Interferon-γ induces cellular senescence through p53-dependent DNA damage signaling in human endothelial cells

Cellular senescence is a stress-response phenomenon in which cells lose the ability to proliferate; it is induced by telomere shortening, activation of oncogenes or tumor suppressor genes, or exposure to a sub-lethal dose of DNA damaging agents or oxidative stresses. cDNA microarray analysis reveals that the levels of interferons (IFNs) and IFN-inducible genes were altered during replicative senescence in human umbilical vascular endothelial cells (HUVECs). However, the role of IFNs in cellular senescence of HUVECs remains unidentified. This study demonstrated that prolonged treatment with IFN-γ induced cellular senescence in HUVECs, as confirmed by G0/G1 cell cycle arrest, up-regulation of p53 and p21 protein levels, increased SA-β-gal staining, and the accumulation of phospho-H₂AX foci. IFN-γ-induced cellular senescence was observed only in p16-knockdown cells or p16-null mouse embryonic fibroblasts (MEFs), but not in p53-knockdown cells or p53-null MEFs. IFN-γ treatment increased ROS production, and an antioxidant, N-acetylcysteine, inhibited IFN-γ-induced cellular senescence. Knockdown of ATM kinase or IFI16 rescued IFN-γ-induced cellular senescence. Therefore, these results suggest that IFN-γ might play an important role in cellular senescence through a p53-dependent DNA damage pathway and contribute to the pathogenesis of atherosclerosis via its pro-senescent activity.     

Comparison of the properties of collagen–chitosan scaffolds after γ-ray irradiation and carbodiimide cross-linking

The property of collagen–chitosan porous scaffold varies according to cross-linking density and scaffold composition. This study was designed to compare the properties of collagen–chitosan porous scaffolds cross-linked with γ-irradiation and carbodiimide (CAR) for the first time. Eleven sets of collagen–chitosan scaffolds containing different concentrations of chitosan at a 5% increasing gradient were fabricated. Fourier transform infrared spectroscopy was performed to confirm the success of cross-linking in the scaffolds. The scaffold morphology was evaluated under scanning electron microscope (SEM). SEM revealed that chitosan was an indispensable material for the fabrication of γ-ray irradiation scaffold. The microstructure of γ-ray irradiation scaffold was less stable than those of alternative scaffolds. Based upon swelling ratio, porosity factor, and collagenase degradation, γ-ray irradiation scaffold was less stable than CAR and 25% proportion of chitosan scaffolds. Mechanical property determines the orientation in γ-irradiation and CAR scaffold. In vitro degradation test indicated that γ-irradiation and CAR cross-linking can elevate the scaffold biocompatibility. Compared with γ-ray irradiation, CAR cross-linked scaffold containing 25% chitosan can more significantly enhance the bio-stability and biocompatibility of collagen–chitosan scaffolds. CAR cross-linked scaffold may be the best choice for future tissue engineering.     

Early IFN-γ production together with decreased expression of TLR3 and TLR9 characterizes EAE development conditional on the presence of myelin

Experimental autoimmune encephalomyelitis (EAE) is a model for the study of multiple sclerosis, which is an inflammatory and demyelinating disease of the central nervous system (CNS). Despite increased efforts to elucidate the function of toll-like receptors (TLRs) in autoimmune diseases of the CNS, the relative contribution of other factors, including the immunomodulatory properties of TLR signaling, role of the innate response and the presence or absence of myelin peptides remain unclear. The aim was to evaluate TLR expression in the CNS during EAE development by investigating the expression of TLRs in the initial phase of EAE and establishing correlations with the modulation of inflammatory factors. Mice were subcutaneously immunized at the tail base with 100?μg of myelin oligodendrocyte glycoprotein peptide (MOG_35–55), emulsified in complete Freund’s adjuvant (CFA) supplemented with 400?μg of attenuated Mycobacterium tuberculosis H37RA. Pertussis toxin (300?ng per animal) was intraperitoneally injected on the day of immunization and 48?h later. Another group (MOG^?) received an equal emulsion of CFA and M. tuberculosis, without MOG_35–55, and the same protocol of Pertussis toxin. The immunized mice presented signs of disease with increased IFN-γ production and presence of NK cells on Day 2 postimmunization and reduced the expression of TLR-3 and TLR-9. In the spinal cord, CCL5 and CCL20 were higher in EAE. This study establishes a correlation between TLR-3 and TLR-9 expression with the development of EAE. In addition, evidence of a role for the myelin peptide in targeting the innate inflammatory response to the CNS is presented.     

甘油二酯激酶γ参与大鼠胚胎脑神经上皮细胞的发育

目的探讨甘油二酯激酶γ(DGKγ)在大鼠胚胎脑神经上皮发育中的作用及可能机制。方法 18只胎龄(E)11.5、E12.5、E14.5、E16.5、E18.5大鼠胚胎用于Western blotting方法检测DGKγ在大鼠胚胎脑组织的表达,E9.5~E18.5大鼠胚胎各5只用于免疫组织化学和免疫荧光染色方法检测DGKγ在大鼠胚胎脑的分布以及DGKγ与Ki67、乙酰胆碱转移酶(Ch AT)及酪氨酸羟化酶(TH)的细胞内共定位。结果 DGKγ蛋白含量在E11.5~E14.5表达逐渐增多,E16.5表达较E14.5显著减少,E18.5表达显著增多且高于E14.5水平。E10.5~E12.5,DGKγ在脑泡壁出现表达,并随着脑泡的发育表达在端脑、除大脑脚外的中脑、后脑和末脑;E13.5~E14.5,DGKγ的强阳性表达从海马及周围的新皮质、苍白球、嗅脑和大脑脚阳性延伸到除新皮质外的脑的各区域;E15.5至出生前,DGKγ在新皮质阳性表达增强,但在大脑脚和脑桥表达减弱。免疫荧光染色显示,E14.5,与海马和苍白球相比,嗅脑的DGKγ阳性细胞中Ki67阳性率较高(87%),Ch AT阳性率较低(9%)(均P0.05),中脑顶盖的DGKγ阳性细胞中62%为TH阳性;E16.5,新皮质的DGKγ阳性细胞中Ki67阳性率为26%,较E14.5时(49%)显著降低(P0.05),Ch AT阳性率为70%,较E14.5时(45%)显著增高(P0.05);DGKγ多位于神经上皮细胞的胞质,也可见于胞膜上,或者同时位于胞质和胞核。结论 DGKγ在胚胎脑的表达模式和亚细胞分布,提示其参与了胚胎脑神经上皮的增殖、迁移和分化后神经元的发育。     

PPAR-γ激活抑制血管紧张素Ⅱ诱导的大鼠心脏成纤维细胞Ets-1表达

目的研究过氧化物酶体增殖物激活受体-γ(PPAR-γ)激活对心肌纤维化的影响是否与血管紧张素Ⅱ(AngⅡ)-Ets-1通路有关。方法体外培养大鼠心脏成纤维细胞(CFs),分为对照组、AngⅡ组、AngⅡ+不同浓度rosiglitazone处理组、AngⅡ+不同PPAR-γ激动剂组、AngⅡ+不同PPAR-γ拮抗剂组,采用实时定量RT-q PCR、Western blot等检测Ets-1、CTGF mRNA及蛋白表达,并测定TGF-β1和Smad2/3的表达及磷酸化水平。结果在CFs中,AngⅡ诱导Ets-1 mRNA及蛋白表达(P0.05),上调Ets-1下游靶基因CTGF的蛋白的表达(P0.05),增加TGF-β1和Smad2/3的表达及磷酸化(P0.05)。PPAR-γ激动剂rosiglitazone,15d-PGJ2抑制AngⅡ诱导的Ets-1 mRNA及蛋白的表达(P0.05),下调AngⅡ诱导的CTGF蛋白表达(P0.05),部分阻断AngⅡ诱导的TGF-β1的表达、Smad2/3的表达及磷酸化(P0.05)。PPAR-γ拮抗剂GW9662及BADGE均可阻断rosiglitazone对Ets-1及CTGF表达的抑制作用(P0.05)。结论 PPAR-γ激活主要通过TGF-β1/Smad2/3通路介导抑制AngⅡ诱导的大鼠CFs转录因子Ets-1的过表达。     

IFN-γ诱导的自噬抑制人胎盘胎儿侧来源间充质干细胞增殖

目的探讨IFN-γ诱导无血清培养人胎盘胎儿侧来源MSCs自噬的发生,并分析自噬对细胞增殖的影响。方法用酶消化法和无血清培养体系分离培养人胎盘胎儿侧来源MSCs,利用流式细胞仪和分化培养体系鉴定细胞属性;用质量浓度50μg/L的IFN-γ处理人胎盘胎儿侧来源MSCs,以未处理细胞作为对照组,3-Ma处理为自噬抑制组;分别提取总蛋白,Western blot检测自噬标志基因LC3Ⅰ/Ⅱ的表达;mRFP-GFP-LC3腺病毒感染细胞,观察细胞内点状聚集的情况;MTT法检测IFN-γ对细胞增殖的影响。结果所分离细胞呈CD73、CD90和CD105阳性细胞,不表达CD14、CD34和CD45,具有向脂肪和成骨细胞分化的能力;IFN-γ可提高LC3Ⅱ的表达量(P0.05),荧光共聚焦显微境观察到IFN-γ处理细胞中的点状聚集显著增加;3-Ma可解除IFN-γ对MSCs增殖能力的抑制(P0.05)。结论 IFN-γ诱导的自噬负调控人胎盘胎儿侧来源MSCs的增殖能力。     

Activation of human neutrophils by the pollutant sodium sulfite: effect on cytokine production,chemotaxis, and cell surface expression of cell adhesion molecules

In chronic beryllium disease (CBD), a granulomatous lung disease characterized by hypersensitivity to beryllium salts (BE), BE challenge of bronchoalveolar lavage cells induces IFNgamma. Although nitric oxide (NO) is elevated in CBD airways, the effects of NO on CBD IFNgamma responses are unknown. Here we report that BE-stimulated IFNgamma production in CBD lavage cells was markedly reduced (74%) by the NO generator DETA NONOate. Investigation of IFNgamma-stimulatory cytokine involvement indicated that lavage cell IL-18 was significantly increased (fourfold) by BE and reduced (64%) by DETA NONOate but IL-12 was undetectable. IL-18 production was caspase-1-dependent but caspase 1 inhibition reduced IFNgamma only partially (43%). Specific antibody depletion of lavage cell IL-18 yielded marginal reduction (19%) of IFNgamma. Data are the first to show that: (1) BE stimulates IL-18 as well as IFNgamma in CBD; (2) BE cytokine responses are NO-sensitive; and (3) NO down-regulation of IFNgamma involves other sites in addition to IL-18.     

TNF-α通过死亡受体途径促进IFN-γ诱导NIT-1细胞凋亡

目的：探讨TNF-α和IFN-γ对小鼠胰岛β细胞株NIT-1细胞凋亡的影响及其可能的分子机制。方法：用MTT法检测TNF-α和IFN-γ单独或联合作用对NIT-1细胞活力的影响,倒置显微镜观察NIT-1细胞形态变化,Hoechst 33258荧光染色后激光共聚焦显微镜观察细胞核形态的变化,Western blot技术检测凋亡相关蛋白Caspase-8,-3和PARP的活化。结果：TNF-α联合IFN-γ处理明显抑制了NIT-1细胞的活力,诱导了细胞的凋亡,促进了凋亡蛋白Caspase-8,-3和PARP的活性。结论：TNF-α通过细胞凋亡的死亡受体途径信号传递促进了IFN-γ诱导的NIT-1细胞凋亡。