后半规管途径导入不同血清型腺相关病毒转染小鼠耳蜗内毛细胞的效果比较

目的 探讨不同血清型的腺相关病毒经后半规管途径导入成年小鼠转染内耳细胞的效率及安全性。 方法 将20只6~8周C57BL/6J小鼠采用数字表法随机分为5组:腺相关病毒(adeno-associated virus,AAV)4组,AAV8病毒注射组、AAV9病毒注射组、AAVie病毒注射组和Anc80L65病毒注射组,均采取后半规管路径进行注射,每只注射病毒溶液2 μL;正常对照组,每只注射0.9%(质量分数)氯化钠注射液2 μL,均以左耳为手术耳。各组于术前1 d及术后2周行听觉脑干反应(auditory brainstem response,ABR)检查,随后取材进行免疫荧光染色观察病毒在基底膜的分布情况。结果 AAV8组:术后2周观察见耳蜗顶中转34.6%±1.8%内毛细胞被转染,中底转39.0%±5.9%内毛细胞表达绿色荧光蛋白(green fluorescent protein,GFP);AAV9组:术后2周见耳蜗顶中转92.5%±1.1%内毛细胞被转染,中底转94.6%±1.0%内毛细胞表达GFP; AAVie组:术后2周见耳蜗顶中转96.7%±1.5%内毛细胞被转染,中底转97.7%±0.8%内毛细胞表达GFP; Anc80L65组:术后2周见耳蜗顶中转62.5%±3.3%内毛细胞转染,中底转63.1%±6.1%内毛细胞表达GFP;正常对照组未见GFP表达。 结论 经半规管途径注射AAVie、AAV9病毒溶液可以高效转染到耳蜗内毛细胞,此结果对耳聋疾病的内耳基因治疗具有一定的参考意义。

Comparison of transfection effects of different serotypes of adeno-associated virus into inner hair cells of mouse cochlea by canalostomy

Objective To investigate the efficiency and safety of adeno-associated virus (AAV) of different serotypes transfected into inner ear cells of adult mice through posterior semicircular canal pathway. Methods Twenty C57BL/6J mice aged 6-8 weeks were randomly divided into five groups: AAV8 virus injection group, AAV9 virus injection group, AAVie virus injection group and Anc80L65 virus injection group. They were injected through the latter half regulatory path, and each mouse was injected with 2 μL of virus solution.In the normal control group, each mouse was injected with 2 μL normal saline, and the left ear was used as the operating ear. Auditory brainstem response (ABR) was examined one day before operation and two weeks after operation in each group, and then samples were taken for immunofluorescence staining to observe the distribution of virus in corti. Results AAV8 group: at 2 weeks after operation, 34.6%±1.8% inner hair cells were transfected at the apical and middle turn of cochlea, and 39%±5.9% inner hair cells at the middle and basal turn of cochlea expressed green fluorescent protein (GFP). AAV9 group: 92.5%±1.1% inner hair cells were transfected at the apical and middle turn of cochlea and 94.6%±1.0% inner hair cells expressed GFP at the middle and basal turn of cochlea 2 weeks after operation.AAVie group: 96.7%±1.5% inner hair cells were transfected at the apical and middle turn of the cochlea and 97.7%±0.8% inner hair cells at the middle and basal turn of the cochlea expressed GFP at 2 weeks after operation.Anc80L65 group: at 2 weeks after operation,62.5%±3.3% inner hair cells were transfected at the apical and middle turn of the cochlea, and 63.1%±6.1% inner hair cells at the middle and basal turn of the cochlea expressed GFP.No GFP expression was found in the normal control group. Conclusion Injecting AAVie and AAV9 virus solution through semicircular canal can efficiently transfect into cochlear inner hair cells. This result has certain reference significance for inner ear gene therapy of deafness.