铁过载对斑马鱼成骨影响的机制

发布时间：2018-04-05 10:51

本文选题：高通量转录组测序　切入点：成骨基因　出处：《中国骨质疏松杂志》2016年06期

【摘要】：目的通过高通量转录组的方法测序高铁环境生存的斑马鱼与和野生型的斑马鱼,通过比较筛选出铁过载对于成骨影响的通道,通过在体以及离体的一系列实验证实铁过载对这些通道的作用。方法通过在斑马鱼高铁环境下生存,造成斑马鱼铁过载,在4天后提取其RNA和野生型斑马鱼提取的RNA进行高通量转录组测序比较,筛选出成骨Bmp2通道,并用定量QPCR验证。在体实验中将Runx2a标记的转基因斑马鱼在高铁环境下与正常环境在第九天进行比较,观察其荧光的变化;离体实验中,用Bmp2a带标签的c DNA和Runx2a带标签的c DNA的细胞进行转染,进行荧光素酶报告基因实验,然后用铁干预,观察其变化。结果通过高通量转录组测序发现高铁对于Bmp2抑制,并用定量验证发现铁过载后Runx2a,Runx2b,sp7等成骨基因均下调,观察发现Runx2a标记的转基因斑马鱼高铁环境下与正常环境相比基因表达明显下调,在细胞转染实验中,荧光素酶报告基因Runx2a在Bmp2a转染下大幅升高,转染铁后Runx2a下降。结论通过高通量转录组测序,我们发现铁过载对于BMP2的抑制影响,运用在体以及离体的一系列实验证实铁过载对Bmp2的抑制作用从而导致成骨代谢的异常。
[Abstract]:Objective to sequence zebrafish, wild type zebrafish and wild type zebrafish by high-throughput transcriptome, and to screen the pathway of the effect of iron overload on osteogenesis.The effect of iron overload on these channels was confirmed by a series of experiments in vivo and in vitro.Methods the zebrafish iron overload was caused by the survival of zebrafish in high-speed iron environment. The RNA extracted from zebrafish and the RNA extracted from wild zebrafish were sequenced by high-throughput transcriptome after 4 days. Osteogenic Bmp2 channels were screened and verified by quantitative QPCR.In vivo, Runx2a labeled transgenic zebrafish were compared with those in normal environment on day 9, and in vitro, c DNA labeled with Bmp2a and c DNA labeled with Runx2a were transfected in vitro.Luciferase reporter gene experiment was carried out, and then iron intervention was used to observe the changes of luciferase report gene.Results High throughput transcriptome sequencing showed that high iron inhibited Bmp2. After iron overload, all osteogenic genes such as Runx2aHX x2bS7 were down-regulated.It was found that the gene expression of Runx2a labeled transgenic zebrafish was significantly down-regulated compared with the normal environment. In the cell transfection experiment, luciferase reporter gene Runx2a increased significantly under Bmp2a transfection, and Runx2a decreased after transfection of iron.Conclusion through high-throughput transcriptome sequencing, we found the effect of iron overload on the inhibition of BMP2. A series of experiments in vivo and in vitro confirmed that the inhibition of iron overload on Bmp2 resulted in abnormal metabolism of osteogenesis.
【作者单位】：南京医科大学附属无锡市第二人民医院;美国俄勒冈州立大学;苏州大学附属第二医院骨科;
【基金】：国家自然科学基金资助项目(81273090;81572179) 省重点专项(BL2014044) 苏州市临床重点专项(LCZX201305) 无锡市科技局项目(CSE31N1506) 江苏省研究生培养创新工程(KYLX_1264)
【分类号】：R580

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