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。
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.
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