摘要
目的:探讨miR-124a对类风湿关节炎(RA)细胞模型小鼠巨噬细胞J774.1细胞中肿瘤坏死因子α(TNF-α)和白细胞介素6(IL-6)表达水平及细胞周期的影响,阐明miR-124a在RA中的作用机制。方法:取对数生长期J774.1细胞,以1×106 mL^(-1)均匀接种于培养皿,每组设3个复孔,当细胞融合度达到60%时进行感染,实验分为miR-124a过表达组(感染miR-124a腺病毒载体)、空载腺病毒组(感染阴性病毒液)和空白对照组(不做任何处理)。ELISA法检测感染48h后3组细胞上清液中TNF-α和IL-6的表达水平,RT-PCR法检测感染48h后J774.1细胞中TNF-α和IL-6mRNA相对表达水平,流式细胞术检测感染48h后3组细胞细胞周期变化。结果:感染48h后,与空白对照组和空载腺病毒组比较,miR-124a过表达组细胞上清中TNF-α和IL-6表达水平明显降低(P=0.038,P=0.042;P=0.043,P=0.044),miR-124a过表达组细胞中TNF-α和IL-6mRNA表达水平明显降低(P=0.001,P=0.002;P=0.001,P=0.003),G1期细胞百分率明显升高(P=0.01),S期和G2期细胞百分率明显降低(P<0.01)。结论:miR-124a感染小鼠巨噬细胞J774.1后可使细胞的炎性因子表达水平下降,抑制细胞增殖,miR-124a有望成为RA治疗的新靶点。
Objective:To explore the effects of miR-124a on the expression levels of tumor necrosis factor alpha(TNF-α)and interleukin-6(IL-6)and cell cycle in the macrophage J774.1 cells of the rheumatoid arthritis(RA)model mice,and to elucidate the mechanism of miR-124a in the pathogenesis of RA.Methods:The J774.1 cells in logarithmic growth phage were obtained and uniformly inoculated in the petri dish with 1×10 6 mL-1,and there were 3 multiple holes in each group;transfection was carried out when the fusion degree of the cells reached 60%.The cells were divided into blank control group(without any treatment),empty vector group(transfected with adenovirus negative fluid)and miR-124a overexpression group(transfected with miR-124a adenovirus vector).The expression levels of TNF-αand IL-6 in supernatant of the cells in three groups were detected by ELISA 48 h after transfection.RT-PCR was used to detect the relative expression levels of TNF-αand IL-6 mRNA in J774.1 cells 48 h after transfection and the changes of cell cycle were detected using flow cytometry.Results:The expression levels of TNF-αand IL-6 in supernatant of the cells in miR-124a overexpression group were lower than those in blank control group and empty vector group 48 h after transfection(P=0.038,P=0.042;P=0.043,P=0.044).The expression levels of TNF-αmRNA and IL-6 mRNA in miR-124a group were significantly lower than those in blank control group and empty vector group(P=0.001,P=0.002;P=0.001,P=0.003).The pecentage of cells in G 1 phase in miR-124a overexpression group was higher than those in blank control group and empty vector group(P<0.01);the percentages of cells in S phase and G 2 phase were lower than those in blank control group and empty vector group(P<0.01).Conclusion:MiR-124a can decrease the expression levels of inflammatory cytokines after transfecting the macrophages J774.1 cells,and inhibit the proliferation of cells.MiR-124a is a potential therapeutic target for the treatment of RA.
作者
尹芳蕊
庞春艳
耿立霞
王永福
YIN Fangrui;PANG Chunyan;GENG Lixia;WANG Yongfu(Department of Rheumatology,First Affiliated Hospital,Baotou Medical College;Institute of Rheumatology and Immunology,Baotou Medical College;Inner Mongolia Key Laboratory of Autoimmunity;Baotou 014010,China)
出处
《吉林大学学报(医学版)》
CAS
CSCD
北大核心
2018年第5期983-987,共5页
Journal of Jilin University:Medicine Edition
基金
内蒙古自治区卫计委医疗卫生科研计划项目资助课题(201301070)