过表达LncRNA DIO3OS通过结合PTBP1调控BDNF表达以缓解氯胺酮诱导的小鼠海马神经元细胞毒性  

LncRNA DIO3OS overexpression alleviates ketamine-induced neurotoxicity in mouse hippocampal neurons by regulating BDNF expression via binding to PTBP1

作　　者：张琳 刘跃丹 李伟 唐富东 Zhang Lin;Liu Yuedan;Li Wei;Tang Fudong(Department of Anesthesiology and Perioperative Medicine,He'nan Provincial People's Hospital,People's Hospital of Zhengzhou University,People's Hospital of Henan University,Zhengzhou 450003,China)

机构地区：[1]河南省人民医院,郑州大学人民医院,河南大学人民医院麻醉与围术期医学科,郑州450003

出　　处：《中华神经医学杂志》2023年第10期973-983,共11页Chinese Journal of Neuromedicine

基　　金：河南省医学科技攻关联合共建项目(LHGJ20220065)。

摘　　要：目的:探讨长链非编码RNA(lncRNA)DIO3OS对氯胺酮诱导的小鼠海马神经元细胞毒性的影响及其作用机制。方法:(1)分离并培养原代小鼠海马神经元,应用CCK-8法检测不同浓度(0、25、50、100、200μmol/L)氯胺酮对细胞活力的影响,qPCR法检测DIO3OS mRNA的表达。(2)将海马神经元分为对照组、氯胺酮组(50μmol/L氯胺酮培养24 h)、氯胺酮+pc组(转染pcDNA3.1质粒48 h后加50μmol/L氯胺酮培养24 h)、氯胺酮+DIO3OS组(转染pcDNA3.1-DIO3OS质粒48 h后加50μmol/L氯胺酮培养24 h),应用CCK-8法检测各组细胞活力,乳酸脱氢酶(LDH)细胞毒性检测试剂盒检测LDH释放量,流式细胞术检测细胞凋亡情况,qPCR法检测DIO3OS、脑源性神经营养因子(BDNF)mRNA的表达,Western blotting实验检测多聚嘧啶区结合蛋白1(PTBP1)、BDNF蛋白的表达。(3)提取正常海马神经元的总蛋白,应用RNA沉降实验检测DIO3OS mRNA及BDNF mRNA与PTBP1蛋白的结合能力。(4)将海马神经元分为氯胺酮+DIO3OS+si-NC组(同时转染pcDNA3.1-DIO3OS和si-NC质粒48 h后加50μmol/L氯胺酮培养24 h)、氯胺酮+DIO3OS+si-PTBP1组(同时转染pcDNA3.1-DIO3OS和si-PTBP1质粒48 h后加50μmol/L氯胺酮培养24 h),应用qPCR法检测DIO3OS、BDNF mRNA的表达,Western blotting实验检测PTBP1、BDNF蛋白的表达。(5)将海马神经元分为氯胺酮组(50μmol/L氯胺酮培养24 h)、氯胺酮+si-DIO3OS组(转染si-DIO3OS质粒48 h后加50μmol/L氯胺酮培养24 h)、氯胺酮+si-PTBP1组(转染si-PTBP1质粒48 h后加50μmol/L氯胺酮培养24 h)、氯胺酮+si-DIO3OS+si-PTBP1组(同时转染si-DIO3OS和si-PTBP1质粒48 h后加50μmol/L氯胺酮培养24 h),应用qPCR法检测DIO3OS、BDNF mRNA的表达,Western blotting实验检测BDNF蛋白的表达。(6)将海马神经元分为氯胺酮+DIO3OS+si-NC组(同时转染pcDNA3.1-DIO3OS和si-NC质粒48 h后加50μmol/L氯胺酮培养24 h)、氯胺酮+DIO3OS+si-BDNF组(同时转染pcDNA3.1-DIO3OS和si-BDNF质粒48 h后加50μmol/L氯胺酮培养24 h),应用Objective To investigate the influence of long non-coding RNA(lncRNA)DIO3OS in ketamine-induced neurotoxicity and its mechanism in mouse hippocampal neurons.Methods(1)Primary mouse hippocampal neurons were isolated and cultured;CCK-8 assay was used to detect the viability of cells treated with different concentrations of ketamine(0,25,50,100,200μmol/L),and qPCR was used to detect DIO3OS mRNA expression.(2)Hippocampal neurons were divided into 4 groups:control group,ketamine group(cultured with 50μmol/L ketamine for 24 h),ketamine+pc group(transfected with pcDNA3.1 plasmid for 48 h,and then cultured with 50μmol/L ketamine for 24 h),and ketamine+DIO3OS group(transfected with pcDNA3.1-DIO3OS plasmid for 48 h,and then cultured with 50μmol/L ketamine for 24 h);cell viability was detected by CCK-8 assay;lactic dehydrogenase(LDH)release was determined by LDH cytotoxicity assay kit;cell apoptosis was detected by flow cytometry;mRNA expressions of DIO3OS and brain-derived neurotrophic factor(BDNF)were examined by qPCR;protein expressions of polypyrimidine tract-binding protein 1(PTBP1)and BDNF were detected by Western blotting.(3)Total proteins of routinely cultured neurons were extracted,and RNA Pull-Down assay was used to detect whether DIO3OS mRNA and BDNF mRNA could directly bind to PTBP1 protein.(4)Hippocampal neurons were divided into ketamine+DIO3OS+si-NC group(co-transfected with pcDNA3.1-DIO3OS plasmid and si-NC plasmid for 48 h,and then cultured with 50μmol/L ketamine for 24 h)and ketamine+DIO3OS+si-PTBP1 group(co-transfected with pcDNA3.1-DIO3OS plasmid and si-PTBP1 plasmid for 48 h,and then cultured with 50μmol/L ketamine for 24 h);qPCR was used to examine the mRNA expressions of DIO3OS and BDNF;Western blotting was used to detect the protein levels of PTBP1 and BDNF.(5)Hippocampal neurons were divided into ketamine group(cultured with 50μmol/L ketamine for 24 h),ketamine+si-DIO3OS group(transfected with si-DIO3OS plasmid for 48 h,and then cultured with 50μmol/L ketamine for 24 h),ketamine+si-PTBP1 gro

关 键 词：长链非编码RNA DIO3OS 氯胺酮 海马神经元 细胞毒性 多聚嘧啶区结合蛋白1 脑源性神经营养因子 

分 类 号：R614[医药卫生—麻醉学]