Kapβ2介导的hnRNPA2/B1核转位与七氟烷脑神经毒性的关系:细胞实验  

Relationship between Kapβ2-mediated nuclear translocation of hnRNPA2/B1 and sevoflurane-induced brain neurotoxicity:a cellular experiment

作　　者：嘉斐宇 汪强 杨陈祎 王晓晴 杨卓[4] 王海云[1,2,3] Jia Feiyu;Wang Qiang;Yang Chenyi;Wang Xiaoqing;Yang Zhuo;Wang Haiyun(The Third Central Clinical College of Tianjin Medical University,Tianjin 300041,China;Tianjin Key Laboratory of Extracorporeal Life Support for Critical Diseases Artificial Cell Engineering Technology Research Center Tianjin Institute of Hepatobiliary Disease,Tianjin 300171,China;Department of Anesthesiology,Tianjin Third Central Hospital,Tianjin 300171,China;College of Medicine,Nankai University,Tianjin 300110,China)

机构地区：[1]天津医科大学三中心临床学院,天津300041 [2]天津市重症疾病体外生命支持重点实验室,天津市人工细胞工程技术研究中心,天津市肝胆疾病研究所,天津300171 [3]天津市第三中心医院麻醉科,天津300171 [4]南开大学医学院,天津300110

出　　处：《中华麻醉学杂志》2023年第3期297-301,共5页Chinese Journal of Anesthesiology

基　　金：国家自然科学基金(82071220);天津市自然科学基金(20JCYBJC01290);天津市卫生健康委员会科技基金(MS20013);天津市医学重点学科(专科)建设项目资助(TJYXZDXK-072C)。

摘　　要：目的采用细胞实验评价核转运蛋白β2(Kapβ2)介导的核不均一性核糖核蛋白A2/B1(hnRNPA2/B1)核转位与七氟烷脑神经毒性的关系。方法采用小鼠海马细胞系HT_(2)2细胞,以2×10^(5/)孔和1×10^(6)/孔的密度接种于共聚焦培养皿和六孔培养板,采用随机数字表法分为4组(n=12):携带绿色荧光蛋白(GFP)空载腺病毒转染的对照组(GFP-C组)、携带GFP空载腺病毒转染的七氟烷组(GFP-Sev组)、Kapβ2基因过表达腺病毒转染的对照组(Kapβ2-C组)和Kapβ2基因过表达腺病毒转染的七氟烷组(Kapβ2-Sev组)。细胞常规培养48 h后,转染携带GFP的空载腺病毒(GFP-C组和GFP-Sev组)和Kapβ2基因过表达的腺病毒(Kapβ2-C组和Kapβ2-Sev组)。转染48 h后GFP-C组和Kapβ2-C组继续常规培养48 h;GFP-Sev组和Kapβ2-Sev组采用3%七氟烷孵育3 h,随后常规培养48 h。采用Western blot法测定细胞Kapβ2、突触素(SYP)、突触后密度蛋白95(PSD95)、hnRNPA2/B1表达,并计算hnRNPA2/B1核质比。采用免疫荧光实验进行hnRNPA2/B1亚细胞定位。结果与GFP-C组比较,GFP-Sev组SYP、PSD95表达下调,hnRNPA2/B1核质比降低,细胞质hnRNPA2/B1表达上调,Kapβ2-C组Kapβ2表达上调(P<0.05);与Kapβ2-C组比较,Kapβ2-Sev组SYP、PSD95表达下调,hnRNPA2/B1核质比降低,细胞质hnRNPA2/B1表达上调(P<0.05);与GFP-Sev组比较,Kapβ2-Sev组Kapβ2、SYP、PSD95表达上调,hnRNPA2/B1核质比升高,细胞质hnRNPA2/B1表达下调(P<0.05)。结论Kapβ2介导的hnRNPA2/B1核转位可能是七氟烷脑神经毒性的内源性保护机制。Objective To evaluate the relationship between Karyopherinβ2(Kapβ2)-mediated nuclear translocation of nuclear inhomogeneous ribonucleoprotein A2/B1(hnRNPA2/B1)and sevoflurane-induced brain neurotoxicity in a cellular experiment.Methods The mouse hippocampal neuronal cell line HT22 cells were inoculated in confocal culture dishes and 6-well culture plates at a density of 2×10^(5) cells/well and 1×10^(6) cells/well and divided into 4 groups(n=12 each)by a random number table method:control group(GFP-C group)carrying green fluorescent protein(GFP)with empty adenovirus transfection,sevoflurane group(GFP-Sev group)carrying GFP with empty adenovirus transfection,control group(GFP-Sev group)transfected with Kapβ2 gene-overexpressing adenovirus,and sevoflurane group(Kapβ2-Sev group)transfected with Kapβ2 gene-overexpressing adenovirus.After 48 h of conventional incubation,empty adenovirus-carrying GFP(GFP-C and GFP-Sev groups)and Kapβ2 gene-overexpressing adenovirus(Kapβ2-C and Kapβ2-Sev groups)were transfected.After 48 h of transfection,the cells were conventionally incubated continuously in GFP-C and Kapβ2-C groups,and the cells were incubated for 3 h with 3%sevoflurane and then were conventionally incubated for 48 h in GFP-Sev and Kapβ2-Sev groups.The expression of Kapβ2,synaptophysin(SYP),postsynaptic density protein 95(PSD95)and hnRNPA2/B1 nucleoplasmic ratio were measured in cells by Western blot.Immunofluorescence assay was used for hnRNPA2/B1 subcellular localization.Results Compared with GFP-C group,the expression of SYP and PSD95 was significantly down-regulated,hnRNPA2/B1 nucleoplasmic ratio was decreased,and cytoplasmic hnRNPA2/B1 expression was up-regulated in GFP-Sev group,and Kapβ2 expression was significantly up-regulated in Kapβ2-C group(P<0.05).Compared with Kapβ2-C group,the expression of SYP and PSD95 was significantly down-regulated,hnRNPA2/B1 nucleoplasmic ratio was decreased,and cytoplasmic hnRNPA2/B1 expression was up-regulated in Kapβ2-Sev group(P<0.05).Compared with GFP-Sev grou

关 键 词：七氟醚 β核胞浆转运蛋白类 核不均一核糖核蛋白A-B组 神经元 

分 类 号：R614[医药卫生—麻醉学]