大鼠骨髓间充质干细胞培养方法的比较及细胞鉴定

发布时间：2018-03-24 16:26

本文选题：骨髓间充质干细胞　切入点：全骨髓培养法　出处：《新疆医科大学》2012年硕士论文

【摘要】：目的：研究大鼠骨髓间充质干细胞(Bone marrow mesenchymal stem cell, BMSCs)的不同培养方法的差异，及对培养出的不同代数的细胞做形态学观察及表面标记物鉴定。为今后向实验提供高纯度的BMSCs提供方法及依据。方法：取150g~180g左右2只雄性SD大鼠后肢股骨、胫骨骨髓，分别行全骨髓培养法和密度密度梯度离心法培养。将2种方法分离出的细胞进行贴壁细胞的筛选并培养，利用BMSCs的易于贴附于塑料制品的特性给予多次传代纯化。观察两种方法培养的不同代数的细胞形态，绘制贴壁的细胞生长曲线，流式细胞仪检测P5代cd34、cd29、cd44、cd45细胞因子，鉴定是否为骨髓间充质干细胞。结果：形态学学观察上可见2种方法所获取的细胞呈长梭形，呈现特征性的漩涡状生长。绘制2种方法P0至P10代的细胞增殖生长曲线图，以每一代单皿传代数量为检测指标进行单样本配对T检验分析结果为：P＜0.05。证明全骨髓培养法细胞增殖比较快。对P3代2组细胞进行流式细胞检测结果CD34、CD45阴性，CD29、CD44阳性。证明2种方法培养的细胞符合BMSCs的特性。结论：全骨髓培养方法相比于密度密度梯度离心法不仅可以成功分离骨髓间充质干细胞而且对试剂要求少，更加经济，操作更简单、易行，，培养所需周期较短。全骨髓培养法培养出的BMSCs并在5~10代时的细胞纯度高，活性好且BMSCs纯度较稳定，可进行BMSCs多向分化诱导及应用BMSCs进行动物实验研究及临床应用。
[Abstract]:Objective: to study the different culture methods of bone marrow mesenchymal stem cells (BMSCs) in rat bone marrow mesenchymal stem cells (BMSCs). Morphological observation and surface marker identification of cultured cells from different generations were performed. Methods: the femur and tibial bone marrow of two male SD rats with 150g~180g were taken from the femur and tibial bone marrow of two male SD rats, and the method and basis of high purity BMSCs were provided for the future experiment. The whole bone marrow culture method and density gradient centrifugation method were used respectively. The cells isolated from the two methods were screened and cultured for adherent cells. The cell morphology of different generations was observed and the cell growth curve of adherent cells was plotted. Flow cytometry was used to detect the cytokines of cd34cd29cd4cd4cd45 in P5 passage. Results: according to the morphological observation, the cells obtained by the two methods were spindle-shaped, showing characteristic whirlpool growth. The cell proliferation and growth curves of P0 to P10 passages of the two methods were plotted. The result of single sample paired T test was: 1: P < 0.05, which proved that the whole bone marrow cell proliferation was faster. Flow cytometry was used to detect CD34, CD45 negative and CD29 CD44 positive in P3 passage 2 cells. Conclusion: compared with density density gradient centrifugation, the whole bone marrow culture method can not only successfully isolate bone marrow mesenchymal stem cells, but also require less reagent. BMSCs cultured by whole bone marrow culture has high purity, good activity and stable purity of BMSCs at 5 ~ 10 passages. It can be used to induce multidirectional differentiation of BMSCs and to use BMSCs for animal experimental study and clinical application.
【学位授予单位】：新疆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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