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neuronatin shRNA真核表达载体的构建及其生物学功能 被引量：1

Construction of shRNA Expression Vector Targeting Neuronatin and Research on Its Biologic Effect
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摘要 目的:获得能持续干扰neuronatin(nnat)基因表达的细胞,观察nnat基因沉默对神经细胞发育与分化的影响,为研究基因功能奠定基础。方法:构建含nnat基因短发夹RNA(shRNA)表达质粒,将质粒转染大鼠肾上腺嗜铬细胞瘤细胞PC12,RT-PCR方法筛选出最有效干扰质粒,稳定转染PC12细胞后观察细胞表型变化,免疫荧光检测nnat蛋白表达,NGF诱导观察nnat表达下调对细胞分化的影响。结果:成功构建并筛选出有效的靶向nnat基因的shRNA真核表达载体;载体稳定转染PC12细胞之后能特异性沉默nnat基因的表达,PC12细胞长出突起,向神经元方向分化,加入诱导因子NGF后能促进突起生长。结论:nnat可能是作为神经分化抑制因子在神经发育与成熟过程中发挥作用。 Aim ： To study the biology function of neuronatin （nnat） gene during the brain development by observing the effect to differentiation of cells, in which nnat mRNA was silenced. Methods ： 4 vectors expressing a short hairpin RNA to nnat were constructed. After the plasmids were transfected into PC12 cells, the expression of nnat were checked by RT-PCR and immunofluorescence. Cellular morphology was observed when nnat expression degraded and after treating with NGF. Results： The shRNA eukaryotic expression vector has been successfully established which can specific inhibit indicated that the expression level of nnat was the expression of nnat mRNA. The in vitro experiment down regulated in RT- PCR and immunofluorescence. Differentiation （neurite outgrowth） was observed after down-regulation of nnat expression and NGF accelerated the differentiating process. Conclusion ： The possible role of nnat is inhibiting the differentiation during mammalian brain development.
作者 余利红 余新炳 项鹏
机构地区 中山大学干细胞与组织工程研究中心 广州医学院生化教研室
出处 《中国生物工程杂志》 CAS CSCD 北大核心 2009年第2期97-102,共6页 China Biotechnology
关键词 neuronatin RNA干扰 SHRNA neuronatin RNA interference shRNA
分类号 Q523 [生物学—生物化学]
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参考文献18

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二级参考文献31

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共引文献1

同被引文献13

  • 1Joseph R, Don D, Tsang W. Moh'cular cloning of a now'l mRNA (neuronatin) that is highly expressed in neonatal mammalian brain [ J ]. Biochem Biopiys Rs Cammun, 1994, 201 : 1227 - 1234.
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  • 4Joseph R, Dou D, Tsang W. Neuronatin mRNA: allemativelv spliced forms of a novel brlin-speeific mammalian dewloprnental gene[J]. Brain Res, 1995,690:92-98.
  • 5Oyang El,, I)avidson BC, l,ee W, et td. Functional characterizatim of tilt" dendritically h,alized mRNA neuronalin in hiplocamtml neu- rons [ J ]. PI..S One, 2011,6 : e24879.
  • 6Dugu I,, Takahara M, Tsuji G, et tt/. Abundant expression of neur- onalin in normal eccrine, apoc'rinc and sebaceous glands and their neoplasms [ J ]. J Dermatol, 2010, 37:846 - 848.
  • 7Joe MK, Lee FIJ, Suh YH, eS a/. Crucial roles of ncuronatin in it> sulin secretion and high glucose-induced apoplosis in panerealic be- ta-cells [ J ]. Cell Signal, 2008, 20:907 - 915,.
  • 8Li X, Thomason PA, Withers DJ, et al. Bio-informatics analysis of a gene co-expression module in adipose tissue containing the diet- responsive gene Nnat [ J]. BMC Syst Biol, 2010, 4:175.
  • 9Siu IM, Bai R, Gallia GL, et al. Coexpression of neuronatin sphce forms promotes medulloblastoma growth [ J ]. Neuro Oncol. 2008, 10:716 -724.
  • 10Dugu L, Hayashida S, Nakahara T, et al. Aberrant expression of tenascin-c and neuronatin in malignant peripheral nerve sheath tumors [J]. Eur J Dermatol, 2010, 20:580-584.

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中国生物工程杂志
2009年 第2期

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