摘要
目的构建抑制自噬基因Beclin 1 shRNA慢病毒载体,建立Beclin 1基因稳定干扰的N2a细胞株,探讨抑制Beclin 1对N2a细胞增殖的影响。方法根据小鼠Beclin 1基因mRNA序列,设计4个Beclin 1靶点并构建shRNA慢病毒质粒。构建好的Beclin 1 shRNA质粒与包装质粒psPAX2及包膜质粒pMD2.G共转染293FT细胞后得到病毒颗粒,将产生的病毒颗粒感染N2a细胞。通过荧光定量PCR和Western blot检测感染的N2a细胞中自噬基因Beclin 1 mRNA及蛋白表达水平,比较shRNA的干扰效率。利用CCK8法检测干扰Beclin 1表达对N2a细胞增殖的影响。结果成功构建了表达Beclin 1 shRNA的慢病毒载体。在稳定感染Beclin 1 shRNA慢病毒载体的N2a细胞中,Beclin 1表达显著降低。抑制Beclin 1表达有促进N2a细胞增殖的趋势,但差异无统计学意义。结论成功构建了自噬基因Beclin 1 shRNA慢病毒表达载体,可显著抑制N2a细胞中Beclin 1表达。抑制Beclin 1表达有可能促进N2a细胞的增殖。
Aim To construct shRNA lentiviral vectors targeting Beclin 1 and establish stable Beclin 1 knockdown N2 a cell lines, and then evaluate its effect on N2 a cell proliferation. Methods Four target regions of Beclin 1 were designed and short hairpin RNA（shRNA） lentiviral plasmids were constructed according to the mRNA sequence of mouse Beclin 1 gene. 293 FT cells were transfected with Beclin 1 shRNA plasmid, psPAX2 packaging plasmid and pMD2.G envelope plasmid to obtain lentiviral particles. Then N2 a cells were infected with lentiviral particles. The expression level of Beclin 1 in infected cells was detected by real time PCR and Western blot, and the silencing efficiency of shRNA were compared with the control. Cholecystokinin octapeptide（CCK8） assay was used to determine the effect of Beclin 1 inhibition on N2 a cell proliferation. Results Lentiviral vectors encoding Beclin 1 shRNAs have beensuccessfully constructed. The expression of Beclin 1 was significantly reduced in N2 a cells that were stably transfected with shRNA lentiviral vectors targeting Beclin 1. The inhibition of Beclin 1 could promote N2 a cell proliferation but it did not attain statistical significance. Conclusion Four shRNA expression plasmids have been successfully constructed, which could effectively inhibit the expression of mouse Beclin 1. And the inhibition of Beclin 1 might promote N2 a cell proliferation.
出处
《中国临床神经科学》
2014年第5期489-495,共7页
Chinese Journal of Clinical Neurosciences
基金
国家自然科学基金资助项目(编号:31171014和编号:31371065)
上海市卫生局科研课题基金资助项目(编号:20134125)
上海市浦东新区卫计委重点专科建设基金资助项目(编号:PDZz2013-10)
