本文選題：骨髓 + 間充質干細胞　； 參考：《蘭州大學》2008年碩士論文

【摘要】： 目的 建立成人骨髓間充質干細胞(MSCs)體外分離培養(yǎng)和鑒定的方法,探討連續(xù)培養(yǎng)的骨髓間充質干細胞應用5-溴脫氧尿嘧啶核苷(BrdU)標記的穩(wěn)定性、最佳時間和劑量,爭取確定應用骨髓間充質干細胞進行研究最好的示蹤指標。 方法 采集成人骨髓10mL,密度梯度法分離出單個核細胞;貼壁篩選法培養(yǎng)純化和擴增MSCs;倒置光學顯微鏡下觀察細胞形態(tài);取生長良好的P3細胞,流式細胞儀(FCM)檢測細胞的表面抗原(CD44、CD71、CD34、HLA-DR);收集生長良好的P1、P3、P5成人骨髓MSCs,用10μmol/L濃度的BrdU標記至細胞生長融合,免疫細胞化學法檢測標記率(LI);選擇P3細胞,以5、10、15μmol/L濃度的BrdU分別標記12、24、48、72、96h;檢測不同時間不同濃度的標記率;將10μmol/LBrdU標記72h的P3細胞連續(xù)傳代,觀察傳代后的細胞形態(tài)及增殖情況。 結果 倒置顯微鏡下觀察體外培養(yǎng)的成人骨髓MSCs形態(tài)均一,為梭形或紡錘形的成纖維細胞樣外觀;FCM檢測細胞表達CD44和CD71,不表達CD34和HLA-DR;大部分MSCs經BrdU標記后核抗BrdU染色陽性,P1、P3、P5各代間不會隨著傳代次數的增加而降低標記率;隨著標記濃度的升高和標記時間的延長,標記率逐漸增高,濃度10μmol/L標記72h標記率在90%以上;標記細胞連續(xù)傳代,細胞形態(tài)無明顯變化。 結論 密度梯度離心、貼壁培養(yǎng)和消化控制相結合的方法是體外分離培養(yǎng)人骨髓MSCs的理想方法;BrdU標記可用于成人骨髓MSCs移植入體內后存活、生長和分化的動態(tài)觀察指標;終濃度10μmol/L標記72h為BrdU最佳標記濃度和時間。
[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.
【學位授予單位】：蘭州大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R329

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