腺相关病毒Anc80L65转染成年小鼠椭圆囊的实验研究  

Gene Transfer of Adeno-Associated Virus Vector Anc80L65 into the Utricle of Adult Mice

作　　者：王国鹏[1] 贺璐[1] 郭婧滢[1] 陈钟壡 龚树生[1] WANG Guopeng;HE Lu;GUO Jingying;CHEN Zhongrui;GONG Shusheng(Department of Otorhinolaryngology Head and Neck Surgery,Beijing Friendship Hospital,Capital Medical University,Beijing 100050,China)

机构地区：[1]首都医科大学附属北京友谊医院耳鼻咽喉头颈外科,北京100050

出　　处：《中华耳科学杂志》2022年第1期21-25,共5页Chinese Journal of Otology

基　　金：国家自然科学基金(81771016);北京市自然科学基金(7212022)。

摘　　要：目的探索腺相关病毒载体Anc80L65对正常和不同程度损伤的成年小鼠椭圆囊前庭感觉上皮的转染特性。方法将6周龄小鼠分为以下三组。1)正常对照组:将携带绿色荧光蛋白(green fluorescent protein,GFP)的Anc80L65病毒液(Anc80L65-GFP),经后半规管注射途径导入小鼠内耳;2)中度损伤组:行水平半规管注射导入150μg链霉素,术后2周通过后半规管注射导入Anc80L65-GFP病毒液;3)重度损伤组:行水平半规管注射导入400μg链霉素,术后2周行后半规管注射导入Anc80L65-GFP病毒液。导入病毒后4周取椭圆囊行免疫荧光染色,观察椭圆囊的形态及GFP在椭圆囊感觉上皮的表达情况。结果正常对照组:Anc80L65-GFP病毒液注射后,正常椭圆囊毛细胞无明显缺失,纤毛形态正常,GFP广泛分布于整个感觉上皮区域。中度损伤组:椭圆囊前庭毛细胞数量明显减少,支持细胞仍存活,GFP广泛分布于整个感觉上皮区域。高倍镜下见前庭支持细胞和毛细胞被转染。重度损伤组:椭圆囊的前庭毛细胞和支持细胞均被损伤,前庭感觉上皮演化为细胞大小不一、形态不规则的扁平上皮结构。在前庭扁平上皮中,GFP阳性细胞数量与对照组及中度损伤组相比明显减少。结论Anc80L65能高效转染正常和中度损伤后的成年小鼠椭圆囊前庭感觉上皮,但对重度损伤后形成的前庭扁平上皮的转染效率较低,这可能与其细胞属性发生改变有关。在未来的前庭基因治疗研究中,须根据前庭感觉上皮的不同损伤程度来选择合适的载体。Objective We aimed to understand transfection characteristics of the adeno-associated virus vector Anc80L65 into the utricular vestibular sensory epithelium in adult mice with or without different degrees of injury.Methods Six-week-old mice were randomly selected into a normal control group to have Anc80L65 viral vector carrying green fluorescent protein(GFP,Anc80L65-GFP)injected into the inner ear via the posterior semicircular canal;moderate injury group to streptomycin(150μg)injected into the horizontal semicircular canal with the Anc80L65 virus solution injected into the posterior semicircular canal 2 weeks later;or a severe injury group to have the same treatment as in moderate injury group but with streptomycin at 400μg.At 4 weeks after virus introduction,utricles were harvested with immunofluorescence staining to examine the morphology of the utricle and distribution of GFP expression in the vestibular sensory epithelium.Results In the normal control group,,there was no evident loss of vestibular hair cells or supporting cells following injection of the Anc80L65-GFP virus solution,demonstrating normal cilia morphology and wide GFP expression throughout the sensory epithelium.In the moderate injury group,there was significant loss of vestibular hair cells in the utricle,with supporting cells surviving and GFP extensively expressed throughout the vestibular sensory epithelium,as well as effective transfection into both supporting and hair cells.In the severe injury group,both hair cells and supporting cells in the utricle were damaged,resulting in a flat epithelial structure showing variable cell sizes with irregular shapes and significantly reduced GFP-positive cells compared with the control and the moderate injury groups.Conclusion Anc80L65 can efficiently transfect both normal and moderately injured utricular vestibular sensory epithelium in adult mice,whereas the transfection efficiency in flat vestibular epithelium is relatively low.This may be caused by changes in cellular properties of the latter f

关 键 词：腺相关病毒 前庭 毛细胞 支持细胞 基因转染 

分 类 号：R764[医药卫生—耳鼻咽喉科]