小发夹RNA抑制Nogo基因表达促进脊髓损伤修复的实验研究

Repair effect of short hairpin RNA on spinal cord injury by inhibiting Nogo gene expression.

目的 利用RNA干涉（RNAi）技术使特定的基因（Nogo）沉默，探索脊髓损伤的治疗方法。方法 设计有小发夹结构的两条DNA序列，PCR扩增带有U6启动子的小发夹，腺病毒包装重组体，转染少突胶质细胞，采用Westernblot法分析Nogo-66的蛋白表达水平。结果成功地构建了靶向Nogo基因RNAi的带有U6启动子的小发夹重组体，Nogo-66蛋白表达水平明显下降。结论 靶向RNAi小发夹重组体经腺病毒包装后，成功转染少突胶质细胞并能有效抑制Nogo-66基因的表达。

Objective To explore the spinal cord injury repair strategies by silencing Nogo gene with RNA interference. Methods Two DNA sequences including short hairpin RNA,U6 promotor were designed and the fragment including Nogo-66 reverse complement of target sequence was obtained by PCR. U6 short hairpin Nogo-66 was constructed into adenovirus vector, and the recombinant adenovirus vectors were transfected into 293 cells. Oligodendroglial cells were infected by the recombinant adenovirus granules. Nogo-66 protein level was assayed by Western blot. Results Short hairpin of U6 promotor and the fragment including Nogo-66 reverse complement of target sequence were successfully constructed, and Nogo-66 protein relative level of oligodendroglial cells was markedly decreased. Conclusion Nogo-66 gene is silenced by short hairpin of U6 promotor and the fragment including reverse complement of target sequence.