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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 observe the relationship between PIAS1 and macrophages through the green fluorescent protein-labeled macrophages transgenic zebrafish (Lyz-EGFP) in order to reveal the mechanism of its action , Thus providing the basis for the PIAS1 involved in the basic research of inflammatory diseases. METHODS: Morpholino-PIAS1 was constructed in vitro and the PIAS1 (213bp) -GFP and PIAS1-m RNA plasmids were verified by in vitro construction. The m RNAs were expressed in vitro. Mismatch with 5 bases mismatch Negative control. Wild-type zebrafish and Lyz-EGFP transgenic zebrafish were cultured and the single cells were co-injected with Morpholino-PIAS1 + PIAS1 (213 bp) -GFP-m RNA and PIAS1 (213 bp) -GFP-m RNA + Mismatch The wild-type zebrafish embryos, as Morpholino group and Mismatch negative control group, were observed under a fluorescence microscope after 6 h of development to determine the efficiency and specificity of knockdown and high expression of targeted genes. Morpholino-PIAS1, PIAS1-m RNA and Mismatch groups were injected separately or co-injected with zebrafish embryos of single-cell stage. After 60 h of development, sterile zebrafish zebrafish were cauterized and confocal microscopy was performed. The phenomenon of migration of macrophages to the wound and counting were observed. The protein expression of ERK / JNK / p38MAPK / MMPs signal transduction pathway was detected by Western blotting. RESULTS: After 6 h of observation in unicellular embryos, it was found that green fluorescence was attenuated in Morpholino-PIAS1 injected wild-type zebrafish embryos but no change was observed in Mismatch negative control group. There was no difference in red fluorescence between groups. GFP-m RNA injection group, green fluorescence cells migrated to the tail in the Morpholino-PIAS1 injection group, while green fluorescent cells migrated to the tail in the PIAS1-GFP-m RNA injection group, Rescure group (Co-injection of Morpholino-PIAS1 + PIAS1-m RNA) caused a decrease in the migration of fluorescent cells, the difference was statistically significant (P <0.05). Western blotting showed that the total protein and phosphorylation of ERK, JNK and p38MAPK, the expression of MMP-9 and the expression of TIMP-1 in Morpholino-PIAS1 injection group, Rescure group and Mismatch negative control group were decreased, while PIAS1- Compared with Morpholino-PIAS1 group, Rescure group and Mismatch negative control group, there were statistically significant differences in the levels of phosphorylated ERK, JNK and p38MAPK in m RNA injection group, followed by a decrease in MMP-9 protein and TIMP-1 protein Significance (P <0.05). Conclusion: PIAS1 has an inhibitory effect on the activation and migration of macrophages. Its mechanism involves PIAS1 regulating ERK / JNK / p38MAPK / MMPs signal transduction pathway. By up-regulating the expression of PIAS1, it can inhibit the activation and migration of inflammatory cells such as macrophages and further improve the severity of inflammatory diseases.