34 staining was negative, indicating that the extracted and purified cells are bone marrow mesenchymal stem cells. Results: BMSCs proliferated and distributed uniformly in long fusiform shape with more uniform morphology than the primary cultured cells, with strong cell growth, rapid proliferation, clear nuclei, clear nucleoli, large proportion of nuclear cytoplasm, and cell morphology Uniform, parallel arrangement was spiral or whirlpool, no significant change from the passage to the fifth generation. With the increase of passage times, cell proliferation decreased gradually, and cell proliferation ability of cells in passage 3 to 5 was stronger. The cultured cells expressed CD44 but not CD34. Conclusion: High purity rabbit bone marrow mesenchymal stem cells can be obtained by density gradient centrifugation and adherent culture in vitro.