TGF-β1表达载体的构建及其在小鼠骨髓间充质干细胞的表达研究

发布时间：2018-03-31 16:23

  本文选题：骨髓　切入点：间充质干细胞　出处：《山西医科大学》2011年硕士论文

【摘要】：第一章小鼠骨髓间充质干细胞(BMSCs)的体外分离扩增及鉴定 目的建立一种简单易行的小鼠骨髓间充质干细胞(bone marrow mesenchymal stem cellsBMscs)分离培养方法，并对其表面标志进行鉴定。方法通过全骨髓贴壁分离法体外分离、扩增小鼠BMSCs。观察原代及传代小鼠BMSCs形态变化，对第3代小鼠BMSCs表面抗原CD34、CD45、CDl05和CDl06进行流式细胞仪检测，并对小鼠BMscs进行纯度测定。 结果用此法进行小鼠BMscs的原代接种培养24 h后可见大量悬浮细胞，少许贴壁细胞，呈小圆形；72h后贴壁细胞逐渐增多，培养至第7d细胞伸展成长梭形，呈集落生长。经反复换液后未贴壁的造血细胞被弃去。BMscs在培养的2～6d细胞增殖较慢，7～10d细胞增殖较快。细胞培养约10—12d左右，可接近75％～80％融合。传代后的小鼠BMscs 24b基本全部贴壁，细胞形态较为均一，呈均匀性分布生长。分离纯化后所得细胞活力为92.6±1.8％。流式细胞术结果显示第3代小鼠BMscs细胞均一性较好，在90%以上。CD34、CD45表达阴性，cDl05、cDl06表达阳性。 结论采用全骨髓贴壁分离法可获得：BMscs，这种方法简单、便捷、实用。所得小鼠BMscs活力及纯度均较高，流式细胞技术可以鉴定体外分离培养的小鼠BMscs。 第二章TGF-β1表达载体的构建及其在小鼠骨髓间充质干细胞中的表达 目的将transforming growth factor beta 1(TGF-β1)序列构建到带GFP的表达载体pcDHl-Mcsl-EFl-copGFP中，并将重组质粒转染入BMscs，使其在BMsc8中表达。 方法以小鼠肺组织cDNA为模板，PcR扩增出TGF-β1基因，并将其插入pcDHl-Mcsl-EFl-copGFP载体质粒中，转化至感受态菌DH5a，抽提质粒，经PcR扩增和测序鉴定后转染入BMscs细胞，利用激光共聚焦显微镜和Real-time PCR方法对其表达位置和表达量进行检测。 结果经PcR及测序鉴定，构建入载体质粒的基因为TGF-β1基因，pcDHl-TGFβ1-EFl-copGFP重组质粒成功构建，，且它能在BMSCs细胞中成功表达。 结论TGF-β1基因在BMSCs细胞成功表达，为进一步研究TGFβ1影响BMSCs细胞的生理功能奠定了实验和理论基础。
[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 卤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.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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