目的:揭示小鼠视神经损伤后视网膜神经节细胞(RGCs)凋亡的分子机制和潜在的治疗靶点.方法:构建视神经损伤(ONC)小鼠模型,对视网膜组织进行HE染色观察组织病理学改变,并使用实时荧光定量PCR(RT-qPCR)检测RGCs标志物—具有多重剪接的RNA结合蛋白(Rbpms)的mRNA表达水平;透射电镜(TEM)观察RGCs线粒体损伤情况;采用转录组测序分析ONC7d组小鼠的视网膜差异表达基因,并对差异表达基因进行Gene Ontology(GO)功能、Kyoto Encyclopedia of Genes and Genomes(KEGG)通路富集和韦恩分析,RT-qPCR检测差异基因mRNA表达水平.结果:与正常组相比,ONC 7 d组RGCs数量显著减少,细胞排列疏松不规则,RGCs标志物Rbpms的mRNA表达水平显著降低(P＜0.001),ONC小鼠模型构建成功.透射电镜结果表明,ONC 7 d组RGCs的线粒体出现肿胀,嵴消失;转录组测序结果表明,ONC 7 d组与正常组比,存在562个差异表达基因,其中152个基因表达下调和410个基因表达上调.GO功能富集分析和KEGG通路富集分析表明ONC后差异表达基因主要富集于神经元损伤修复的多个重要信号通路;RT-qPCR结果显示,与正常组相比,与神经元损伤修复相关的基因Ecel1、Atf3、Sprr1a的表达均在损伤后第4天显著上调(均P＜0.05),与测序结果一致.结论:ONC小鼠RGCs凋亡与线粒体损伤有关,而神经元损伤修复相关基因Atf3、Ecel1、Sprr1a在视神经损伤的早期参与了小鼠视神经损伤后RGCs的保护.
Analysis of gene expression profile of mouse retinal ganglion cells after optic nerve crush
Objective:To explore the molecular mechanism and potential therapeutic targets of retinal ganglion cells(RGCs)apoptosis after optic nerve crush in mice.Methods:An optic nerve crush(ONC)mouse model was constructed,the retinal tissue was stained with HE staining to observe the histamathological changes,and the mRNA expression level of RGCs marker,RNA binding proteins with multiple splicing(Rbpms)was detected by reverse transcription-quantitative PCR(RT-qPCR).The mitochondrial damage of RGCs was observed by trans-mission electron microscopy(TEM).Transcriptomic sequencing was used to analyze the retinal differentially ex-pressed genes of ONC 7 d group mice,and the differentially expressed genes were analyzed by Gene Ontology(GO)function,Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment and Wayne analysis.The mRNA expression levels of differentially expressed genes were detected by RT-qPCR.Results:Compared with the normal group,the number of RGCs in the ONC 7 d group was significantly reduced,the cell arrange-ment was loose and irregular,and the mRNA expression level of RGCs marker Rbpms was significantly de-creased(P＜0.001).The ONC mouse model was successfully constructed.The results of TEM showed that the mitochondria of RGCs in the ONC 7 d group were swollen and the ridge disappeared.Transcriptome sequencing results showed that there were 562 differentially expressed genes in the ONC 7 d group compared with the nor-mal group,among which 152 genes were down-regulated and 410 genes were up-regulated.GO functional enrich-ment analysis and KEGG pathway enrichment analysis showed that the differentially expressed genes after ONC were mainly concentrated in several important signaling pathways involved in neuronal damage repair.The RT-qPCR results showed that compared with the normal group,the expression of Ecel1,Atf3,and Sprr1a genes relat-ed to neuronal damage repair was significantly up-regulated on the 4th day after injury(all P＜0.05),which was consistent with the sequencing results.Conclusion:The apoptosis of RGCs in ONC mice is related to mitochon-drial injury,and the Atf3,Ecel1 and Sprr1a genes related to neuronal damage repair are involved in the the early protection of RGCs after ONC in mice.
万方数据
维普
