氯胺酮致小鼠发育期神经毒性中长链非编码RNA的表达变化  

Changes of long non-coding RNAs in developmental neurotoxicity induced by ketamine

作　　者：李春竹 李文龙 王皓 严佳[1] 周徐慧[1] 马晓凡 徐安妮[2] 姜虹[1] LI Chunzhu;LI Wenlong;WANG Hao;YAN Jia;ZHOU Xuhui;MA Xiaofan;XU Anni;JIANG Hong(Department of Anesthesiology,Shanghai Ninth People’s Hospital,Shanghai JiaoTong University School of Medicine,Shanghai 200011,China)

机构地区：[1]上海交通大学医学院附属第九人民医院麻醉科,上海200011 [2]上海交通大学医学院附属新华医院药学部

出　　处：《上海医学》2022年第3期153-159,共7页Shanghai Medical Journal

基　　金：国家自然科学基金(81901070);上海市科学技术委员会科研计划(17DZ1205403);上海市青年科技英才扬帆计划(19YF1427700);上海交通大学医学院附属第九人民医院基础研究助推计划(JYZZ047、JYZZ120)。

摘　　要：目的探讨多次或长时间给予氯胺酮麻醉对发育期小鼠海马神经的影响,以及长链非编码RNA(lncRNAs)表达谱的改变。方法选择健康无特定病原体(SPF)级孕14~16 d的雌性C57BL/6小鼠,以及出生后第7~9天的C57BL/6新生小鼠,将新生小鼠随机分为氯胺酮组(予小鼠腹腔内注射氯胺酮100 mg/kg)、对照组(予小鼠腹腔内注射等体积0.9%氯化钠溶液),连续给药3 d,末次给药后24 h取小鼠海马组织。原代培养海马神经元种板后第3天以氯胺酮处理6 h。采用蛋白质印迹法(Western blotting)检测海马神经元凋亡蛋白表达,激光共聚焦显微镜观察海马神经元树突分支。使用高通量测序检测并通过实时荧光定量聚合酶链反应(RT-qPCR)验证海马lncRNAs表达变化,对差异表达的lncRNAs功能进行基因本体(GO)分析和京都基因与基因组百科全书(KEGG)通路数据库分析。结果蛋白质印迹法检测结果显示,氯胺酮组小鼠海马组织中bcl-2/Bax相对表达量显著低于对照组(P<0.01),cleaved-caspase3相对表达量显著高于对照组(P<0.01);在体外培养神经元中,氯胺酮50μmol/L组和氯胺酮100μmol/L组的bcl-2/Bax相对表达量均显著低于对照组(P值均<0.01),cleaved-caspase3相对表达量均显著高于对照组(P值均<0.01)。氯胺酮组的树突一级分支和树突二级分支数的最大值分别显著小于对照组(P值均<0.01)。与对照组比较,氯胺酮组lncRNAs相对表达量发生明显改变(降低或升高),有67种lncRNAs显著上调和7种lncRNAs显著下调。GO分析显示,差异表达的lncRNAs主要分布于细胞内结构。KEGG通路分析显示,与差异表达的lncRNAs相关的主要通路中改变最显著的通路为氧化磷酸化。RT-qPCR验证显示,氯胺酮组Six3os1相对表达量显著高于对照组(P<0.01),D930019O06Rik、4833438C02Rik相对表达量均显著低于对照组(P值均<0.05);两组间TUG1、Snhg14、Snrpert、Gm33239相对表达量的差异均无统计学意义(P值均>0.05)。�Objective To explore the effects of ketamine on hippocampal nerve development and the changes of long non-coding RNAs(lncRNAs)expression profile in developing mice.Methods P7-9 C57BL/6 neonatal mice were intraperitoneally given 100 mg/kg ketamine(ketamine group)or normal saline(control group)7-9 days after birth.The hippocampus was isolated 24 h after the final injection.Hippocampal neurons were treated with ketamine for 6 h on the 3rd day after primary culture.Western blotting was used to detect the expression of apoptotic protein,and immunofluorescence was used to observe the morphological changes of neuronal dendrites in the hippocampus.The expression of lncRNAs was detected and verified by high-throughput sequencing and real-time quantitative polymerase chain reaction(RT-qPCR),respectively.Gene Ontology(GO)analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis were used to study the role of lncRNAs in developmental neurotoxicity induced by ketamine.Results Compared with control group,the ratio of bcl2/Bax was decreased(P<0.01),and the expression of cleaved-caspase3 was increased(P<0.01)in the hippocampal tissues of ketamine group.Moreover,in vitro results showed that the ratio of Bcl2/Bax was decreased(both P<0.01),and the expression of cleaved-caspase3 was increased(both P<0.01)in two ketamine-treated groups(50μmol/L and 100μmol/L).Ketamine reduced the number of primary and secondary dendritic branches(both P<0.01).In sequencing,67 significantly up-regulated and 7 down-regulated lncRNAs were identified in ketamine group.GO analysis showed that differentially expressed lncRNAs were mainly distributed in intracellular structures.KEGG pathway analysis revealed that oxidative phosphorylation was the most significantly altered pathway related to these lncRNAs.The expression level of Six3os1 in ketamine group was significantly higher than that in control group(P<0.01),and the expression levels of D930019O06Rik and 4833438C02Rik were significantly lower than those in control group(both P<0.05)

关 键 词：氯胺酮 RNA 长链非编码 神经发育 

分 类 号：R614[医药卫生—麻醉学]