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  

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

作　　者：王唯一[1] 章永平[1] 袁耀宗[1] 吴云林[1] 陈平[1] 

机构地区：[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

出　　处：《诊断学理论与实践》2017年第1期60-65,共6页Journal of Diagnostics Concepts & Practice

基　　金：国家自然科学基金面上项目(81170437)

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-m RNA表达质粒并体外表达m RNA,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-m RNA、PIAS1(213bp)-GFP-m RNA+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-m RNA、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-m RNA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-m RNA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-m RNA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Objective： To investigate the effect of PIAS1 （protein inhibitor of activated STAT1） on regulating the mi- gration of macrophage via transgenie zebrafish （Lyz-EGFP） with green fluorescent protein labelling maerophages, so as to provide evidences for basic research on anti-inflammatory role of PIAS1. Methods： Morpholino-PIAS1 （checking plasmid PIAS1 （213bp） GFP）., PIASI-mRNA （mRNA expressing plasmid PIAS1）, and mismatch plasmid （as negative control） were established. The morpholino-PIAS1, PIASI-mRNA, and mismatch plasmid were injected separately or together into ze- brafish 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 ze- brafish 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/JNFUp38MAPK/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- PIAS! 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-PIASl＋PIASl-mRNA）, while it was reduced in PIASI-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 PIASI-mR- NA injected group （P〈0.05）. Conclusions： PIAS1 is an inhibitor for macrophage activation, and is involved in

关 键 词：活化STAT蛋白抑制剂-1 巨噬细胞 炎症 斑马鱼 

分 类 号：R574.62[医药卫生—消化系统]