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| 铁过载对斑马鱼的成骨影响的机制 |
| The effect and mechanism of iron overload on zebra fish osteogenesis |
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| DOI：10.3969/j.issn.1006-7108.2016.06.012 |
| 中文关键词: 高通量转录组测序 成骨基因 转基因鱼 BMP2通道 |
| 英文关键词:High-throughput transcriptome sequencing Osteogenesis genes Transgenic fish BMP2 channel |
| 基金项目: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） |
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| 中文摘要: |
| 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cDNA和Runx2a带标签的cDNA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。 |
| 英文摘要: |
| Objective Using high-throughput transcriptome sequencing method to compared the effect of iron overload on zebra fish RNA, and to confirm the effect of iron overload on osteogenesis channel through a series of in vivo and in vitro experiments. Methods The zebra fish was overloaded in high-iron environment. RNA of the overloaded and wild type zebra fish was extracted after 4 days. The extracted RNA was compared using high-throughput transcriptome sequencing. BMP2 signal channel was screened and confirmed with quantitative QPCR. Runx2a transgenic zebra fish was fed in high-iron environment and normal environment, respectively for 9 days. The change of fluorescence was observed. Bmp2a-labeled cDNA and Runx2a-labeled cDNA were transfected to cells. Luciferase report gene experiments were conducted, and the change after iron intervention was observed. Results Using high-throughput transcriptome sequencing method, we found that Bmp2 was suppressed by high iron, and the suppression of Runx2a, Runx2b, and sp7 gene were down-regulated after iron overloading. Runx2a was down-regulated in Runx2a transgenic zebra fish in high-iron environment, compared to those in normal environment. Luciferase reporter gene of Runx2a increased in Bmp2a-transfected zebra fish, but declined after iron intervention. Conclusion Through high-throughput transcriptome sequencing, we find that iron overload inhibits BMP2. Using a series in vivo and in vitro experiments we confirm that iron overload has inhibitory effect on Bmp2, resulting in bone metabolic abnormalities. |
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