| 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 |
| Alternative Title | Human Bone Marrow-derived Mesenchymal Stem Cells Labeled with 5-Bromodeoxyuridine
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| 王小蕊 |
| Thesis Advisor | 侯相麟
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| 2008-05-21
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| Degree Grantor | 兰州大学
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| Place of Conferral | 兰州
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| Degree Name | 硕士
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| Keyword | 骨髓
骨髓间充质干细胞
细胞培养
流式细胞术
5-溴脱氧尿嘧啶核苷
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| Abstract | 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；贴壁筛选法培养分离、纯化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；选择P3细胞，以5、10、15 µmol/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：体外培养的成人骨髓MSCs形态均一，为梭形或纺锤形外观；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，细胞形态及增殖无明显变化。
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。
关键词：骨髓；间充质干细胞；细胞培养；流式细胞术； 5-溴脱氧尿嘧啶核苷 |
| Other Abstract | Aim:To establish the methods of culture,expansion and identification of adult bone marrow mesenchymal stem cells(MSCs) and explore the best time and concentration for labeling the MSCs with 5-bromodeoxyuridine(5-BrdU) in vitro,so as to attain the best tracing index of applying MSCs to future research.Methods:10mL bone marrow was collected from the healthy volunteer,obtaining monocyte by density gradient centrifugation.MSCs were isolated and purified by adhering to the culture glassware wall.Observing cell morphology by phase-contrast micros- cope.The surface antigen that CD44,CD71,CD34 and HLA-DR were detected with Flow cytometry (FCM), tracing P1,P3 and P5 of the MSCs with 10µmol/L BrdU a- nd detecting labeling rate with Immunocytochemistry,the same method was used to detecte P3 of the MSCs with 5,10 and 15 µmol/L BrdU at 12,24,48,72 and 96h of which P3 with 10µmol/L BrdU at 72h was passaged,so as to observe cell morpholo- gy and proliferation.
Results:The human marrow MSCs had a fusiform or similar spindle-like morphology.FCM showed that MSCs expressed CD44 and CD71 but not expressed CD34 and HLA-DR. Most MSCs had a positive nuclear stain when labeled by 5-BrdU and the labeled effect was Stability among passage cells. The labeling rate increased gradually with the elongation of concentration and time.The incubation of the MSCs with 5-BrdU 10µmol/L and 72h appeared to achieve the highest labeling rate, both of which exceeded 90%.5-BrdU has little effect on morphology and proliferation of labeled cells after serial passage.
Conclusion:The combination of density gradient centrifugation and adherent culture and digestion control is an ideal method for culture and purify MSCs in vitro;5-BrdU labeling provides a feasible means for a dynamic observation of the survival, growth and differentiation of the implanted MSCs.the incubation of which with 10µmol/L 5-BrdU for 72h is the optimal dosage and timing.
Key words:bone marrow;mesenchymal stem cells;cell culture;flow cytometry;bromodeoxyuridine |
| URL | 查看原文
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| Language | 中文
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| Document Type | 学位论文
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| Identifier | https://ir.lzu.edu.cn/handle/262010/224261
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| Collection | 学院待认领
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| Affiliation | 临床医学院
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Recommended Citation
GB/T 7714 |
王小蕊. 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[D]. 兰州. 兰州大学,2008.
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