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目的:构建人特异性受体Notch1短发夹RNA重组腺病毒载体,探讨其对人脐带间充质干细胞中Notch1表达的影响,以便进一步研究Notch1的生物学功能。方法:设计合成Notch1序列干扰序列,插入到pGenesil-1.1上构建重组质粒,取阳性克隆进行酶切及DNA测序鉴定。通过同源重组,构建含目的基因的重组腺病毒质粒载体pGsadeno-Notch1-shRNA。经Pac I线性化后脂质体介导转染到HEK 293细胞,包装后得到腺病毒颗粒。产生的重组腺病毒体外转染人脐带间充质干细胞,RT-PCR和Western blot检测特异性shRNA对人脐带间充质干细胞中Notch1蛋白在mRNA及蛋白表达水平的抑制效果。结果:经酶切及DNA测序重组腺病毒质粒构建正确。重组腺病毒转染人脐带间充质干细胞3 d后,RT-PCR及Western blot检测,可显著下调细胞内Notch1的转录及蛋白表达水平。对Notch1 mRNA和蛋白表达的抑制率分别为70.31%和86.97%,具有统计学意义(P<0.05)。结论:成功构建了表达人Notch1受体shRNA的重组腺病毒载体,为研究Notch1在肿瘤学中的生物学作用及机制奠定基础。
OBJECTIVE: To construct a recombinant adenovirus vector containing human Notch1 short hairpin RNA to investigate its effect on Notch1 expression in human umbilical cord mesenchymal stem cells, in order to further study the biological function of Notch1. Methods: The Notch1 sequence was designed and synthesized. The recombinant plasmids were inserted into pGenesil-1.1. The positive clones were digested with restriction endonucleases and identified by DNA sequencing. Through homologous recombination, a recombinant adenovirus plasmid vector pGsadeno-Notch1-shRNA containing the gene of interest was constructed. After Pac I linearization, lipofectamine was transfected into HEK 293 cells and packaged to obtain adenovirus particles. The recombinant adenovirus was transfected into human umbilical cord mesenchymal stem cells in vitro. The inhibitory effect of specific shRNA on mRNA and protein expression of Notch1 in human umbilical cord mesenchymal stem cells was detected by RT-PCR and Western blot. Results: Recombinant adenovirus plasmid was constructed correctly by restriction enzyme digestion and DNA sequencing. The recombinant adenovirus transfected human umbilical cord mesenchymal stem cells 3 d, RT-PCR and Western blot detection, can significantly downregulate Notch1 intracellular transcription and protein expression levels. The inhibitory rates of Notch1 mRNA and protein expression were 70.31% and 86.97%, respectively, with statistical significance (P <0.05). Conclusion: The recombinant adenovirus vector expressing shRNA of Notch1 receptor was successfully constructed, which laid the foundation for studying the biological role of Notch1 in oncology.