THP1细胞过表达腺病毒介导的          抑制炎症过程中关键基因的鉴别

张媛媛; 吴红莲; 徐嫚鸿; 李筱荣; 邵彦

doi:10.3760/cma.j.cn115989-20230607-00219

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中华实验眼科杂志

• 实验研究 •

ENGLISH ABSTRACT

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

作者及单位信息

出版日期： 2024 -10 -10 ·

DOI： 10.3760/cma.j.cn115989-20230607-00219

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

Authors Info & Affiliations

Zhang Yuanyuan

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

Wu Honglian

Xu Manhong

Li Xiaorong

Shao Yan

Published： 2024 -10 -10 ·

DOI： 10.3760/cma.j.cn115989-20230607-00219

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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相对表达量降低，CSF1R、RNPS1和IBA57 mRNA相对表达量升高，差异均有统计学意义(均 P<0.05)。Set1数据集与GSE5504数据集交集后得到20个差异蛋白，主要富集于基因表达的正向调控、ERK1和ERK2级联的正向调节、胰岛素分泌的正向调节参与细胞对葡萄糖刺激的反应和抗原加工与递呈通路上。通过构建PPI网络和Cytoscape软件中MCODE插件筛选出关键基因 CSF1R。Western blot结果显示，高糖组和高糖＋GFP组中细胞CSF1R相对表达量分别为1.961±0.085和1.000±0.069，分别高于甘露醇组的1.000±0.072和高糖＋PEDF组的0.469±0.079，差异均有统计学意义( t＝14.940、8.765，均 P<0.01)； Pedf ^－/－糖尿病组中视网膜CSF1R相对表达量为1.633±0.192，高于 Pedf ^－/－组的1.000±0.050，差异有统计学意义( t＝5.537， P<0.01)。

结论 CSF1R可能为THP1细胞过表达腺病毒介导的 PEDF抑制炎症过程中的关键基因和治疗靶点。

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

ABSTRACT

ObjectiveTo 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.

MethodsProteomic 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).

ResultsThrough 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 significantly lower in PEDF group than in GFP group, showing statistically significant differences (all at P<0.001).There were significant overall differences in the relative expression levels of down-regulated TCF25, KCTD9 and ARF5 mRNA and up-regulated CSF1R, RNPS1 and IBA57 mRNA among control group, Pedf ^-/- group and Pedf ^-/- diabetes group ( F=64.057, 27.561, 37.179, 65.757, 44.024, 34.248; all at P<0.001).Compared with control group, the relative expression levels of TCF25, KCTD9 and ARF5 mRNA were decreased and the relative expression levels of CSF1R and RNPS1 mRNA were increased in Pedf ^-/- group, showing statistically significant differences (all at P<0.05).Compared with control group, the relative expression levels of TCF25, KCTD9 and ARF5 mRNA were decreased and the relative expression levels of CSF1R, RNPS1 and IBA57 mRNA were increased in Pedf ^-/- diabetes group, showing statistically significant differences (all at P<0.05).Compared with Pedf ^-/- group, the relative expression level of TCF25 mRNA was decreased and the relative expression levels of CSF1R, RNPS1 and IBA57 mRNA were increased in Pedf ^-/- diabetes group, showing statistically significant differences (all at P<0.05).After intersection with the GSE5504 dataset, 20 differential proteins were obtained, which were mainly enriched in positive regulation of gene expression, positive regulation of ERK1 and ERK2 cascade, positive regulation of insulin secretion involved in cell response to glucose stimulation and antigen processing and presentation pathways.The key gene CSF1R was screened by constructing PPI network and MCODE plugin in Cytoscape software.Western blot results showed that the expression levels of CSF1R in high glucose group and high glucose+ GFP group were 1.961±0.085 and 1.000±0.069, which were higher than 1.000±0.072 in mannitol group and 0.469±0.079 in high glucose+ PEDF group, respectively, and the differences were statistically significant ( t=14.940, 8.765; both at P < 0.01).The expression of CSF1R in the retina of Pedf ^-/- diabetes group was 1.633±0.192, which was higher than 1.000±0.050 in Pedf ^-/- group, and the difference was statistically significant ( t=5.537, P<0.01).

ConclusionsCSF1R may be a key gene and therapeutic target for the inhibition of inflammation by overexpression of adenovirus-mediated PEDF gene in THP1 cell.

KEYWORDS： Diabetic retinopathy;Inflammation;Pigment epithelium-derived factor;Bioinformatics; CSF1R gene ;THP1 cell

Corresponding author: Shao Yan, Email: mocdef.3ab61heumtys

FUNDING:

National Natural Science Foundation of China（81900891）

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All articles published represent the opinions of the authors, and do not reflect the official policy of the Chinese Medical Association or the Editorial Board, unless this is clearly specified.

引用本文

张媛媛,吴红莲,徐嫚鸿,等. 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 [J]. 中华实验眼科杂志,2024,42(10)：887-897.

DOI:10.3760/cma.j.cn115989-20230607-00219

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糖尿病视网膜病变(diabetic retinopathy，DR)和年龄相关性黄斑变性(age-related macular degeneration，AMD)是主要的致盲眼病。炎症是视网膜水肿和损伤的重要原因，也是视网膜疾病进展的潜在因素 ^{[
1
]}。DR会影响视网膜的神经元和微血管系统，其发展与炎症因子的作用有关，而这些炎症因子与小胶质细胞的激活有关 ^{[
2
]}。小胶质细胞是单核吞噬细胞，可视为视网膜的组织驻留巨噬细胞。在静止状态下，小胶质细胞通过吞噬作用和控制低度炎症来维持视网膜中的组织稳态 ^{[
3
]}。然而，长期高血糖引起的组织应激会引发小胶质细胞过度反应，同时产生促炎细胞因子和趋化因子，从而导致慢性炎症 ^{[
4
]}。人单核细胞白血病细胞THP1可被诱导分化为单核/巨噬细胞，已作为体外细胞模型被广泛用于单核/巨噬细胞功能、机制和信号通路等相关研究 ^{[
5
]}。色素上皮衍生因子(pigment epithelium-derived factor，PEDF)是一种由视网膜色素上皮分泌的内源性糖蛋白，最初被确定为视网膜色素上皮细胞中的神经营养因子 ^{[
6
]}。PEDF已被证明在多种类型细胞和组织中可以减轻炎症反应，在介导细胞免受氧化应激损伤方面发挥核心作用 ^{[
7
]}。据报道，PEDF可通过抑制血管内皮生长因子(vascular endothelial growth factor，VEGF)诱导的视网膜微血管内皮细胞增殖来抑制视网膜新生血管形成 ^{[
8
]}。然而，PEDF在早期DR中的保护作用尚不完全清楚，因此深入阐明PEDF抑制炎症的机制，寻找视网膜疾病的关键治疗靶点，对临床诊疗以及改善患者预后具有重要意义。高通量蛋白质组学技术可用于探索细胞内蛋白的种类和动态变化，找到涉及视网膜炎症过程中的差异蛋白 ^{[
9
]}。随着蛋白质组学的发展，生物信息学在视网膜疾病研究领域中的应用也越来越广泛，结合大数据整合挖掘与DR和AMD等视网膜疾病发生和发展相关的关键hub基因，对筛选潜在的生物标志物及寻找新的视网膜疾病治疗靶点具有重要的临床意义 ^{[
10
]}。由于蛋白质组学技术受限于仪器的精度，存在一定的假阳性率，故需要在基因水平和蛋白水平对DEGs进行验证。本研究借助蛋白质组学技术和生物信息学技术寻找PEDF抗炎过程中的关键基因并对其进行验证，为深入探索DR等视网膜疾病的发生机制、后续临床诊疗及靶向干预提供理论依据。

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参考文献

参考文献

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