腺相关病毒AAV2/Anc80L65对原代培养的螺旋神经元形态标记效能的探究  

Effects of a neuron specific AAV2 viral vector on the imaging efficiency of primary cultured spiral ganglion neurons in vitro

作　　者：刘耀谦 张振[1,2,3] 张博雅 陈正侬 LIU Yaoqian;ZHANG Zhen;ZHANG Boya;CHEN Zhengnong(Otolaryngology Research Institute,Shanghai Jiaotong University School of Medicine,Shanghai 200233,China;Department of Otorhinolaryngology,Affiliated Sixth People's Hospital,Shanghai Jiaotong University School of Medicine,Shanghai 200233,China;Shanghai Key Laboratory of Sleep Disordered Breathing,Shanghai 200233,China)

机构地区：[1]上海交通大学医学院耳鼻咽喉及头颈外科研究所,上海200233 [2]上海交通大学医学院附属上海市第六人民医院耳鼻咽喉科,上海200233 [3]上海市睡眠呼吸障碍重点实验室,上海200233

出　　处：《山东大学耳鼻喉眼学报》2023年第3期1-6,20,共7页Journal of Otolaryngology and Ophthalmology of Shandong University

基　　金：国家自然科学基金面上项目(82071040)。

摘　　要：目的探究含有神经元特异性启动子(human synapsin promoter,hSyn)及绿色荧光蛋白(enhanced green fluorescent protein,eGFP)标记的AAV2/Anc80L65-hSyn-eGFP病毒对体外培养的螺旋神经元(spiral ganglion neuron,SGN)形态的标记效果。方法向体外培养的SGNs中加入一定量的AAV2/Anc80L65-hSyn-eGFP病毒,通过荧光显微镜在低倍镜下计数荧光神经元的比例以评估其对螺旋神经元细胞的转染的特异性和效率,在高倍镜下观察荧光神经元胞体及神经突的精细形态。并通过膜片钳实验验证该病毒在活体SGNs中的实际应用。结果AAV2/Anc80L65-hSyn-eGFP病毒对体外培养的SGNs具有很高的特异性和转染率,且不会产生明显的细胞毒性。同时,该病毒可以快速清晰地标记螺旋神经元胞体和神经突的形态且对神经元形态没有明显损伤。相比Tuj1荧光染色还可以标记更加精细的神经突结构,如丝状伪足。当应用于膜片钳实验中时,AAV2/Anc80L65-hSyn-eGFP病毒有助于神经元识别和定位,可极大提高SGNs膜片钳实验的效率和成功率。结论AAV2/Anc80L65-hSyn-eGFP适合作为标记体外培养的活体SGNs形态的理想工具。Objective To explore the effects of AAV2/Anc80L65-hSyn-eGFP virus containing a neuron-specific human synapsin promoter(hSyn)and enhanced green fluorescent protein(eGFP)on the imaging of spiral ganglion neuron(SGN)morphologies in vitro.Methods Dissociated SGNs were transfected with AAV2/Anc80L65-hSyn-eGFP in vitro.Using fluorescence microscopy,the transfection specificity and efficiency of the virus were evaluated by calculating the proportion of fluorescent neurons as observed under low magnification.Elaborate morphologies of single neuron were then observed under high magnification.The imaging application of AAV2/Anc80L65-hSyn-eGFP on living SGNs was further verified by patch clamp studies.Results The AAV2/Anc80L65-hSyn-eGFP virus had both high transfection specificity and efficiency for SGNs in vitro,and did not produce obvious cytotoxicity.Importantly,the virus could reveal the morphology of somas and neurites both rapidly and clearly without causing damage to their structures.Compared with neuron-specific Tuj1 fluorescent staining,AAV2/Anc80L65-hSyn-eGFP was able to mark delicate neurite structures such as filopodia.When utilized for the identification and localization of SGNs in patch clamp studies,AAV2/Anc80L65-hSyn-eGFP significantly promoted the efficiency and accuracy of the experiments.Conclusion AAV2/Anc80L65-hSyn-eGFP is a competent and ideal tool for highlighting the morphology of living SGNs in vitro.

关 键 词：AAV2/Anc80L65-hSyn-eGFP病毒 螺旋神经元形态 原代培养 标记效果 

分 类 号：R764.431[医药卫生—耳鼻咽喉科]