本文選題：骨髓　切入點：間充質干細胞　出處：《山西醫(yī)科大學》2011年碩士論文

【摘要】：第一章小鼠骨髓間充質干細胞(BMSCs)的體外分離擴增及鑒定 目的建立一種簡單易行的小鼠骨髓間充質干細胞(bone marrow mesenchymal stem cellsBMscs)分離培養(yǎng)方法，并對其表面標志進行鑒定。方法通過全骨髓貼壁分離法體外分離、擴增小鼠BMSCs。觀察原代及傳代小鼠BMSCs形態(tài)變化，對第3代小鼠BMSCs表面抗原CD34、CD45、CDl05和CDl06進行流式細胞儀檢測，并對小鼠BMscs進行純度測定。 結果用此法進行小鼠BMscs的原代接種培養(yǎng)24 h后可見大量懸浮細胞，少許貼壁細胞，呈小圓形；72h后貼壁細胞逐漸增多，培養(yǎng)至第7d細胞伸展成長梭形，呈集落生長。經反復換液后未貼壁的造血細胞被棄去。BMscs在培養(yǎng)的2～6d細胞增殖較慢，7～10d細胞增殖較快。細胞培養(yǎng)約10—12d左右，可接近75％～80％融合。傳代后的小鼠BMscs 24b基本全部貼壁，細胞形態(tài)較為均一，呈均勻性分布生長。分離純化后所得細胞活力為92.6±1.8％。流式細胞術結果顯示第3代小鼠BMscs細胞均一性較好，在90%以上。CD34、CD45表達陰性，cDl05、cDl06表達陽性。 結論采用全骨髓貼壁分離法可獲得：BMscs，這種方法簡單、便捷、實用。所得小鼠BMscs活力及純度均較高，流式細胞技術可以鑒定體外分離培養(yǎng)的小鼠BMscs。 第二章TGF-β1表達載體的構建及其在小鼠骨髓間充質干細胞中的表達 目的將transforming growth factor beta 1(TGF-β1)序列構建到帶GFP的表達載體pcDHl-Mcsl-EFl-copGFP中，并將重組質粒轉染入BMscs，使其在BMsc8中表達。 方法以小鼠肺組織cDNA為模板，PcR擴增出TGF-β1基因，并將其插入pcDHl-Mcsl-EFl-copGFP載體質粒中，轉化至感受態(tài)菌DH5a，抽提質粒，經PcR擴增和測序鑒定后轉染入BMscs細胞，利用激光共聚焦顯微鏡和Real-time PCR方法對其表達位置和表達量進行檢測。 結果經PcR及測序鑒定，構建入載體質粒的基因為TGF-β1基因，pcDHl-TGFβ1-EFl-copGFP重組質粒成功構建，，且它能在BMSCs細胞中成功表達。 結論TGF-β1基因在BMSCs細胞成功表達，為進一步研究TGFβ1影響B(tài)MSCs細胞的生理功能奠定了實驗和理論基礎。
[Abstract]:In vitro isolation, amplification and identification of mouse bone marrow mesenchymal stem cells (BMSCs)Objective to establish a simple method for isolation and culture of marrow mesenchymal stem cells BMscs from mouse bone marrow mesenchymal stem cells, and to identify its surface markers.Methods BMSCs were isolated by whole bone marrow adherent method in vitro.The morphological changes of BMSCs in primary and subculture mice were observed. The surface antigens CD34, CD45, CDl05 and CDl06 were detected by flow cytometry, and the purity of BMscs in mice was determined by flow cytometry.Results after 24 hours of primary inoculation and culture of mouse BMscs, a large number of suspension cells were observed, a few adherent cells were observed, and the adherent cells increased gradually after 72 hours of small circle. After 7 days of culture, the cells expanded and spindle-shaped, and showed colony growth.After repeated fluid exchange, the unadherent hematopoietic cells were abandoned. BMscs proliferated more slowly on the 6th day of culture than on the 7th day after 10 days.Cell culture was about 10-12 days, close to 75% fusion.After passage, the BMscs 24b of mice was almost adherent to the wall, and the cell morphology was uniform, and the cells grew homogeneously.The cell viability after purification was 92.6 鹵1.8.Conclusion the whole bone marrow adherent separation method can be used to obtain bone marrow BMscs. This method is simple, convenient and practical.The BMscs activity and purity of the obtained mice were high. Flow cytometry could be used to identify the mouse BMSCs isolated and cultured in vitro.Construction of TGF- 尾 1 expression Vector and its expression in Mouse Bone Marrow Mesenchymal Stem cellsObjective to construct the transforming growth factor beta 1hTGF- 尾 1) sequence into the expression vector pcDHl-Mcsl-EFl-copGFP with GFP, and to transfect the recombinant plasmid into BMscs and make it express in BMsc8.Methods the TGF- 尾 1 gene was amplified from mouse lung tissue cDNA and inserted into the plasmid of pcDHl-Mcsl-EFl-copGFP vector. The plasmid was extracted from the plasmid DH 5a. The plasmid was transfected into BMscs cells by PcR amplification and sequencing.Laser confocal microscopy and Real-time PCR were used to detect its expression position and quantity.Results the recombinant plasmid of TGF- 尾 1 gene, pcDHl-TGF 尾 1-EFl-copGFP, was successfully constructed by PcR and sequencing, and it was successfully expressed in BMSCs cells.Conclusion TGF- 尾 1 gene was successfully expressed in BMSCs cells, which laid an experimental and theoretical foundation for further study on the effect of TGF 尾 1 on the physiological function of BMSCs cells.
【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R329

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