Dicer及其miRNAs是血管平滑肌细胞分化和增殖所必需的基因和调节因子
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河南科技学院,河南科技学院,河南科技学院,河南科技学院,田纳西大学,生理学系

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国家自然科学基金资助项目(31372407)


Dicer and Its miRNAs are Necessary Gene and Regulatory Factors for Differentiation and Proliferation of Vascular Smooth Muscle Cell
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Henan Institute fo Science and Technology,Henan Institute fo Science and Technology,Henan Institute fo Science and Technology,Henan Institute fo Science and Technology,Department of Physiology, University of Tennessee Health Science Center

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This work was supported by a grant from The National Natural Science Foundation of China (31372407)

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    摘要:

    为了研究Dicer及其所产生的miRNAs在血管平滑肌细胞 (VSMC)中的作用,本研究采用条件性基因打靶法敲除了小鼠VSMC中的Dicer外显子23,通过采用连续性剖检妊娠小鼠法、病理组织学、免疫荧光、PCR、Western blot和实时PCR等技术对条件性敲除Dicer(Dicer cKO)胚胎的血管病变和VSMC中的Dicer、miRNAs和信号转导通路蛋白变化进行了详细研究.结果发现,在培育条件性敲除Dicer小鼠的过程可产生三种不同基因型小鼠,即野生型、杂合型和纯合型(Dicer cKO)小鼠.其中野生型和杂合型小鼠出生后无明显临床异常,而Dicer cKO小鼠却死于腹中而不能出生.Dicer cKO胚胎在胚胎发育的第12.5天(E12.5)就出现发育迟滞变化,在E14.5,皮肤、骨骼肌和肝脏的血管极度扩张、血液淤滞和广泛的弥漫性出血,在E15.5死亡.Dicer cKO胚胎血管壁的病变于E13.5即出现,主要表现为血管中膜的VSMC排列不整,增生减少;E14.5血管壁变薄、塌陷,管腔不规则,细胞增生明显减少;E15.5血管壁的结构完全破坏,细胞增生停止,血管壁的屏障作用破坏,通透性增强,向外渗血.在胚胎发育的E14.5,VSMC标志性基因的表达明显下调,VSMC中大部分受检miRNAs的表达也明显降低,磷酸化的信号转导通路蛋白,即细胞外信号调节激酶和蛋白激酶明显衰减.研究证明,Dicer是血管发育所必需的基因,它可通过控制miRNA产生和成熟来调节VSMC标志性基因的表达,借以促进VSMC的增殖与分化,保障血管壁结构的完整.

    Abstract:

    To research the role of the Dicer and miRNA in vascular smooth muscle cell (VSMC), the conditional gene targeting method, sequential biopsy pregnancy mice, histopathology, immunofluorescence, PCR, Western blot and Real time PCR were used to examine carefully the vasculopathy, the changes of Dicer, miRNAs and proteins of signal transduction pathway in conditional knock out the Dicer (Dicer cKO) embryos. Results indicated that three types of mice with different genotype that was wild type mice, heterozygotic type mice and homozygotic type mice were generated during breeding Dicer cKO mice. In them, wild type mice and heterozygotic mice were able to born, and no evident clinical abnormal showed when they were born, but homozygotic mice was not able to born and died in mother's abdomen. The development delay of Dicer cKO embryos was found in embryonic growth 12.5days (E12.5), the obvious vessels expansion, blood stasis and far-ranging diffuse hemorrhage in E14.5 and death in E15.5. The lesions of Dicer cKO embryos' vessel walls appeared as early as at E13.5, mainly showed irregular arrangement and lower proliferative cells; the pathologic changes of vessel walls thinning, collapse and irregular lumen appeared at E14.5. All destroyed structure of vessel wall, few cell proliferation, damaged barrier function of vessel wall, permeability enhancement and exudation of red blood cell occured at E15.5. The expression of VSMC marker genes, most detected miRNAs and phosphorylated signal transduction pathway proteins that were extracellular signal-regulated kinase and protein kinase showed distinct down regulation at E14.5. This experiment proves that the Dicer is necessary gene for vessel development. The Dicer regulates the expression of VSMC marker genes through commanding miRNA generation and maturation, so as to promote proliferation and differentiation of VSMC and guarantee the integrity of vessel wall structure.

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潘耀谦,潘博,刘兴友,李瑞珍,岳军明. Dicer及其miRNAs是血管平滑肌细胞分化和增殖所必需的基因和调节因子[J].生物化学与生物物理进展,2014,41(12):1255-1264

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历史
  • 收稿日期:2014-04-24
  • 最后修改日期:2014-07-21
  • 接受日期:2014-08-11
  • 在线发布日期: 2014-12-19
  • 出版日期: 2014-12-20