杆状病毒介导内耳基因治疗的实验研究

Experimental study of baculovirus-mediated inner ear gene therapy

目的探讨重组杆状病毒作为内耳基因治疗载体的可行性及其转染特点。方法应用Bac-to-Bac杆状病毒表达系统制备重组杆状病毒Bac-GFP,建立新生大鼠耳蜗Corti器体外模型,加入携带报告基因的病毒悬液,观察外源基因表达情况,并将其通过圆窗直接显微注射进入豚鼠内耳,观察杆状病毒在体内的转染特点。结果Bac-GFP联合丁酸钠可以高效转染毛细胞和螺旋神经节细胞,但并没有改变Corti器的正常结构。体内试验显示杆状病毒可以忠实和长效转染螺旋神经节细胞,而未被补体系统灭活。结论实验证实杆状病毒有希望成为内耳基因治疗的新型载体。

Objective To study the feasibility and characteristics of recombinant baculovirus as the inner ear gene transfer vectors. Methods After the generation of recombinant baculovirus Bac-GFP according to Bac-to-Bac baculovirus expression system, the in vitro model of the organ of Corti was established and the cochlear explants were infected by Bac-GFP. The expression of exogenous gene was observed. Bac-GFP was directly injected into the inner ear of guinea pigs, and the in vivo transfection characteristics of baculovirus were investigated. Results Baculovirus was able to infect spiral ganglion neurons （SGNs） cultures and hair cells, and the expression level could be up-regulated by sodium butyrate. However, Bac-GFP and sodium butyrate didn＇t change the organized structure of the organ of Corti. Bac-GFP could transduce SGNs in vivo and was not inactivated by the complement system. Conclusion Baculovirus is a novel and promising tool for inner ear gene therapy.