利用斑马鱼模型研究miR-191对髓系造血分化的调控

发布时间：2018-06-16 10:20

本文选题：斑马鱼模型 + miR-　；参考：《中国实验血液学杂志》2017年04期

【摘要】：目的:利用斑马鱼造血发育模型探索microRNA-191(miR-191)对髓系造血分化的调控。方法:将miR-191和对照(cel-miR-67)通过显微注射斑马鱼受精卵,收集发育到10 s、24 hpf、36 hpf和48 hpf的胚胎,分别提取对照及实验组胚胎的RNA,利用荧光定量qRT-PCR比较斑马鱼胚胎红系细胞分化标记基因(gata1,scl,hbbe3,lmo2)和粒-单系细胞分化标记基因(pu.1,L-plastin,mpx,cebpα)的表达水平;选取24 hpf胚胎进行上述基因的整胚原位杂交,比较注射miR-191对髓系分化标记基因的原位时空表达的影响。结果:通过分子克隆及T7 RNA聚合酶体外转录成功制备了斑马鱼红系、粒-单系细胞分化标记基因的RNA探针且特异性良好;与注射对照的受精卵相比较,荧光定量qRT-PCR检测发现,注射miR-191的24 hpf和36 hpf斑马鱼胚胎整体中髓系分化基因的水平均有微弱的上调趋势;而发育10 s的胚胎中髓系基因mpx的mRNA表达出现10-20倍的显著上调;整胚原位杂交结果发现24 hpf的胚胎中髓系特异基因L-plastin的原位表达有所上调。结论:本研究建立了斑马鱼受精卵显微注射技术及分析胚胎髓系分化基因表达的两种方法;miR-191对斑马鱼胚胎髓系分化调控功能的研究结果显示,该miRNA可能参与调控粒-单核系和红系分化,其对髓系基因mpx表达调控的分子机制及相应功能有待进一步的研究。
[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 伪) 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.
【作者单位】：实验血液学国家重点实验室中国医学科学院协和医学院血液学研究所血液病医院;医学分子生物学国家重点实验室中国医学科学院协和医学院基础医学研究所;
【分类号】：R-332;R55

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