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  本文選題：干細胞　切入點：細胞分化　出處：《安徽醫(yī)科大學》2008年碩士論文　論文類型：學位論文

【摘要】： 目的:研究5-溴脫氧尿嘧啶核苷(BrdU)標記脂肪間充質干細胞(ADSCs)的效果,確定體外標記的最佳濃度和時間,評價BrdU標記的ADSCs的適用性。 方法:取6月齡新西蘭白兔頸后皮下脂肪,采用Ⅰ型膠原酶消化方法體外分離培養(yǎng)脂肪干細胞,利用成骨、成脂誘導培養(yǎng)基定向向成脂、成骨細胞誘導并用組織化學方法鑒定其誘導結果,從而鑒定其具有多向分化的干細胞潛能。選取第3代ADSCs,分別采用5、10、15、20μmol/L濃度的BrdU體外標記干細胞24、48、72小時,利用免疫組化實驗計算不同濃度和標記時間下脂肪干細胞的標記率,確定BrdU對ADSCs的最佳標記方法。通過臺盼藍染色和細胞倍增時間的測量確定最佳標記方法的安全性。對第3代脂肪干細胞采用最佳標記方法標記后更換為普通培養(yǎng)液繼續(xù)培養(yǎng),適時傳代,連續(xù)檢測第5、7、9、11代脂肪干細胞的BrdU標記率,了解該標記方法在脂肪干細胞體外擴增培養(yǎng)過程中的衰減情況。同時將標記后的第3代脂肪干細胞作為實驗組,未標記細胞作為對照組,分別種植于乳酸-羥基乙酸共聚物(PLGA)支架材料上,體外培養(yǎng)3天后自體回植于動物皮下,4周后取出支架材料,制作石蠟切片采用免疫組化法鑒定BrdU標記的脂肪干細胞在體內標記的效果。 結果:兔脂肪干細胞體外培養(yǎng)具有成纖維細胞樣外形,增埴迅速,連續(xù)傳代14代細胞形態(tài)無明顯改變,無自發(fā)性向脂肪細胞分化現象。成脂誘導12天后油紅O染色胞漿內發(fā)現紅染的油滴,成骨誘導14天后Von kossa染色陽性,由此證明在體外定向誘導后脂肪干細胞可以向脂肪細胞和成骨細胞分化。BrdU可標記脂肪干細胞的核,采用10μmol/L的BrdU體外標記48小時是適宜的標記方法,該方法對脂肪干細胞的增殖無明顯影響。初測標記率達95%,標記率隨傳代次數而降低,傳代8次(約體外培養(yǎng)4周)后標記率仍達41%。同期進行的體內實驗證實,BrdU標記后的脂肪干細胞移植入體內4周后免疫組化檢測有BrdU陽性細胞存在,從而證明移植的脂肪干細胞可以在體內存活。 結論:兔脂肪干細胞易于體外分離培養(yǎng),具有較強的自我更新能力及多向分化的潛能.BrdU標記脂肪干細胞的操作簡單易行,10μmol/L的BrdU標記脂肪干細胞48h后,體外標記效果滿意.標記細胞移植體內1月后BrdU仍顯示較好的示蹤作用,因此BrdU可以用于脂肪干細胞的標記。
[Abstract]:Aim: to study the effect of 5-bromodeoxyuridine BrdU (BrdU) on the labeling of adipose mesenchymal stem cells (ADSCs), determine the optimal concentration and time of in vitro labeling, and evaluate the applicability of BrdU labeled ADSCs. Methods: adipose stem cells were isolated and cultured in vitro from 6-month-old New Zealand white rabbits with posterior subcutaneous fat. Adipose stem cells were isolated and cultured by type I collagenase digestion. Adipogenic medium was used to induce adipogenesis. Osteoblasts were induced and identified by histochemical method, so as to identify their multidirectional stem cell potential. The third generation of ADSCs was labeled with BrdU at the concentration of 5 10 ~ 1520 渭 mol/L for 24872 hours in vitro. The labeling rate of adipose stem cells at different concentrations and labeling time was calculated by immunohistochemistry. The best labeling method for ADSCs by BrdU was determined. The safety of the best labeling method was determined by trypan blue staining and cell doubling time measurement. The third generation adipose stem cells were labeled with the best labeling method and then replaced with normal culture medium. After timely passage, the BrdU labeling rate of adipose stem cells (ASCs) of generation 5, 7, 9 and 11 was continuously detected, and the attenuation of the labeling method in the process of proliferation and culture of adipose stem cells in vitro was investigated. At the same time, the third generation of labeled adipose stem cells were used as experimental group. The unlabeled cells were used as the control group and planted on the lactic acid-glycolic acid copolymer (PLGA) scaffold. After 3 days of culture in vitro, autologous cells were implanted subcutaneously in the animals for 4 weeks, then the scaffolds were removed. Paraffin sections were made to evaluate the effect of BrdU labeled adipose stem cells in vivo by immunohistochemical method. Results: rabbit adipose stem cells cultured in vitro showed fibroblast-like appearance, rapid growth, and no obvious changes in cell morphology in successive passage 14 passages. There was no spontaneous differentiation into adipocytes. After 12 days of lipogenesis, red oil droplets were found in the cytoplasm of oil red O staining, and Von kossa staining was positive after 14 days of osteogenic induction. The results showed that adipose stem cells could differentiate into adipocytes and osteoblasts after directional induction in vitro. BrdU could label the nucleus of adipose stem cells. It was suitable to label adipose stem cells with 10 渭 mol/L BrdU for 48 hours in vitro. This method had no significant effect on the proliferation of adipose stem cells. The initial labeling rate was 95%, and the labeling rate decreased with the passage times. After 8 passages (about 4 weeks in vitro culture), the labeling rate was still 41%. The results of in vivo experiments confirmed that there were BrdU positive cells in adipose stem cells after transplantation with BrdU in vivo for 4 weeks. This proves that transplanted adipose stem cells can survive in vivo. Conclusion: rabbit adipose stem cells are easy to be isolated and cultured in vitro, and have strong self-renewal ability and multidirectional differentiation potential. BrdU labeling of adipose stem cells is simple and easy to be used to label adipose stem cells with 10 渭 mol/L BrdU for 48 h. The labeling effect in vitro was satisfactory. BrdU still showed a good tracer effect after in vivo transplantation of labeled cells, so BrdU could be used as a marker of adipose stem cells.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R329

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