构建pcDNA3.1-BMP-2重组载体及转染兔BMSCs的研究

发布时间：2018-10-21 19:24

【摘要】： 目的：构建骨形态发生蛋白-2(BMP-2)真核表达质粒pcDNA3.1-BMP-2,转染兔骨髓间充质干细胞(BMSCs)。检测转染后BMP-2基因的表达。方法：(1)利用人外周血中抽提的基因组DNA,通过聚合酶链式反应(PCR)合成BMP-2基因,将其与pcDNA3.1载体进行基因重组,构建表达质粒pcDNA3.1-BMP-2。再通过PCR及DNA测序的方法检测重组体。(2)利用pcDNA3.1-BMP-2转染兔BMSCs,再通过RT-PCR、免疫组化和蛋白印迹法(Western blot)检测BMP-2基因的表达。结果：(1)经PCR扩增和DNA测序证实,本实验构建的重组质粒目的基因片段为人BMP-2-cDNA。(2)经RT-PCR、免疫组化和Western blot证实,转染pcDNA3.1-BMP-2后的兔BMSCs内有大量BMP-2 mRNA的转录和蛋白的表达,而未转染的兔BMSCs未见阳性表达。结论：(1)本实验成功构建BMP-2真核表达质粒并在兔BMSCs中得到表达。(2)本研究为进一步研究用BMP-2基因转染的方法来加速牵张成骨(Distraction osteogenesis DO)新骨形成的实验奠定了基础。
[Abstract]:Objective: to construct eukaryotic expression plasmid pcDNA3.1-BMP-2, of bone morphogenetic protein-2 (BMP-2) and transfect (BMSCs). Into rabbit bone marrow mesenchymal stem cells (BMSCs). The expression of BMP-2 gene after transfection was detected. Methods: (1) the genomic DNA, extracted from human peripheral blood was used to synthesize BMP-2 gene by polymerase chain reaction (PCR). The BMP-2 gene was recombined with pcDNA3.1 vector to construct the expression plasmid pcDNA3.1-BMP-2.. Then the recombinant was detected by PCR and DNA sequencing. (2) the expression of BMP-2 gene was detected by RT-PCR, immunohistochemistry and Western blotting (Western blot). (2) Rabbit BMSCs, was transfected with pcDNA3.1-BMP-2. Results: (1) it was confirmed by PCR amplification and DNA sequencing that the target gene fragment of recombinant plasmid constructed in this study was human BMP-2-cDNA. (2) by RT-PCR, immunohistochemistry and Western blot, a large number of BMP-2 mRNA transcription and protein expression were found in the BMSCs transfected with pcDNA3.1-BMP-2. No positive expression was found in untransfected rabbit BMSCs. Conclusion: (1) the eukaryotic expression plasmid of BMP-2 was successfully constructed and expressed in rabbit BMSCs. (2) this study laid a foundation for further study on the method of BMP-2 gene transfection to accelerate the bone formation of distraction osteogenesis (Distraction osteogenesis DO).
【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】：R341

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