5-溴脱氧尿嘧啶核苷对成人骨髓间充质干细胞体外标记的研究

发布时间：2018-06-03 23:06

  本文选题：骨髓 + 间充质干细胞　； 参考：《兰州大学》2008年硕士论文

【摘要】： 目的 建立成人骨髓间充质干细胞(MSCs)体外分离培养和鉴定的方法,探讨连续培养的骨髓间充质干细胞应用5-溴脱氧尿嘧啶核苷(BrdU)标记的稳定性、最佳时间和剂量,争取确定应用骨髓间充质干细胞进行研究最好的示踪指标。 方法 采集成人骨髓10mL,密度梯度法分离出单个核细胞;贴壁筛选法培养纯化和扩增MSCs;倒置光学显微镜下观察细胞形态;取生长良好的P3细胞,流式细胞仪(FCM)检测细胞的表面抗原(CD44、CD71、CD34、HLA-DR);收集生长良好的P1、P3、P5成人骨髓MSCs,用10μmol/L浓度的BrdU标记至细胞生长融合,免疫细胞化学法检测标记率(LI);选择P3细胞,以5、10、15μmol/L浓度的BrdU分别标记12、24、48、72、96h;检测不同时间不同浓度的标记率;将10μmol/LBrdU标记72h的P3细胞连续传代,观察传代后的细胞形态及增殖情况。 结果 倒置显微镜下观察体外培养的成人骨髓MSCs形态均一,为梭形或纺锤形的成纤维细胞样外观;FCM检测细胞表达CD44和CD71,不表达CD34和HLA-DR;大部分MSCs经BrdU标记后核抗BrdU染色阳性,P1、P3、P5各代间不会随着传代次数的增加而降低标记率;随着标记浓度的升高和标记时间的延长,标记率逐渐增高,浓度10μmol/L标记72h标记率在90%以上;标记细胞连续传代,细胞形态无明显变化。 结论 密度梯度离心、贴壁培养和消化控制相结合的方法是体外分离培养人骨髓MSCs的理想方法;BrdU标记可用于成人骨髓MSCs移植入体内后存活、生长和分化的动态观察指标;终浓度10μmol/L标记72h为BrdU最佳标记浓度和时间。
[Abstract]:Purpose To establish a method for isolation, culture and identification of adult bone marrow mesenchymal stem cells (MSCs) in vitro, and to investigate the stability, optimal time and dose of 5-bromodeoxyuridine BrdU labeling of bone marrow mesenchymal stem cells in continuous culture. To identify the best tracer index for the study of bone marrow mesenchymal stem cells. Method Mononuclear cells were isolated by density gradient method from adult bone marrow, MSCs were purified and amplified by adherent screening, morphology of cells was observed under inverted optical microscope, and P3 cells were obtained. Flow cytometry (FCM) was used to detect HLA-DRN of the cell surface antigen (CD44T / CD71T / CD34N). MSCs of adult bone marrow were collected and labeled with 10 渭 mol/L concentration of BrdU. The labeling rate was detected by immunocytochemistry, and P3 cells were selected. The labeling rate of P3 cells labeled with 10 渭 mol/LBrdU for 72 hours was detected at different time and different concentration, and the morphology and proliferation of P3 cells were observed after being labeled with 10 渭 mol/LBrdU for 72 hours. Result The morphology of adult bone marrow MSCs cultured in vitro was observed under inverted microscope. For fusiform or fusiform fibroblast-like cells, CD44 and CD71were detected by FCM, but CD34 and HLA-DR1 were not expressed in most of MSCs cells, and the labeling rate was not decreased with the increase of passage times after most MSCs were labeled with BrdU. With the increase of labeling concentration and the prolongation of labeling time, the labeling rate increased gradually, and the labeling rate of 10 渭 mol/L for 72 h was more than 90%. Conclusion The method of density gradient centrifugation, adherent culture and digestion control is an ideal method for isolation and culture of human bone marrow MSCs in vitro. BrdU labeling can be used to observe the survival, growth and differentiation of adult bone marrow MSCs after transplantation in vivo. The best concentration and time of BrdU labeling was 10 渭 mol/L for 72 h.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329

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