N-甲基-D-天冬氨酸诱导的视网膜兴奋性毒性大鼠模型中5-甲基胞嘧啶的表观转录组学分析  

The m5C-methylated epi-transcriptomic analysis in a rat model of N-methyl-D-aspartate-induced retinal excitotoxicity

作　　者：王森楠 李亚红 刘勋 张琰 Wang Sennan;Li Yahong;Liu Xun;Zhang Yan(Tianjin Key Laboratory of Retinal Functions and Diseases,Tianjin Branch of National Clinical Research Center for Ocular Disease,Eye Institute and School of Optometry,Tianjin Medical University Eye Hospital,Tianjin 300384,China)

机构地区：[1]天津医科大学眼科医院、眼视光学院、眼科研究所、国家眼耳鼻喉疾病临床医学研究中心、天津市分中心、天津市视网膜功能与疾病重点实验室,天津300384

出　　处：《中华眼底病杂志》2023年第10期836-843,共8页Chinese Journal of Ocular Fundus Diseases

基　　金：国家自然科学基金面上项目(81970827);天津市医学重点学科(专科)建设项目(TJYXZDXK-037A)。

摘　　要：目的分析和验证N-甲基-D-天冬氨酸(NMDA)诱导的视网膜兴奋性毒性大鼠模型中经5-甲基胞嘧啶(m5C)甲基化修饰的转录本的差异性表达。方法7~8周龄雄性Sprague Dawley大鼠65只,随机分为正常对照组、NMDA组。NMDA组大鼠右眼(模型眼)玻璃体腔注射浓度为50.0 mmol/L的NMDA 3μl,正常对照组大鼠右眼玻璃体腔注射等体积生理盐水。建模后7 d,采用视觉诱发电位检测大鼠视神经传导功能;苏木精-伊红染色观察大鼠视网膜整体结构,检测视网膜各层厚度以及视网膜神经节细胞层的细胞数量;视网膜铺片免疫荧光染色检测β3微管蛋白免疫荧光染色阳性细胞个数。收集正常对照组、NMDA组大鼠视网膜,提取总RNA,进行高通量m5C修饰RNA测序,并进行生物信息学分析;实时定量聚合酶链反应检测视网膜中SLFN3、PLXNB3、CD36、HIC2 mRNA相对表达量。组间比较行非配对t检验。结果正常对照组、NMDA组P1波潜伏期分别为(117.86±6.48)、(148.46±3.78)ms,振幅分别为(42.57±2.41)、(8.68±0.63)μV;与正常对照组比较,NMDA组潜伏期延长,振幅显著下降,差异均有统计学意义(P<0.001)。正常对照组大鼠视网膜神经节细胞(RGC)排列均匀,细胞核大而圆;NMDA组大鼠视网膜RGC体积萎缩,数量减少,细胞核固缩。正常对照组、NMDA组视网膜总厚度分别为(207.51±12.76)、(187.51±12.54)μm;β3微管蛋白阳性细胞数分别为(79.86±6.56)、(29.36±2.16)个。与正常对照组比较,NMDA组视网膜总厚度、β3微管蛋白阳性细胞数均降低,差异有统计学意义(P<0.01)。与正常对照组比较,NMDA组筛选出差异表达m5C mRNA 576个,其中上调、下调基因分别为230、346个。生物信息分析结果显示,与正常对照组比较,NMDA组表达上调的m5C mRNA主要参与感知力、细胞-细胞黏附等生物过程,主要富集于细胞因子-细胞因子受体的相互作用、神经活性配体-受体相互作用通路中;表达下调的m5C mRNA参�Objective To study the differential expression profiling of the transcripts modified by m5C methylation in a rat model of N-methyl-D-aspartate(NMDA)-induced retinal excitotoxicity.Methods A total of 65 Sprague Dawley male rats aged 7-8 weeks were randomly divided into two groups:normal control group and NMDA group.The right eye(model eye)of rats in the NMDA group were injected with 50.0 mmol/L of NMDA 3μl in the vitreous cavity,while in the normal control group,equal volume of normal saline was injected into the vitreous cavity.After 1 week of the injection,the optic nerve conduction function of rats was detected by visual evoked potential.The whole structure of rat retina was observed by hematoxylin-eosin staining,and the thickness of each retinal layer and the number of retinal ganglion cell layer were detected.The number ofβ3 tubulin immunofluorescence positive cells was detected by immunofluorescence staining on retinal stretched preparation.Total RNA was extracted from the retinas of normal control group and NMDA group,and highthroughput m5C modified RNA was sequenced,and bioinformatics analysis was performed.The relative expression levels of SLFN3,PLXNB3,CD36 and HIC2 mRNA in retina were detected by real-time quantitative polymerase chain reaction.The comparison between the two groups was performed using an unpaired t test.Results The P1 latency of control group and NMDA group were(117.86±6.48)and(148.46±3.78)ms,and the amplitudes were(42.57±2.41)and(8.68±0.63)μV,respectively.Compared with the normal control group,the latency period was prolonged and the amplitude was significantly decreased in the NMDA group,with statistical significance(P<0.001).In normal control group,retinal ganglion cells(RGC)were uniformly arranged with large round nuclei.In NMDA group,the volume of retinal RGC was atrophied and the number of RGC was reduced.The total retinal thickness in the control group and NMDA group was(207.51±12.76)μm and(187.51±12.54)μm,respectively.The number ofβ3 tubulin positive cells was 79.86±

关 键 词：N-甲基-D-天冬氨酸 5-甲基胞嘧啶 视网膜兴奋性毒性 转录组学分析 动物实验 

分 类 号：R774[医药卫生—眼科] R-332[医药卫生—临床医学]