CAJ | 학술논문

目的通过血清5型重组腺病毒和血清2型重组腺相关病毒感染大鼠内耳组织的比较研究,筛选出更适合内耳基因治疗的病毒载体.方法一定体积的血清5型重组腺病毒液和血清2型重组腺相关病毒液经圆窗膜注入鼓阶外淋巴液,通过荧光共聚焦显微镜、免疫组化、Western免疫印迹及听性脑干反应等方法对两种载体所携带报告基因的表达部位、表达时程以及两种病毒本身对内耳组织细胞生活状态的影响加以比较.结果血清5型重组腺病毒和血清2型重组腺相关病毒所携带的增强型绿色荧光蛋白报告基因均可在前庭膜、血管纹、基底膜、盖膜、螺旋神经节区及转染对侧耳表达.rAAV2携带的EGFP蛋白第14天表达开始明显增多,第60天仍可检测到高表达.rAd5携带的EGFP蛋白在耳蜗组织内的表达高峰维持在第1～21天,在30天时表达明显减弱.结论腺病毒的优势在于其基因表达的迅速和高效,而腺相关病毒载体以其长的表达时程,低组织细胞毒性在内耳基因转染中表现出独特的优势.
목적 통과혈청5형중조선병독화혈청2형중조선상관병독감염대서내이조직적비교연구,사선출경괄합내이기인치료적병독재체.방법 일정체적적혈청5형중조선병독액화혈청2형중조선상관병독액경원창막주입고계외림파액,통과형광공취초현미경、면역조화、Western면역인적급은성뇌간반응등방법 대량충재체소휴대보고기인적표체부위、표체시정이급량충병독본신대내이조직세포생활상태적영향가이비교.결과 혈청5형중조선병독화혈청2형중조선상관병독소휴대적증강형록색형광단백보고기인균가재전정막、혈관문、기저막、개막、라선신경절구급전염대측이표체.rAAV2휴대적EGFP단백제14천표체개시명현증다,제60천잉가검측도고표체.rAd5휴대적EGFP단백재이와조직내적표체고봉유지재제1～21천,재30천시표체명현감약.결론 선병독적우세재우기기인표체적신속화고효,이선상관병독재체이기장적표체시정,저조직세포독성재내이기인전염중표현출독특적우세.
Objective To determine which of the two,recombinant adeno- associated viral vector 2(rAAV2) and recombinant adenovirus vector 5(rAd5),is more suitable for gene transfer in rodent cochlea.Methods The rAAV2-EGFP and rAd5-EGFP particls were injected into the perilymph through round window membrane.The target tissue accessibility, time course of expression, tissue toxicity of gene transfer and effects on hearing were evaluated.Results The expression of EGFP was detected in spiral ligament,strial vascukarises, Reissner membrane,basilar membrane, spiral ganglion,and contralateral cochlea.EGFP expression in the rAAV2 lasted over 60 days, with peak expression between days 14 and 60. EGFP in the rAd5 was detected within 24 h of transfection, and peak expression was observed between days 1 to 21. EGFP activity decreased sharply on day 30 after transfection with rAd5, while high EGFP expression was observed 60 days after transfection with rAAV2.Conclusion AAV has significant advantages for long-term transgene expression and no ototoxicity in the cochlea compared to adenovirus vectors.