基于三叉神经节神经元电生理特征的单细胞转录组测序技术方法构建  

Construction of single cell RNA sequencing technique based on electrophysiological characteristics of trigeminal ganglion neurons

作　　者：李腊梅 杨晶 刘苑莹 史云欣 胡文超 解柔刚 姜鸣 LI Lamei;YANG Jing;LIU Yuanying;SHI Yunxin;HU Wenchao;XIE Rougang;JIANG Ming(School of Life Sciences&Research Center for Resource Peptide Drugs,Shaanxi Engineering&Technological Research Center for Conversation&Utilization of Regional Biological Resources,Yan an University,Yan an 716099,China;Department of Neurobiology,Air Force Medical University,Xi'an 710032,China;Heart Hospital,Xi'an International Medical Center Hospital,Xi'an 710109,China;No.6 Cadet Regiment,School of Basic Medical Sciences,Air Force Medical University,Xi'an 710032,China)

机构地区：[1]延安大学生命科学学院多肽资源药物研究中心,陕西省区域生物资源保育与利用工程技术研究中心,陕西延安716099 [2]空军军医大学基础医学院神经生物学教研室,陕西西安710032 [3]西安国际医学中心心脏病医院,陕西西安710109 [4]空军军医大学基础医学院学员六大队,陕西西安710032

出　　处：《空军军医大学学报》2022年第8期939-945,951,共8页Journal of Air Force Medical University

基　　金：国家自然科学基金(82171212,81870867,81400949)。

摘　　要：目的建立小鼠三叉神经节(TRG)神经元电生理特征匹配单细胞转录组测序(scRNA-seq)技术方法。方法制备小鼠TRG标本,通过膜片钳技术检测并记录神经元电活动特征。记录完成后的神经元细胞,用玻璃微电极获取并收集,进行scRNA-seq分析。结果膜片钳记录到小鼠TRG神经元中73%的中大型神经元具有特征性放电模式,给予去极化的方波刺激只诱发产生单个动作电位,具有较高的基强度(800.00±129.10)pA,给予斜波刺激不能诱发动作电位。将这种放电特征的神经元细胞通过膜片钳记录电极获取并收集后进行单细胞转录测序,结果显示该方法获取的单细胞符合测序要求,进一步测序分析该类神经元的基因表达分布,发现与神经元兴奋性相关的Na^(+)和K^(+)通道相关基因表达比例无明显差异,其中Na^(+)通道中Na_(V)β1为主要类型。结论本研究成功建立了TRG神经元电活动检测匹配scRNA-seq技术方法,并为感觉神经元电活动检测匹配转录组学特征的研究提供了重要工具。Objective To establish a single cell RNA sequencing(scRNA-seq)technique for matching electrophysiological characteristics of trigeminal ganglion(TRG)neurons in mice.Methods TRG specimens of mice were prepared,and the electrical activity characteristics of neurons were detected and recorded by patch clamp technique.After recording,the neurons were obtained and collected with glass microelectrode and analyzed by scRNA-seq.Results Patch clamp recorded that 73%of the medium and large neurons in mouse TRG neurons had characteristic discharge patterns.Depolarized current steps wave stimulation only induced a single action potential with a high rheobase(800.00±129.10)pA.Current ramps stimulation could not induce action potential.The neurons with these discharge characteristics were obtained by patch clamp recording electrode and collected for scRNA-seq.The results showed that the single cell obtained by this method met the requirements of sequencing.Further sequencing analysis of gene expression distribution of these neurons showed that there was no significant difference in the proportion of Na^(+) and K^(+) channel related genes related to neuronal excitability,and Na_(V)β1 was the main type in Na^(+) channel.Conclusion The technique of matching scRNA-seq for electrical activity detection of TRG neurons established in this study is successful.The successful establishment of this method provides an important tool for the study of electrical activity detection and matching transcriptome characteristics of sensory neurons.

关 键 词：三叉神经节 神经元电活动 单细胞转录组测序 

分 类 号：R338[医药卫生—人体生理学]