THP1细胞过表达腺病毒介导的PEDF抑制炎症过程中关键基因的鉴别  

Identification of key genes involved in the inhibition of inflammation by overexpression of PEDF adenovirus in THP1 cells

作　　者：张媛媛 吴红莲 徐嫚鸿 李筱荣[1] 邵彦[1] Zhang Yuanyuan;Wu Honglian;Xu Manhong;Li Xiaorong;Shao Yan(Tianjin Key Laboratory of Retinal Function and Diseases,Tianjin International Joint Research Center of Ophthalmology and Vision Science,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、天津医科大学眼视光学院、天津医科大学眼科研究所、天津市视网膜功能与疾病重点实验室、天津市眼科学与视觉科学国际联合研究中心,天津300384

出　　处：《中华实验眼科杂志》2024年第10期887-897,共11页Chinese Journal Of Experimental Ophthalmology

基　　金：国家自然科学基金青年项目(81900891)。

摘　　要：目的鉴别人单核细胞白血病细胞THP1过表达腺病毒介导的色素上皮衍生因子(PEDF)抑制炎症过程中的关键基因。方法对THP1细胞过表达腺病毒介导的PEDF进行蛋白质组学分析。将THP1细胞分为GFP组和PEDF组,分别用GFP腺病毒和PEDF腺病毒感染细胞;将THP1细胞分为甘露醇组、高糖组、高糖+GFP组和高糖+PEDF组,分别用D-甘露醇、D-无水葡萄糖、GFP腺病毒和PEDF腺病毒培养4、4、3和3 d;将Pedf^(-/-)小鼠采用随机数表法分为Pedf^(-/-)组和Pedf^(-/-)糖尿病组,每组12只,另取10只C57BL/6小鼠为对照组,取小鼠视网膜进行实验。采用实时荧光定量PCR验证差异表达基因(DEGs)的mRNA在视网膜组织和THP1细胞系中的表达水平。与GSE5504数据集进行DEGs交集,使用String数据库构建蛋白-蛋白相互作用(PPI)网络,Cytoscape软件及MCODE应用程序提取PPI网络模块,与Set1数据集取交集并找到关键基因。采用Western blot在THP1细胞和Pedf^(-/-)小鼠中验证关键基因的表达水平。结果通过蛋白质组学和生物信息学分析,筛选出Set1数据集中的105个差异蛋白。实时荧光定量PCR结果显示,PEDF组细胞中ARF5、TCF25和KCTD9 mRNA相对表达量明显高于GFP组,RNPS1、CSF1R、OGA、IBA57和MGST2 mRNA相对表达量明显低于GFP组,差异均有统计学意义(均P<0.001)。对照组、Pedf^(-/-)组和Pedf^(-/-)糖尿病组视网膜组织中表达显著下调的TCF25、KCTD9、ARF5 mRNA和表达显著上调的CSF1R、RNPS1、IBA57 mRNA相对表达量总体比较,差异均有统计学意义(F=64.057、27.561、37.179、65.757、44.024、34.248,均P<0.001);与对照组相比,Pedf^(-/-)组TCF25、KCTD9、ARF5 mRNA相对表达量降低,CSF1R、RNPS1 mRNA相对表达量升高,差异均有统计学意义(均P<0.05);Pedf^(-/-)糖尿病组TCF25、KCTD9、ARF5 mRNA相对表达量降低,CSF1R、RNPS1、IBA57 mRNA相对表达量升高,差异均有统计学意义(均P<0.05)。与Pedf^(-/-)组相比,Pedf^(-/-)糖尿病组TCF25 mRNA相对Objective To identify the key genes in the process inhibiting inflammation by overexpression adenovirus-mediated pigment epithelium-derived factor(PEDF)gene in human monocytic leukemia cells THP1.Methods Proteomic analysis of THP1 overexpressing adenovirus-mediated PEDF gene was performed.The THP1 cells were divided into GFP and PEDF groups,transfected with GFP and PEDF adenovirus,respectively.The THP1 cells were divided into mannitol group,high glucose group,high glucose+GFP group,and high glucose+PEDF group,which were cultured with mannitol for 4 days,anhydrous glucose for 4 days,GFP adenovirus for 3 days,and PEDF adenovirus for 3 days,respectively.The Pedf^(-/-)mice were divided into Pedf^(-/-)group and Pedf^(-/-)diabetes group according to the random table method,with 12 mice in each group.Another 10 C57BL/6 mice were taken as the control group.Mouse retinas were collected for experiments.The mRNA expression levels of differentially expressed genes(DEGs)in retina and THP1 cells were verified by real-time fluorescence quantitative PCR.The DEGs were intersected with the GSE5504 dataset,and the protein-protein interaction(PPI)network was built using the String database.Modules of the PPI were extracted using the Cytoscape software and the MCODE application.Intersections were taken with the Set1 dataset and key genes were found.The expression levels of key genes in THP1 cells and Pedf^(-/-)mice were verified by Western blot.The feeding and operation of experimental animals were in accordance with the regulations of the State Science and Technology Commission on the management of experimental animals and approved by the Animal Management and Use Committee of Tianjin Medical University(No.TTYY2023120217).Results Through proteomics and bioinformatics analysis,105 DEGs in the Set1 dataset were screened.The results of real-time PCR showed that the relative expression levels of ARF5,TCF25 and KCTD9 mRNA were significantly higher and the relative expression levels of RNPS1,CSF1R,OGA,IBA57 and MGST2 mRNA were significant

关 键 词：糖尿病视网膜病变 炎症 色素上皮衍生因子 生物信息学 CSF1R基因 THP1细胞 

分 类 号：R587.2[医药卫生—内分泌] R774.1[医药卫生—内科学]