兔骨髓间充质干细胞体外培养扩增及BMP-2真核表达质粒转染的实验研究

发布时间：2018-07-26 17:09

【摘要】： 目的(1)培养转染所需兔骨髓间充质干细胞(bone marrow mesenchymal stem cells, BMSCs)。(2)通过采用阳性脂质体pcDNA3.1(-)介导的转染到BMSCs方法,获得稳定转染的BMSCs细胞。(3)使用免疫组化、WesternBlot法检测pcDNA3.1-hBMP2在兔BMSCs中蛋白的表达情况。 方法(1)贴壁法培养扩增所需BMSCs并从形态学、流式细胞仪测量表面标记物。(2)将已成功构建的pcDNA3.1-hBMP2重组质粒进行体外靶细胞转染并使用G418筛选稳定表达细胞。(3)通过免疫组化、WesternBlot法检测其在BMSCs中的蛋白表达。 结果(1)经培养,扩增兔骨髓间充质干细胞,P2、P3可作为hBMP2转染的靶细胞。(2)用阳离子脂质体转染pcDNA3.1-hBMP2的MSCs经G418筛选后,经免疫组化、WesternBlot法检测,转染pcDNA3.1-hBMP2后的兔骨髓间充质干细胞内有大量hBMP2 mRNA的转录和相关蛋白的表达,而未转染的骨髓间充质干细胞未见阳性表达。 结论(1)兔骨髓间充质干细胞可作为hBMP2转染的靶细胞。(2)本实验成功构建hBMP2真核表达质粒并在兔骨髓间充质干细胞中得到相关表达。(3)本研究为进一步研究BMP2基因转染MSCs的体外增殖并稳定表达和在动物试验中来加速牵张成骨新骨形成的实验奠定了基础。
[Abstract]:Objective (1) to culture rabbit bone marrow mesenchymal stem cells (bone marrow mesenchymal stem cells, BMSCs). (2) for transfection to BMSCs by using positive liposome pcDNA3.1 (-)-mediated transfection. Stable transfected BMSCs cells were obtained. (3) the expression of pcDNA3.1-hBMP2 in rabbit BMSCs was detected by immunohistochemistry and Western blot. Methods (1) BMSCs was cultured and amplified by adherent method. Flow cytometry was used to measure the surface markers. (2) the successfully constructed pcDNA3.1-hBMP2 recombinant plasmid was transfected into target cells in vitro and the stable expression cells were screened by G418. (3) the protein expression in BMSCs was detected by immunohistochemistry and Western Blot. Results (1) cultured rabbit bone marrow mesenchymal stem cells (BMSCs) could be used as target cells for hBMP2 transfection. (2) MSCs transfected with pcDNA3.1-hBMP2 by cationic liposome was screened by G418 and detected by immunohistochemistry with Western blot. A large number of hBMP2 mRNA transcripts and related proteins were expressed in rabbit bone marrow mesenchymal stem cells after pcDNA3.1-hBMP2 transfection, but no positive expression was found in untransfected bone marrow mesenchymal stem cells. Conclusion (1) Rabbit bone marrow mesenchymal stem cells can be used as target cells for hBMP2 transfection. (2) the eukaryotic expression plasmid of hBMP2 was successfully constructed and expressed in rabbit bone marrow mesenchymal stem cells. (3) in order to further study the transfection of BMP2 gene in rabbit bone marrow mesenchymal stem cells. The proliferation and stable expression of MSCs in vitro and the experiment of accelerating the formation of new bone in distraction osteogenesis were established in animal experiments.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

【引证文献】

相关硕士学位论文 前1条

1 高晓燕;金葡液在兔下颌骨牵张成骨中作用的研究[D];辽宁医学院;2011年

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