ISG15过表达抑制THP-1细胞的增殖与吞噬

目的:干扰素刺激基因15蛋白(Interferon-stimulated Gene 15,ISG15)是由I型干扰素诱导产生的类泛素蛋白,在先天免疫中起重要作用,本文旨在阐明ISG15的表达水平对巨噬细胞功能的影响并进一步探究其作用机制。方法:构建ISG15过表达质粒并通过慢病毒感染的方法整合进入THP-1细胞中,通过流式细胞仪分选出单克隆ISG15过表达细胞系,利用Western Blotting的方法验证ISG15在细胞内的过表达效果。在构建成功的细胞系中进行CCK8细胞增殖实验和Latex Beads细胞吞噬实验,最后通过定量蛋白质组学的方法观察细胞内蛋白质水平上变化。结果:Western Blotting的结果验证了ISG15在THP-1巨噬细胞系中的过表达效果,证明了ISG15过表达巨噬细胞系的成功构建。CCK8细胞增殖实验的结果表明,ISG15过表达的细胞系与对照组细胞系相比其增殖能力减弱;Latex beads细胞吞噬实验显示ISG15过表达细胞系的吞噬能力发生下降,并在蛋白质组学数据中找到糖酵解相关酶和膜转运蛋白下调的证据。结论:ISG15过表达能够降低与糖酵解相关的蛋白从而导致增殖能力的下降;同时也引起膜转运相关蛋白下调造成吞噬能力降低。

Overexpression of ISG15 in THP-1 Inhibits Cell Proliferation and Phagocytosis

ABSTRACT Objective: ISG15 (Interferon-stimulated Gene 15) is a type I IFN-stimulated ubiquitin-like protein and plays an impor- tant role in innate immunity. Here, we investigated the effect of ISG15 overexpression on cell proliferation and endocytosis of THP-1 cells, then futher speculated underlying machanism. Methods: ISG15 overexpresing plasmids were transfected into THP-1 by using lentivirus infection and cytometry was used to selecte monoclonal cell line. The ISG15 overexpression efficiency was confirmed by West- ern Blotting. We employed CCK8 proliferation assay and Latex Beads phagocytosis assay to analyze phenotype alteration. Quantitative proteomics was carried out to investigate the effects of ISG15 overexpression on cell proteome. Results: Using CCK8 assay, we showed that ISG15 overexpression decreased the cell growth. We performed the endocytosis assay and showed that ISG15 overexpression de- creased phagocytosis in THP-1 cells. Proteomic analysis indicated that glycolytic enzymes and proteins associated with membrane trans- port were downregulated. Conclusion: ISG15 overexpression decreases cell proliferation by downregulating glycolytic enzymes and the downregulation of membrane transport-associated proteins cause the decrease of endocytosis.