| 共表达腺病毒载体构建鉴定及其转染骨髓间充质干细胞观察 |
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投稿时间：2020-08-18
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| 期刊信息：《中国骨伤》2021年，第34卷，第7期，第674-679页 |
| DOI：10.12200/j.issn.1003-0034.2021.07.017 |
| 基金项目:中央高校基本科研业务专项资金（编号：2242020K40156）；国家自然科学基金（编号：81871810） |
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中文摘要:
目的：构建并鉴定绿色荧光蛋白标记的人骨形态发生蛋白（bone morphogenetic protein，BMP）2和血管内皮生长因子（vascular endotheliel growth factor，VEGF）165双基因共表达的重组腺病毒，为研究该双基因对骨髓间充质干细胞成骨方向诱导和体内骨缺损修复作用奠定基础。
方法：从cDNA文库中PCR扩增BMP2和VEGF165目的基因，并将其插入腺病毒穿梭质粒pAd-MCMV-GFP的多克隆位点，将构建的重组穿梭质粒pAd-MCMV-BMP2-VEGF165和腺病毒辅助质粒pBHGloxΔE1，3Cre共转染细胞HEK293细胞内，经历腺病毒基因重组及出毒后收集细胞，多次冻融离心获取病毒溶液（Ad-BMP2-VEGF165）。进一步纯化并测定病毒滴度，随后通过转染兔骨髓间充质干细胞并检测BMP2和VEGF165双目的基因表达和倒置荧光显微镜下观察绿色荧光蛋白表达。
结果：基因测序、菌落PCR、Western blotting、Real-time PCR、绿色荧光蛋白表达均表明载体Ad-BMP2-VEGF165构建成功，可稳定转染骨髓间充质干细胞内并稳定表达，经测定腺病毒载体滴度达到1×1010 PFU/ml。
结论：携带人BMP2和VEGF165双基因共表达重组腺病毒载体构建成功，并能获得高滴度的病毒感染液。 |
| 【关键词】骨形态发生蛋白 血管内皮生长因子 腺病毒载体 骨髓间充质干细胞 |
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| Construction and identification of recombinant adenovirus vector and observation of its transfecting rabbit bone marrow mesenchymal stem cells |
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ABSTRACT
Objective: To construct and identify adenovirus vector co-expressing hBMP2 and hVEGF165 fusion protein which labeled with green fluorescence protein,and laying the foundtion of the effect of hBMP2 and hVEGF165 gene inducing BMMSCs differentiation to osteoblast and bone defect repaired in the body.
Methods: BMP2 and VEGF165 gene was amplified from cDNA library by PCR and inserted to the polyclonal site of adenovirus shuttle plasmid pAd-MCMV-GFP. Ad-BMP2-VEGF165 was recombinated and propagated in HEK293 cells by co-transfecting with the constructed recombinant shuttle plasmid pAd-MCMV-BMP2-VEGF165 and adenovirus helper plasmid pBHGloxΔ E1,3Cre. The recombinant adenovirus was purified and virus titer was determined,and then to research GFP expression and to calculate the adenovirus transfection rate in rabbit BMMSCs.
Results: The recombinant adenovirus vector Ad-BMP2-VEGF165 was successfully constructed by the methods of gene analyzing,colony PCR,Western blotting and observing GFP expression,and the titer of the adenovirus was 1×1010 PFU/ml.
Conclusion: Recombinant adenovirus vector containing hBMP2 and hVEGF165 gene was successfully constructed and its high titer was obtained. |
| KEY WORDS Bone morphogenetic protein Vascular endotheliel growth factor Adenovirus vector Bone marrow mesenchymal stem cells |
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| 引用本文，请按以下格式著录参考文献： |
| 中文格式: | 李千会,孟纯阳,麻凤玉,张聪.共表达腺病毒载体构建鉴定及其转染骨髓间充质干细胞观察[J].中国骨伤,2021,34(7):674~679 |
| 英文格式: | LI Qian-hui,MENG Chun-yang,MA Feng-yu,ZHANG Cong.Construction and identification of recombinant adenovirus vector and observation of its transfecting rabbit bone marrow mesenchymal stem cells[J].zhongguo gu shang / China J Orthop Trauma ,2021,34(7):674~679 |
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