兰州大学机构库 >学院待认领
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 TitleHuman Bone Marrow-derived Mesenchymal Stem Cells Labeled with 5-Bromodeoxyuridine
王小蕊
Thesis Advisor侯相麟
2008-05-21
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name硕士
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
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 AbstractAim: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
Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/224261
Collection学院待认领
Affiliation临床医学院
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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