發(fā)布時(shí)間：2018-06-03 23:06

  本文選題：骨髓 + 間充質(zhì)干細(xì)胞�。� 參考：《蘭州大學(xué)》2008年碩士論文

【摘要】： 目的 建立成人骨髓間充質(zhì)干細(xì)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(zhì)干細(xì)胞應(yīng)用5-溴脫氧尿嘧啶核苷(BrdU)標(biāo)記的穩(wěn)定性、最佳時(shí)間和劑量,爭(zhēng)取確定應(yīng)用骨髓間充質(zhì)干細(xì)胞進(jìn)行研究最好的示蹤指標(biāo)。 方法 采集成人骨髓10mL,密度梯度法分離出單個(gè)核細(xì)胞;貼壁篩選法培養(yǎng)純化和擴(kuò)增MSCs;倒置光學(xué)顯微鏡下觀察細(xì)胞形態(tài);取生長(zhǎng)良好的P3細(xì)胞,流式細(xì)胞儀(FCM)檢測(cè)細(xì)胞的表面抗原(CD44、CD71、CD34、HLA-DR);收集生長(zhǎng)良好的P1、P3、P5成人骨髓MSCs,用10μmol/L濃度的BrdU標(biāo)記至細(xì)胞生長(zhǎng)融合,免疫細(xì)胞化學(xué)法檢測(cè)標(biāo)記率(LI);選擇P3細(xì)胞,以5、10、15μmol/L濃度的BrdU分別標(biāo)記12、24、48、72、96h;檢測(cè)不同時(shí)間不同濃度的標(biāo)記率;將10μmol/LBrdU標(biāo)記72h的P3細(xì)胞連續(xù)傳代,觀察傳代后的細(xì)胞形態(tài)及增殖情況。 結(jié)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(xì)胞樣外觀;FCM檢測(cè)細(xì)胞表達(dá)CD44和CD71,不表達(dá)CD34和HLA-DR;大部分MSCs經(jīng)BrdU標(biāo)記后核抗BrdU染色陽(yáng)性,P1、P3、P5各代間不會(huì)隨著傳代次數(shù)的增加而降低標(biāo)記率;隨著標(biāo)記濃度的升高和標(biāo)記時(shí)間的延長(zhǎng),標(biāo)記率逐漸增高,濃度10μmol/L標(biāo)記72h標(biāo)記率在90%以上;標(biāo)記細(xì)胞連續(xù)傳代,細(xì)胞形態(tài)無(wú)明顯變化。 結(jié)論 密度梯度離心、貼壁培養(yǎng)和消化控制相結(jié)合的方法是體外分離培養(yǎng)人骨髓MSCs的理想方法;BrdU標(biāo)記可用于成人骨髓MSCs移植入體內(nèi)后存活、生長(zhǎng)和分化的動(dòng)態(tài)觀察指標(biāo);終濃度10μmol/L標(biāo)記72h為BrdU最佳標(biāo)記濃度和時(shí)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(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類(lèi)號(hào)】：R329

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