摘要
目的 研究肝纤维化小鼠脾脏对肝脏中巨噬细胞活化肝星状细胞作用的影响。方法 18只雄性C57BL/6小鼠随机分为3组,A、B组注射CCl4制备肝纤维化模型,C组注射玉米油作为正常对照组。4周后,A、B组分别行脾切除术(切脾组)或假手术(有脾组)。继续注射2周后取材3组小鼠肝脏,制备肝脏匀浆(L-Homo)并分离肝脏细胞。Luminex检测3组L-Homo中IL-1β、IL-13、TGF-β、TNF-α、PDGF-β、VEGF的表达;RT-qPCR与流式细胞术进一步观察如上因子在A、B组肝脏巨噬细胞(L-Mφ)及肝脏其他单个核细胞中的表达。利用A/B组L-Homo分别进行体外处理,以模拟脾脏存在与否时肝脏微环境对巨噬细胞的影响。收集接受不同L-Homo处理的巨噬细胞,一方面利用RT-qPCR比较其中细胞因子与谷氨酰胺合成/分解酶与谷氨酰胺转运蛋白的表达差异;另一方面与肝星状细胞JS1进行共培养,分析其对JS1存活与胞外基质表达的影响。组间比较使用Student’s t检验(两组间)或单因素方差分析(多组间)。结果 与正常对照组相比,IL-1β、IL-13、TGF-β与TNF-α的浓度在模型组L-Homo中显著升高,且在模型有脾组中显著高于切脾组;其中巨噬细胞是表达这些细胞因子的主要细胞类型群。相较于切脾组,有脾组L-Homo体外处理上调巨噬细胞中IL-1β、TGF-β、TNF-α等细胞因子以及谷氨酰胺酶的表达,并且促进巨噬细胞发挥活化肝星状细胞胞外基质分子表达的能力。结论 脾脏参与调控L-Mφ炎性因子表达,增强其活化肝星状细胞的能力。
Objective To investigate the effect of spleen on hepatic macrophages mediated activation of hepatic stellate cells(HSCs)in mice with liver fibrosis.Methods Eighteen male C57BL/6 mice were randomly divided into three groups.Mice in Group A and Group B were injected intraperitoneally with CCl 4 to establish liver fibrosis mouse model,while those in Group C were injected with corn oil as normal control.Four weeks later,mice with liver fibrosis received splenectomy(Spx)or sham operation(Sham),respectively.After continuous injection for 2 weeks,liver homogenates(L-Homo)were prepared and liver cells were isolated from the three groups.Expressions of IL-1β,IL-13,TGF-β,TNF-α,PDGF-βand VEGF in the liver homogenates of the three groups were detected by Luminex multifactor analysis.The expressions of these cytokines in liver macrophages(L-Mφ)and other non-parenchymal cells of Sham and Spx mice were analyzed by Real-time quantitative PCR(RT-qPCR)and flow cytometry.Macrophage cell line RAW264.7 or bone marrow-derived macrophages(BMDMs)were treated with liver homogenates from the Sham and Spx groups.Then the differently treated RAW264.7 cells were analyzed for mRNA expressions of cytokines and glutamine metabolism-related molecules by RT-qPCR,or transwell co-cultured with hepatic stellate cell line JS1.After co-culture,the survival and extracellular matrix expression of JS1 cells were analyzed.For comparison,Student’s t test(between two groups)or one-way analysis of variance(among multiple groups)were used.Results Compared with normal control group,the concentrations of IL-1β,IL-13,TGF-βand TNF-αin the L-Homo of model group were significantly increased and showed higher levels in Sham group than in Spx group.Moreover,the hepatic macrophages were indicated as the major source of these cytokines.Consistently,macrophages treated with liver homogenate of Sham mice had increased expressions of IL-1β,TGF-βand TNF-αand glutaminase(GLS).After co-culture with macrophages treated with liver homogenate of Sham group rather t
作者
张少颖
万丹
邓熙
梁肖
梁凡凡
张冲宇
朱佳真
赵阳
李宗芳
ZHANG Shaoying;WAN Dan;DENG Xi;LIANG Xiao;LIANG Fanfan;ZHANG Chongyu;ZHU Jiazhen;ZHAO Yang;LI Zongfang(National&Local Joint Engineering Research Center of Biodiagnosis&Biotherapy,The Second Affiliated Hospital of Xi’an Jiaotong University,Xi’an 710004,China;Shaanxi Provincial Clinical Medical Research Center for Liver and Spleen Diseases,The Second Affiliated Hospital of Xi’an Jiaotong University,Xi’an 710004,China;Department of Geriatric General Surgery,The Second Affiliated Hospital of Xi’an Jiaotong University,Xi’an 710004,China)
出处
《西安交通大学学报（医学版）》
CAS
CSCD
北大核心
2024年第4期575-581,共7页
Journal of Xi’an Jiaotong University（Medical Sciences）
基金
国家自然科学基金资助项目(No.82101915)
西安交大基础临床融合项目(No.YXJLRH2022099)
西安交大二附院自由探索项目[No.2020YJ(ZYTS)546-01]。
