文章摘要
廖军义,杜婷婷,黄 伟,何通川,徐 伟.重组腺病毒过表达Notch1受体胞内结合域(NICD1)外源性激活Notch信号通路[J].南京医科大学学报,2019,(3):320~325
重组腺病毒过表达Notch1受体胞内结合域(NICD1)外源性激活Notch信号通路
Recombinant adenovirus mediated overexpression of Notch1 intracellular domain(NICD1)stimulates the activation of Notch signaling pathway
投稿时间:2018-05-11  
DOI:10.7655/NYDXBNS20190302
中文关键词: Notch1受体胞内结合域  重组腺病毒  Notch信号通路
英文关键词: Notch1 intracellular domain  recombinant adenovirus  Notch signaling pathway
基金项目:国家自然科学基金面上项目(81371972,81572142);重庆市科委基础研究与前沿探索项目(cstc2018jcyjAX0088);重庆市研究生科研创新项目(CYB15098);重庆医科大学附属第一医院院内培育基金资助项目(2018PYJJ?11)
作者单位
廖军义 重庆医科大学附属第一医院骨科重庆 400016 
杜婷婷 重庆市第七人民医院内分泌与肾病科重庆 400054 
黄 伟 重庆医科大学附属第一医院骨科重庆 400016 
何通川 芝加哥大学医学中心分子肿瘤实验室伊利诺伊州 芝加哥 60637美国 
徐 伟 重庆医科大学附属第一医院骨科重庆 400016 
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中文摘要:
      目的:构建Notch1受体胞内结合域(Notch1 intracellular domain,NICD1)的重组腺病毒AdNICD1,探讨外源性过表达AdNICD1对Notch信号通路激活的影响。方法:利用高保真PCR(high fidelity PCR,Hi?Fi PCR)扩增NICD1编码区,并通过Gibson Assembly方法将其克隆至腺病毒载体中,经过菌落PCR、测序鉴定后,在大肠杆菌BJ5183中进行同源重组,再次通过质粒PCR鉴定,然后运用PacⅠ酶切线性化后用重组腺病毒载体转染过表达pTP基因的人胚肾293细胞(human embryo kidney 293 cell line,HEK293)中进行包装,收取病毒后使用过表达pTP基因的HEK293细胞扩增重组腺病毒AdNICD1。运用AdNICD1感染脂肪来源的间充质干细胞(immortalized multipotent adipose?derived mesenchymal stem cells,iMAD),检测病毒感染效率;通过Q?PCR检测NICD1和Notch下游基因表达情况;利用Western blot检测NICD1在蛋白水平的表达情况。结果:成功构建过表达NICD1的重组腺病毒并有效感染iMAD;利用AdNICD1可明显增加NICD1在基因和蛋白水平的表达,并激活Notch信号下游基因的表达。结论:成功构建过表达NICD1的重组腺病毒,并有效激活Notch信号通路。
英文摘要:
      Objective:To construct and identify the recombinant adenovirus AdNICD1 carrying Notch1 intracellular domain(NICD1)and observe the influence of the overexpression of AdNICD1 on the activation of Notch signaling pathway. Methods:NICD1 coding area was amplified by high fidelity PCR(Hi?Fi PCR)and then subcloned to recombinant adenovirus vector by Gibson assembly. Bacteria screening and gene sequencing were used to make sure NICD1 was subcloned to recombinant adenovirus vector accurately. And then the plasmid was transfected into BJ5183 to complete homogenous recombination and generate recombinant adenovirus plasmid pAdNICD1. pAdNICD1 was identified by plasmid screening and linearized by PacⅠ enzyme,and then transfected to human embryo kidney 293 cell line(HEK293)for packaging. The packaged AdNICD1 were collected and amplified stepwisely. Immortalized multipotent adipose?derived mesenchymal stem cells(iMAD)were infected by AdNICD1,and the infection efficiency was tested. QPCR and Western blot were used to detect the expression of NICD1,and the downstream gene expression of Notch signaling pathway was determinated by QPCR. Results:NICD1 was successfully subcloned to the recombinant adenovirus vector,and AdNICD1 was successfully packaged and amplified in HEK293 cell line. The overexpression of NICD1 was confirmed by QPCR and Western blot in MSCs. Notch signaling pathway downstream gene expressions were also upregulated by AdNICD1. Conclusion:The recombinant adenovirus AdNICD1 was successfully constructed,and overexpression of NICD1 can exogenously activate Notch signaling pathway.
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