本文選題：高通量轉(zhuǎn)錄組測序　切入點：成骨基因　出處：《中國骨質(zhì)疏松雜志》2016年06期

【摘要】：目的通過高通量轉(zhuǎn)錄組的方法測序高鐵環(huán)境生存的斑馬魚與和野生型的斑馬魚,通過比較篩選出鐵過載對于成骨影響的通道,通過在體以及離體的一系列實驗證實鐵過載對這些通道的作用。方法通過在斑馬魚高鐵環(huán)境下生存,造成斑馬魚鐵過載,在4天后提取其RNA和野生型斑馬魚提取的RNA進行高通量轉(zhuǎn)錄組測序比較,篩選出成骨Bmp2通道,并用定量QPCR驗證。在體實驗中將Runx2a標記的轉(zhuǎn)基因斑馬魚在高鐵環(huán)境下與正常環(huán)境在第九天進行比較,觀察其熒光的變化;離體實驗中,用Bmp2a帶標簽的c DNA和Runx2a帶標簽的c DNA的細胞進行轉(zhuǎn)染,進行熒光素酶報告基因?qū)嶒?然后用鐵干預,觀察其變化。結(jié)果通過高通量轉(zhuǎn)錄組測序發(fā)現(xiàn)高鐵對于Bmp2抑制,并用定量驗證發(fā)現(xiàn)鐵過載后Runx2a,Runx2b,sp7等成骨基因均下調(diào),觀察發(fā)現(xiàn)Runx2a標記的轉(zhuǎn)基因斑馬魚高鐵環(huán)境下與正常環(huán)境相比基因表達明顯下調(diào),在細胞轉(zhuǎn)染實驗中,熒光素酶報告基因Runx2a在Bmp2a轉(zhuǎn)染下大幅升高,轉(zhuǎn)染鐵后Runx2a下降。結(jié)論通過高通量轉(zhuǎn)錄組測序,我們發(fā)現(xiàn)鐵過載對于BMP2的抑制影響,運用在體以及離體的一系列實驗證實鐵過載對Bmp2的抑制作用從而導致成骨代謝的異常。
[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.
【作者單位】： 南京醫(yī)科大學附屬無錫市第二人民醫(yī)院;美國俄勒岡州立大學;蘇州大學附屬第二醫(yī)院骨科;
【基金】：國家自然科學基金資助項目(81273090;81572179) 省重點專項(BL2014044) 蘇州市臨床重點專項(LCZX201305) 無錫市科技局項目(CSE31N1506) 江蘇省研究生培養(yǎng)創(chuàng)新工程(KYLX_1264)
【分類號】：R580

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