本文選題：斑馬魚模型 + miR-�。� 參考：《中國實驗血液學(xué)雜志》2017年04期

【摘要】：目的:利用斑馬魚造血發(fā)育模型探索microRNA-191(miR-191)對髓系造血分化的調(diào)控。方法:將miR-191和對照(cel-miR-67)通過顯微注射斑馬魚受精卵,收集發(fā)育到10 s、24 hpf、36 hpf和48 hpf的胚胎,分別提取對照及實驗組胚胎的RNA,利用熒光定量qRT-PCR比較斑馬魚胚胎紅系細胞分化標記基因(gata1,scl,hbbe3,lmo2)和粒-單系細胞分化標記基因(pu.1,L-plastin,mpx,cebpα)的表達水平;選取24 hpf胚胎進行上述基因的整胚原位雜交,比較注射miR-191對髓系分化標記基因的原位時空表達的影響。結(jié)果:通過分子克隆及T7 RNA聚合酶體外轉(zhuǎn)錄成功制備了斑馬魚紅系、粒-單系細胞分化標記基因的RNA探針且特異性良好;與注射對照的受精卵相比較,熒光定量qRT-PCR檢測發(fā)現(xiàn),注射miR-191的24 hpf和36 hpf斑馬魚胚胎整體中髓系分化基因的水平均有微弱的上調(diào)趨勢;而發(fā)育10 s的胚胎中髓系基因mpx的mRNA表達出現(xiàn)10-20倍的顯著上調(diào);整胚原位雜交結(jié)果發(fā)現(xiàn)24 hpf的胚胎中髓系特異基因L-plastin的原位表達有所上調(diào)。結(jié)論:本研究建立了斑馬魚受精卵顯微注射技術(shù)及分析胚胎髓系分化基因表達的兩種方法;miR-191對斑馬魚胚胎髓系分化調(diào)控功能的研究結(jié)果顯示,該miRNA可能參與調(diào)控粒-單核系和紅系分化,其對髓系基因mpx表達調(diào)控的分子機制及相應(yīng)功能有待進一步的研究。
[Abstract]:Aim: to explore the regulation of hematopoietic differentiation in medullary system by microRNA-191 miR-191 using zebrafish hematopoietic development model. Methods: microinjected zebrafish fertilized eggs with miR-191 and control group were used to collect 36 hpf and 48 hpf embryos developed to 10 sg 24 h. RNA was extracted from the embryos of control group and experimental group, and the expression levels of red cell differentiation marker gene (gata1hbbe3hbbe3mmo2) and granulocyte differentiation marker gene pu.1( L-plastininmmpxancebp 偽) of zebrafish embryos were compared by using fluorescence quantitative RT-PCR. The effect of miR-191 injection on in situ expression of myeloid differentiation marker gene was compared by in situ hybridization of 24 hpf embryos. Results: zebrafish erythrocytes were successfully prepared by molecular cloning and T7 RNA polymerase transcription in vitro. The level of myeloid differentiation genes in 24 hpf and 36 hpf zebrafish embryos injected with miR-191 showed a slight up-regulation trend, while the expression of myeloid gene mpx in 10 s embryos showed a 10-20 fold up-regulation. The in situ expression of myeloid specific gene L-plastin was up-regulated in 24 hpf embryos. Conclusion: in this study, microinjection of zebrafish fertilized eggs and two methods for analyzing the gene expression of embryonic medullary differentiation were established. The results showed that miR-191 could regulate the differentiation of zebrafish embryo. These miRNAs may be involved in the regulation of granulocyte-monocyte and erythroid differentiation. The molecular mechanism and function of the mpx expression of myeloid genes need to be further studied.
【作者單位】： 實驗血液學(xué)國家重點實驗室中國醫(yī)學(xué)科學(xué)院協(xié)和醫(yī)學(xué)院血液學(xué)研究所血液病醫(yī)院;醫(yī)學(xué)分子生物學(xué)國家重點實驗室中國醫(yī)學(xué)科學(xué)院協(xié)和醫(yī)學(xué)院基礎(chǔ)醫(yī)學(xué)研究所;
【分類號】：R-332;R55

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