低氧诱导因子1α慢病毒载体转染骨髓间充质干细胞的成骨基因表达

发布时间：2017-12-30 21:47

本文关键词：低氧诱导因子1α慢病毒载体转染骨髓间充质干细胞的成骨基因表达　出处：《中国组织工程研究》2014年01期 　论文类型：期刊论文

【摘要】：背景:成骨作用和血管发生在骨的形成和修复过程中紧密结合,而低氧诱导因子1α被认为是促血管新生相关基因调控中最重要的核心转录因子,其可促进缺氧部位血管的形成,进而促进骨的形成。目的:构建野生型和点突变型两个低氧诱导因子1α慢病毒真核表达载体Lenti-HIF1α-IRES-EGFP,并比较其对兔骨髓间充质干细胞成骨基因表达的影响。方法:首先根据野生型人源低氧诱导因子1α基因序列信息和确定酶切位点的点突变型序列信息构建低氧诱导因子1α基因野生型和点突变型两个慢病毒真核表达载体;然后采用制备的病毒液转染兔骨髓间充质干细胞。结果与结论:免疫荧光显微镜观察显示,转染7 d后野生型组和点突变型组细胞均未见明显荧光,转染14 d后两组细胞均呈现明显的绿色荧光;qPCR定量分析结果表明,转染7 d后即有低氧诱导因子1α和骨形态发生蛋白2基因的显著表达,转染14 d后,两基因仍然显示较高的表达水平。说明实验成功构建野生型和点突变型低氧诱导因子1α基因慢病毒真核表达载体,转染后可以促进兔骨髓间充质干细胞成骨基因的表达。
[Abstract]:Background: osteogenesis and angiogenesis are closely associated with bone formation and repair. Hypoxia inducible factor-1 伪 is considered to be the most important core transcription factor in the regulation of angiogenesis related genes. It can promote the formation of blood vessels at anoxic site. Objective: to construct the eukaryotic expression vector Lenti-HIF1 伪 -IRES-EGFP of wild type and point mutant type of hypoxia inducible factor 1 伪 lentivirus. To compare its effect on osteogenic gene expression of rabbit bone marrow mesenchymal stem cells. Firstly, two eukaryotic expression vectors of wild type human hypoxia inducible factor 1 伪 gene and point mutant gene were constructed based on the sequence information of wild type human hypoxia inducible factor 1 伪 gene and the point mutation sequence information of restriction enzyme digestion site. ; Rabbit bone marrow mesenchymal stem cells (BMSCs) were transfected with the prepared virus solution. Results and conclusion: immunofluorescence microscopy showed that there was no obvious fluorescence in both wild-type and point-mutant cells after 7 days of transfection. After 14 days of transfection, the cells in both groups showed obvious green fluorescence. The results of qPCR quantitative analysis showed that hypoxia inducible factor 1 伪 and bone morphogenetic protein-2 gene were significantly expressed at 7 days after transfection and 14 days after transfection. The two genes still showed high expression level, which indicated that the eukaryotic expression vector of wild type and point mutant hypoxia inducible factor-1 伪 gene lentivirus was constructed successfully. Transfection can promote the expression of osteogenic gene in rabbit bone marrow mesenchymal stem cells.
【作者单位】：同济大学口腔医学院;同济大学附属东方医院口腔科;杭州市第一人民医院口腔科;
【基金】：国家自然科学基金重点项目(81070806) 浙江省自然科学基金(LQ12H14002) 上海市科委医学重点课题(10JC1413300) 杭州市医药卫生科技计划项目(2012A015)~~
【分类号】：R363
【正文快照】： 0引言Introduction充足的血供是维持细胞正常生命活动的前提,也是缺损区组织再生修复的首要条件。由于创伤、肿瘤等原因引起大面积颌骨缺损时,邻近区域由于骨膜和牙龈组织萎缩、血管网络受损甚至缺失而处于低氧状态,严重影响组织再生[1-3]。1992年Semenza等[4]首次发现低氧诱

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