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目的构建针对大鼠neuritin基因的短发夹状小干扰RNA(shRNA)表达载体,并研究该载体对neuritin基因表达的沉默效应及雪旺细胞生存的影响。方法设计并合成neuritin靶向siRNA序列,连接pGCSIL-GFP慢病毒表达载体后,将neuritin基因干扰质粒和重组表达质粒共转染入293T细胞,包装成慢病毒,以此感染大鼠雪旺细胞,分为空白对照组(A组)、无意义干扰组(B组)和干扰组(C组)。RT-qPCR、Western blot及Annexin V/PI方法分别检测细胞neuritinmRNA、蛋白表达及细胞凋亡情况。结果与A、B组相比,C组雪旺细胞neuritin mRNA和蛋白表达水平降低,而细胞凋亡明显增加(P<0.01或P<0.05)。结论成功构建neuritin基因shRNA慢病毒表达载体。其转染的雪旺细胞neuritin基因表达受抑制,并导致细胞凋亡增加。
Objective To construct short hairpin RNA (shRNA) expression vector targeting rat neuritin gene and study the effect of this vector on the silencing effect of neuritin gene and the survival of Schwann cell. Methods The neuritin targeting siRNA sequence was designed and synthesized. After connecting with the lentiviral vector pGCSIL-GFP, the neuritin gene interference plasmid and the recombinant plasmid were co-transfected into 293T cells and packaged into lentivirus to infect rat Schwann cells. Divided into blank control group (A group), nonsense interference group (B group) and interference group (C group). RT-qPCR, Western blot and Annexin V / PI methods were used to detect the expression of neuritin mRNA, protein and cell apoptosis respectively. Results Compared with group A and B, the expression of neuritin mRNA and protein in Schwann cells was decreased in group C, and the apoptosis was significantly increased in group C (P <0.01 or P <0.05). Conclusion The neuritin gene shRNA lentiviral vector was successfully constructed. The transfected Schwann cell neuritin gene expression was inhibited and lead to increased apoptosis.