诊断学理论与实践 ›› 2017, Vol. 16 ›› Issue (01): 60-65.doi: 10.16150/j.1671-2870.2017.01.012

• 论著 • 上一篇    下一篇

活化STAT蛋白抑制剂1调控巨噬细胞迁移能力及机制的实验研究

王唯一, 章永平, 袁耀宗, 吴云林, 陈平   

  1. 上海交通大学医学医学院附属瑞金医院北院消化内科,上海 201801
  • 收稿日期:2016-10-10 出版日期:2017-02-25 发布日期:2022-06-20
  • 通讯作者: 陈平 E-mail: chenping714@aliyun.com
  • 基金资助:
    国家自然科学基金面上项目(81170437)

Experimental study on effect of PIAS1 in regulating migration of macrophage and its mechanism

WANG Weiyi, ZHANG Yongping, YUAN Yaozong, WU Yunlin, CHEN Ping   

  1. Department of Gastroenterology, Ruijin Hospital North, Shanghai Jiao Tong University School of Medicine, Shanghai 201801, China
  • Received:2016-10-10 Online:2017-02-25 Published:2022-06-20

摘要: 目的 通过绿色荧光蛋白标记巨噬细胞的转基因斑马鱼 (Lyz-EGFP)实时观察活化STAT蛋白抑制剂1(protein inhibitor of activated STAT1, PIAS1)与巨噬细胞间的关系,揭示其作用的相关机制,从而为PIAS1参与炎性疾病的基础研究提供依据。方法 以PCSⅡ质粒为基础,体外构建Morpholino-PIAS1验证质粒PIAS1(213bp)-GFP、PIAS1-mRNA表达质粒并体外表达mRNA,并以随机错配5个碱基的错配(Mismatch)作为阴性对照。培养野生型斑马鱼和Lyz-EGFP转基因斑马鱼,利用显微注射技术,将Morpholino-PIAS1+ PIAS1(213bp)-GFP-mRNA、PIAS1(213bp)-GFP-mRNA+Mismatch分别共注射单细胞期野生型斑马鱼胚胎,作为Morpholino组及Mismatch阴性对照组,发育6 h后在荧光显微镜下观察荧光表达变化,确定敲减和高表达靶向基因的效率和特异性。然后将Morpholino-PIAS1、PIAS1-mRNA、Mismatch组分别或共同注射单细胞期斑马鱼胚胎,发育60 h后行无菌手术刀斑马鱼切尾,采用共聚焦显微镜,实时动态观察在炎症区域巨噬细胞向伤口处迁移现象及计数,采用蛋白印迹法检测ERK/JNK/p38MAPK/MMPs信号转导通路蛋白的表达情况。结果 单细胞胚胎期观察6 h后发现,Morpholino-PIAS1注射组野生型斑马鱼胚胎可见绿色荧光的减灭,而Mismatch阴性对照组无改变,组间红色荧光无差异。Lyz-EGFP转基因斑马鱼胚胎发育60 h尾巴切除6 h后见Morpholino-PIAS1注射组绿色荧光细胞向尾部迁移增加,而PIAS1-GFP-mRNA注射组绿色荧光细胞向尾部迁移减少,拯救实组(Rescure)组(Morpholino-PIAS1+PIAS1- mRNA共同注射)引起荧光细胞的迁移增加有所减少,差异有统计学意义(P<0.05)。蛋白印迹检测结果显示,Morpholino-PIAS1注射组、Rescure组及Mismatch阴性对照组的ERK、JNK、p38MAPK总蛋白及磷酸化水平及MMP-9蛋白表达升高,TIMP-1蛋白表达下降;而PIAS1-mRNA注射组的ERK、JNK及p38MAPK磷酸化蛋白水平下降,随后MMP-9蛋白水平下降,TIMP-1蛋白水平升高,与Morpholino-PIAS1组、Rescure组及Mismatch阴性对照组比较差异有统计学意义(P<0.05)。结论 PIAS1对巨噬细胞活化迁移具有抑制作用,其机制涉及PIAS1调控ERK/JNK/p38MAPK/MMPs信号转导途径。通过上调PIAS1的表达,可抑制巨噬细胞等炎症细胞活化、迁移,进而改善炎性疾病的严重程度。

关键词: 活化STAT蛋白抑制剂-1, 巨噬细胞, 炎症, 斑马鱼

Abstract: Objective: To investigate the effect of PIAS1 (protein inhibitor of activated STAT1) on regulating the migration of macrophage via transgenic zebrafish (Lyz-EGFP) with green fluorescent protein labelling macrophages, so as to provide evidences for basic research on anti-inflammatory role of PIAS1. Methods: Morpholino-PIAS1 (checking plasmid PIAS1 (213bp) GFP), PIAS1-mRNA (mRNA expressing plasmid PIAS1), and mismatch plasmid (as negative control) were established. The morpholino-PIAS1, PIAS1-mRNA, and mismatch plasmid were injected separately or together into zebrafish embryo using micro-injection method. When cultured for 6 h, the fluorescent protein expression was assessed under fluorescence microscopy to determine the efficiency and specificity of transfection. After cultured for 60 h, the tail of zebrafish was cut with a sterile scapel, and the migration and count of macrophages at the cutting site were observed by confocal microscope. The activation of ERK/JNK/p38MAPK/MMPs signal protein and MMP-9/TIMP-1 protein expression was detected by western blotting. Results: In single cell of 6 h embryo, the green fluorescence was reduced in morpholino-PIAS1 injected group, while no change was seen in mismatch control group. There was no significant difference in red fluo-rescence between these groups. And in the cutting tail of zebrafish, the migration of green fluorescent cells was increased in morpholino-PIAS1 injected group, mismatch control group and rescue group (morpholino-PIAS1+PIAS1-mRNA), while it was reduced in PIAS1-mRNA injected group, the difference was statistically significant(P<0.05). Western blotting showed that the phosphorylation levels of ERK/JNK/p38MAPK proteins and the ratio of MMP-9/TIMP1 proteins were increased in morpholino-PIAS1 injected group, mismatch control group and the rescue group, while were decreased in the PIAS1-mRNA injected group(P<0.05). Conclusions: PIAS1 is an inhibitor for macrophage activation, and is involved in ERK/JNK/p38MAPK/MMPs signaling transduction pathways. The up-regulation of PIAS1 expression could inhibit activation of macrophage and to ameliorate the severity of inflammatory diseases.

Key words: Protein inhibitor of activated STAT1, Macrophages, Inflammation, Zebrafish

中图分类号: