摘要
目的分析和验证结缔组织生长因子(CTGF)刺激对视网膜Müller细胞基因表达谱的影响。方法将体外培养的视网膜Müller细胞分为对照组、CTGF组。对照组细胞采用正常DMEM完全培养基培养;CTGF组细胞采用含有10 ng/ml CTGF的DMEM完全培养基持续培养24 h。收集处于对数生长期的细胞行细胞划痕实验,对比分析两组细胞的细胞迁移率;应用HiSeq测序技术对两组细胞进行全转录组测序,获得生物学大数据,在此基础上分析差异表达的miRNA;通过基因注释(GO)功能显著性富集分析和京都基因与基因组百科全书(KEGG)通路显著性富集分析对差异miRNA的功能及信号通路进行分析。基于转录组数据筛选出两组间差异表达倍数位于前10名的基因,分析其基因特征。采用实时荧光定量PCR(qRT-PCR)、Western blot和免疫荧光定性检测对骨形态发生蛋白4(BMP4)的mRNA和蛋白表达进行验证。结果CTGF组细胞迁移率较对照组明显升高,差异有统计学意义(t=3.453,P=0.026)。对照组与CTGF组之间共有325个差异表达基因,其中上调者152个,下调者173个。GO功能显著性富集分析结果显示,差异miRNA的功能主要分为生物学过程、细胞成分和分子功能3类。KEGG通路显著性富集分析结果显示,差异miRNA表达高度富集在神经系统间信号传递、细胞间黏附以及细胞因子与其受体相互作用等相关的通路。分析两组间差异表达倍数位于前10名的基因,这些差异表达基因参与组织炎症反应及纤维化过程等不同的代谢通路及生物学过程。qRT-PCR、Western blot及免疫荧光定性检测结果显示,CTGF组细胞中BMP4 mRNA及蛋白表达均较对照组明显提高,差异有统计学意义(t=39.490、10.110、5.470,P=0.004、0.001、0.006)。结论CTGF通过上调Müller细胞中BMP4的表达而促进细胞的增生和迁移导致组织纤维化并可诱导炎症反应。
Objective To study the effects of connective tissue growth factor(CTGF)on retinal Müller cells based on transcriptome analysis of RNA-seq technology.Methods Retinal Müller cells were divided into the control group and the CTGF treatment group which was continuously cultured with 10 ng/ml of CTGF for 24 h.The influence of CTGF on the migration of Müller cells were tested by scratching experiments.The RNA transcriptome analysis was applied to complete transcriptome sequencing,differentially expressed genes and functional enrichment analysis of the two groups of cells.HiSeq sequencing technology was used to sequence the whole transcriptome of the two groups of cells to obtain biological big data,and analyze the differentially expressed miRNAs on this basis.The functions and signal pathways of differential miRNAs were analyzed through gene annotation(GO)functional significance enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway significant enrichment analysis.Based on transcriptome data,genes with differential expression multiples in the top ten between the two groups were screened out,and the expression of bone morphogenetic protein 4(BMP4)gene was verified by real time fluorescence quantification PCR(qRT-PCR),immunofluorescence and Western blot.Results After CTGF stimulation of Müller cells,cell viability and mobility which compared with the control group were significantly increased,with statistically significant differences(t=3.453,P<0.05).The differential gene expression profile of CTGF induced Müller cells was obtained by RNA transcriptome analysis.Comparing the sequencing results of the two groups,it was found that 325 differentially expressed genes included 152 up-regulated genes and 173 down-regulated genes.The results of GO functional significance enrichment analysis showed that the functions of differential miRNA were mainly divided into three categories:biological processes,cellular components,and molecular functions.These differentially expressed genes were involved in signa
作者
步绍翀
张哲
王琼
洪亚茹
李筱荣
东莉洁
Bu Shaochong;Zhang Zhe;Wang Qiong;Hong Yaru;Li Xiaorong;Dong Lijie(Tianjin Key Laboratory of Retinal Functions and Diseases,Tianjin International Joint Research and Development Centre of Ophthalmology and Vision Science,Eye Institute and School of Optometry,Tianjin Medical University Eye Hospital,Tianjin 300384,China)
出处
《中华眼底病杂志》
CAS
CSCD
北大核心
2020年第12期964-970,共7页
Chinese Journal of Ocular Fundus Diseases
基金
国家自然科学基金面上项目(81570872)
天津医科大学眼科医院青年创新人才项目(YDYYRCXM-C2018-02)
天津市教委科研计划一般项目(2017KJ214)
天津医科大学眼科医院高水平创新型人才培养基金(YDYYRCXM-C2018-01)。
