microRNA 20a通过核因子-κB信号通路影响肺炎模型幼鼠炎症水平的机制研究  

The mechanism of microRNA 20a affecting T cell subsets and inflammatory level of pneumonia model mice through NF-κB signaling pathway

作　　者：胡正权[1] 孙欣娜 杨青[1] HU Zheng-quan;SUN Xin-na;YANG Qing(Department of Pediatrics,Hebei Seventh People's Hospital,Baoding Hebei 073000,China.)

机构地区：[1]河北省第七人民医院儿科,河北保定073000

出　　处：《临床和实验医学杂志》2020年第8期815-819,共5页Journal of Clinical and Experimental Medicine

基　　金：河北省科技局基金(编号:15ZF091)。

摘　　要：目的 探讨microRNA 20a通过核因子-κB信号通路影响肺炎模型幼鼠炎症反应和T细胞亚群分型的机制。方法 30只SPF级雄性BALB/c小鼠随机分为两组:肺炎组和健康组,每组各15只。肺炎模型组采用刺破鼻黏膜后,从鼻腔滴入50μl链球菌液法制备小鼠链球菌性肺炎模型;健康组刺破鼻黏膜后,从鼻腔滴入50μl生理盐水。将肺炎细胞分成4组:对照组、脂多糖(LSP)组、LPS+miR-20a组、LSP+miR-20a+PDTC组,分别用模拟对照物、脂多糖、mir-20a模拟物转染细胞,过表达miR-20A的细胞在37℃时用NF-κB抑制剂PDTC(100μg/L)处理30 min。用逆转录定量聚合酶链反应检测miR-20a的血清表达水平。用酶联免疫吸附(ELISA)法检测临床血清和组织细胞中白细胞介素-6(IL-6)、肿瘤坏死因子(TNF)α和C-反应蛋白(CRP)的浓度。用Western blotting法检测核因子(NF)-κBα(iκbα)和磷酸化p-NF-κB的蛋白表达水平。分析NF-κB抑制剂PDTC对miR-20a诱导的细胞炎症反应的影响。结果 肺炎组miR-20a的表达水平510. 75±50. 21,高于健康组202. 36±39. 58(P <0. 05)。肺炎组血清IL-6、TNF-α、CRP水平均高于健康组,差异有统计学意义(F <0. 05)。与对照组相比,LPS组的炎症因子IL-6、TNF-α和CRP浓度增加(F <0. 05),IκBα和p-NF-κB蛋白表达水平升高(P <0. 05)。与LPS组相比,LPS+miR-20a组细胞IL-6、TNF-α和CRP的表达水平,IκBα和p-NF-κB蛋白的表达水进一步升高(P <0. 05)。与LPS+miR-20a组比较,NF-κB抑制剂PDTC对炎症因子表达有抑制作用(P <0. 05)。结论 miR-20a在肺炎小鼠模型中表达上调,miR-20a的过表达可能通过激活NF-κB信号通路促进炎症反应。Objective To explore the mechanism of microRNA 20a influencing inflammatory response and T cell subsets in pneumonia model mice through nuclear factor-kappa B signaling pathway.Methods Animal grouping and model were established.30 SPF BALB/c male mice were randomly divided into two groups:pneumonia group and healthy group,with 15 mice in each group.The pneumonia model group was used to pierce the nasal mucosa and then instill 50μl of streptococcus solution from the nasal cavity to prepare a mouse streptococcus pneumonia model.In the healthy group,50μl saline was given from the nasal cavity after puncturing the nasal mucosa.Pneumonia cells were divided into 4 groups:control group,lipopolysaccharide(LSP)group,LPS+miR-20a group,and LSP+miR-20a+PDTC group.Cells were transfected with mock control,lipopolysaccharide,and mir-20a mimic respectively.Cells expressing miR-20A were treated with NF-κB inhibitor PDTC(100μg/L)for 30 min at 37℃.Serum expression level of microRNA-20a was detected preliminarily,and reverse transcription quantitative polymerase chain reaction(RT-QPCR)was used.The concentrations of interleukin-6(IL-6),tumor necrosis factor(TNF)alpha and C-reactive protein(CRP)in clinical serum and A549 cells were detected by ELISA.In addition,Western blotting was used to detect the protein expression levels of nuclear factor(NF)-κBα(IκBα)inhibitors and phosphorylation(p)-NF-κB inhibitors.Results The expression level of miR-20a in the pneumonia group was 510.75±50.21,which was higher than that of the healthy group 202.36±39.58(P<0.05).Serum levels of IL-6,TNF-α,and CRP were higher in the pneumonia group than those in the control group,and the differences were statistically significant(P<0.05).Compared with the control group,the concentrations of inflammatory factors IL-6,TNF-αand CRP in the LPS group increased(P<0.05),and the expression levels of IκBαand p-NF-κB protein increased(P<0.05).Compared with the LPS group,the expression levels of IL-6,TNF-αand CRP,and the expression of IκBαand p-NF-κB

关 键 词：小鼠 肺炎 microRNA-20a 炎症因子 核因子-ΚB 信号通路 脂多糖 

分 类 号：R725.6[医药卫生—儿科] R-332[医药卫生—临床医学]