吉林大学学报(医学版)  2019, Vol. 45 Issue (01): 111-116

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胡博, 王小稳, 曹建晖, 孙晓敏, 崔亚杰, 石聪聪
HU Bo, WANG Xiaowen, CAO Jianhui, SUN Xiaomin, CUI Yajie, SHI Congcong
哮喘患儿外周血单个核细胞中NLRP3炎症小体及血清中IL-1β和IL-18表达变化及其意义
Changes of expressions of NLRP3 inflammasome in peripheral blood mononuclear cells and IL-1β and IL-18 in serum in children with asthma and their significances
吉林大学学报(医学版), 2019, 45(01): 111-116
Journal of Jilin University (Medicine Edition), 2019, 45(01): 111-116
10.13481/j.1671-587x.20190121

文章历史

收稿日期: 2018-05-27
哮喘患儿外周血单个核细胞中NLRP3炎症小体及血清中IL-1β和IL-18表达变化及其意义
胡博1 , 王小稳1 , 曹建晖1 , 孙晓敏2 , 崔亚杰3 , 石聪聪1     
1. 郑州大学附属儿童医院急诊科, 河南 郑州 450000;
2. 郑州大学附属儿童医院普内科一病区, 河南 郑州 450000;
3. 郑州大学附属儿童医院重症监护室, 河南 郑州 450000
[摘要]: 目的: 观察哮喘患儿外周血单个核细胞中NOD样受体热蛋白结构域相关蛋白3(NLRP3)炎症小体表达水平及血清中下游因子白细胞介素1β(IL-1β)和白细胞介素18(IL-18)水平变化,探讨其对患儿病情评估的意义。方法: 176例哮喘患儿根据临床表现分为急性发作期组(n=91)、慢性持续期组(n=49)和缓解期组(n=36),同期从门诊体检中心选取健康儿童60名作为对照组,采用肺功能仪检查各组研究对象肺功能。采用实时荧光定量PCR法检测各组研究对象外周血单个核细胞中NLRP3、含有CARD结构域的凋亡相关斑点样蛋白(ASC)和含半胱氨酸的天冬氨酸蛋白水解酶1(Caspase-1)mRNA表达水平,采用酶联免疫吸附(ELISA)实验检测各组研究对象血清中IL-1β和IL-18水平。结果: 与对照组比较,急性发作期组、慢性持续期组和缓解期组哮喘患儿第1秒用力呼气容积占预计值百分比(FEV1%)和第1秒用力呼气容积所占肺活量比值(FEV1/FVC)均降低,且急性发作期组 < 慢性持续期组 < 缓解期组,组间比较差异有统计学意义(P < 0.05);哮喘患儿外周血单个核细胞中NLRP3、ASC和Caspase-1 mRNA表达水平及血清中IL-1β和IL-18水平均高于对照组(P < 0.01);急性发作期组患儿外周血单个核细胞中NLRP3、ASC和Caspase-1 mRNA表达水平及血清中IL-1β和IL-18水平高于慢性持续期组和缓解期组(P < 0.05),且慢性持续期组患儿高于缓解期组(P < 0.05)。Pearson相关分析,哮喘患儿外周血单个核细胞中NLRP3 mRNA表达水平与ASC、Caspase-1 mRNA表达水平和血清中IL-1β、IL-18水平均呈正相关关系(P < 0.05),而与FEV1%和FEV1/FVC呈负相关关系(P < 0.05);ASC mRNA表达水平与Caspase-1 mRNA表达水平和血清中IL-1β、IL-18水平呈正相关(P < 0.05),而与FEV1%和FEV1/FVC呈负相关关系(P < 0.05);Caspase-1 mRNA表达水平与血清中IL-1β和IL-18水平呈正相关关系(P < 0.05),而与FEV1%和FEV1/FVC呈负相关关系(P < 0.05);血清中IL-1β水平与FEV1%和FEV1/FVC呈负相关关系(P < 0.05);IL-18水平与FEV1%和FEV1/FVC呈负相关关系(P < 0.05)。结论: 哮喘患儿外周血NLRP3炎症小体及下游因子IL-1β和IL-18水平升高,且与哮喘患儿的临床分期有关,NLRP3炎症小体通路可能促进了儿童哮喘的发病过程。
关键词: 支气管哮喘    NOD样受体热蛋白结构域相关蛋白3    炎症小体    白细胞介素1β    白细胞介素18    
Changes of expressions of NLRP3 inflammasome in peripheral blood mononuclear cells and IL-1β and IL-18 in serum in children with asthma and their significances
HU Bo1, WANG Xiaowen1, CAO Jianhui1, SUN Xiaomin2, CUI Yajie3, SHI Congcong1     
1. Department of Emergency, AffiliatedChildren's Hospital, Zhengzhou University, Zhengzhou 450000, China;
2. Department of Internal Medicine, Affiliated Children's Hospital, Zhengzhou University, Zhengzhou 450000, China;
3. Department of Intensive Care Unit, Affiliated Children's Hospital, Zhengzhou University, Zhengzhou 450000, China
[ABSTRACT]: Objective: To investigate the changes of expressions of NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome in the peripheral blood mononuclear cells and downstream factors interleukin-1β (IL-1β) and interleukin-18 (IL-18) in serum in the children with asthma, and to explore their significances on assessing the condition of the children. Methods: A total of 176 cases of children with asthma were divided into acute exacerbation group (n=91), chronic persistent group (n=49) and clinical remission group (n=36) according to the clinical manifestation. During the same period, 60 healthy children were selected from the outpatient physical examination center as control group.The pulmonary function of children was checked with lung function instrument. The expression levels of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC) and cysteinyl aspartate-specific proteinase-1 (Caspase-1) mRNA in peripheral blood mononuclear cells of the subjects in various groups were detected by using real-time quantitative PCR. The serum levels of IL-1β and IL-18 of the subjects in various groups were detected by using enzyme-linked immunosorbent assay (ELISA). Results: Compared with control group, the forced expiratory volume in 1 second percentage of predicted value (FEV1%) and fixed ratio of forced expiratory volume in the first second/forced vital capacity (FEV1/FVC) of the children in acute exacerbation, chronic persistent and clinical remission groups were decreased (P < 0.05);acute exacerbation group < chronic persistent group < clinical remission group, and there were significant differences between various groups(P < 0.05).The levels of NLRP3, ASC and Caspase-1 mRNA in the peripheral blood mononuclear cells and serum levels of IL-1β and IL-18 in the children with asthma were higher than those in control group (P < 0.01).The expression levels of NLRP3, ASC and Caspase-1 mRNA in the peripheral blood mononuclear cells of the children in acute exacerbation group were higher than those in chronic persistent and clinical remission groups (P < 0.05), and the expression levels of NLRP3, ASC and Caspase-1 mRNA in chronic persistent group were higher than those in clinical remission group (P < 0.05).Pearson correlation analysis showed that the expression level of NLRP3 mRNA in the peripheral blood mononuclear cells of asthmatic children was positively correlated with the expression levels of ASC, Caspase-1 mRNA and the serum levels of IL-1β and IL-18 (P < 0.05), while it was negatively correlated with FEV1% and FEV1/FVC; the expression level of ASC mRNA was positively correlated with the expression level of Caspase-1 mRNA and the serum levels of IL-1β and IL-18 (P < 0.05), while it was negatively correlated with FEV1% and FEV1/FVC (P < 0.05); the expression level of Caspase-1 mRNA was positively correlated with the expression level of Caspase-1 mRNA and the serum levels of IL-1β and IL-18 (P < 0.05), while it was negatively correlated with FEV1% and FEV1/FVC (P < 0.05); the serum level of IL-1β was negatively correlated with FEV1% and FEV1/FVC (P < 0.05), and the serum level of IL-18 was negatively correlated with FEV1% and FEV1/FVC (P < 0.05). Conclusion: The expression levels of NLRP3 inflammasome and the downstream factor IL-1β and IL-18 in peripheral blood of the children with asthma are increased, and they are related to the clinical stage of the children with asthma. NLRP3 inflammasome pathway might promote the pathogenesis of asthma in the children.
KEYWORDS: children     bronchial asthma     NOD-like receptor family pyrin domain containing 3     inflammasome     interleukin-1β     interleukin-18    

支气管哮喘(简称哮喘)作为儿童期呼吸系统常见病,是影响患儿健康的主要慢性病,且发病率呈逐年升高趋势,特别是年幼儿童高发[1]。研究[2]表明:哮喘作为多种炎症细胞参与的慢性炎症性疾病,长期的气道炎症可导致气道黏膜损伤、气道反应性增加及气道重构。炎症小体作为机体内多种蛋白在内外刺激下形成的复合体,可通过调控促炎因子释放而对内外刺激应答[3],其中,NOD样受体热蛋白结构域相关蛋白3(NOD-like receptor family pyrin domain containing 3,NLRP3)炎症小体是重要类型之一,由NLRP3、含有CARD结构域的凋亡相关斑点样蛋白(apoptosis-associated speck-like protein containing a CARD,ASC)和含半胱氨酸的天冬氨酸蛋白水解酶1(cysteinyl aspartate-specific proteinase-1,Caspase-1)相互作用组成,不仅在细菌等病原体导致的固有免疫应答中发挥重要作用,而且与非感染性炎症反应关系密切[4]。研究[5]表明:NLRP3炎症小体可通过调控Caspase-1活性而促进白细胞介素1β(interleukin-1β,IL-1β)和白细胞介素18(interleukin-18,IL-18)大量成熟和释放,在炎症反应中发挥重要作用。目前,NLRP3炎症小体通路及其下游细胞因子在儿童哮喘发病及病情进展中的表达变化鲜有报道。本研究旨在分析哮喘患儿外周血单个核细胞中NLRP3炎症小体通路及下游炎症因子IL-1β和IL-18水平的变化,探讨其在哮喘患儿病情评估及发病中的意义。

1 资料与方法 1.1 临床资料

选取2016年4月—2017年12月在郑州大学附属儿童医院治疗的哮喘患儿176例作为哮喘组,其中男性98例,女性78例,年龄1~14岁,平均年龄(6.43±2.74)岁,均符合中华医学会儿科学分会呼吸学组制定的《儿童支气管哮喘诊断与防治指南(2016年版)》[6]中的相关诊断标准;排除心、肝、肾等重要脏器严重功能障碍及肺结核、恶性肿瘤、风湿性疾病和肾小球肾炎者,以及发病前30d内使用肾上腺皮质激素或免疫调节剂者。根据哮喘患儿临床表现,分为急性发作期组、慢性持续期组和缓解期组;其中急性发作期组患儿91例,男性54例,女性37例,年龄(1~13)岁,平均年龄(6.44±2.54)岁;慢性持续期组患儿49例,男性27例,女性22例,年龄(1~14)岁,平均年龄(6.18±2.90)岁;缓解期组患儿36例,男性19例,女性17例,年龄(1~14)岁,平均年龄(6.73±3.05)岁。从同期门诊体检中心选取60名健康儿童作为对照组,均排除心、肝和肾等重要脏器严重功能障碍者和有特异性疾病史及呼吸道感染史者,其中男性38名,女性22名,年龄(1~14)岁,平均年龄(6.71±3.14)岁。本研究通过医院伦理委员会批准,所有患儿监护人均知情同意。

1.2 标本采集

所有研究对象均采取晨起空腹肘静脉血10mL,分装于含EDTA抗凝管和无抗凝血管内,各5mL。含EDTA抗凝管中的静脉血利用人淋巴细胞分离液(购自上海索宝生物科技公司)分离外周血单个核细胞,加入细胞裂解液,利用Trizol总RNA提取试剂盒(购自美国Invitrogen公司)提取总RNA,使用紫外分光光度计(购自上海美谱达仪器公司)检测总RNA纯度和浓度,合格标准为吸光度[A(260)]值/A(280)为1.80~2.20,按照逆转录试剂盒(购自日本TaKaRa公司)将总RNA逆转录为cDNA,-70℃冰箱保存;无抗凝血管中的静脉血采用离心机以2500r·min-1离心15 min,留取血清,-70℃冰箱保存备用。

1.3 实时荧光定量PCR法检测各组研究对象外周血单个核细胞中

NLRP3、ASC和Caspase-1 mRNA表达水平以cDNA为模板,按PCR扩增试剂盒(购自日本TaKaRa公司)说明书采用实时荧光定量PCR仪(购自瑞士Roche公司)对引物进行扩增。引物序列见表 1,均由上海生工生物公司设计合成。PCR反应条件:92℃、5 min,92℃、30s,56℃、60s,70℃、30s,连续循环38次,每个样品设6个平行复孔。采用2-△△Ct法计算外周血单个核细胞中NLRP3、ASC和Caspase-1 mRNA表达水平。

表 1 引物序列 Tab. 1 Sequences of primers
Primer Sequence (5′-3′)
NLRP3 Forward: GCTGGTCTTGAATTCCTCA
Reverse: GGCACACGGATGAGTCTTT
ASC Forward: AACCCAAGCAAGATGCGGAAG
Reverse: TTAGGGCCTGGAGGAGCAAG
Caspase-1 Forward: CCAGGACATTAAAATAAGGAAACTGT
Reverse: CCAAAAACCTTTACAGAAGAATCTC
β-actin Forward: ACTCTTCCAGCCTTCCTTCC
Reverse: CGTACAGGTCTTTGCGGATG
1.4 酶联免疫吸附(ELISA)实验检测各组研究对象血清中IL-1β和IL-18水平

采用ELISA实验检测各组研究对象血清中IL-1β和IL-18水平,试剂盒购自南京信帆生物技术公司,所有操作均按试剂盒说明书在标准实验室完成。

1.5 肺功能检测

采用肺功能仪(购自德国JAEGER公司)对各组研究对象肺功能重复检查3次,选最稳定的1次结果作为最终肺功能检查结果,包括第1秒用力呼气容积占预计值百分比(FEV1%)和第1秒用力呼气容积所占肺活量比值(FEV1/FVC)。检查前停止使用激素类、茶碱类及抗胆碱类药物。

1.6 统计学分析

采用SPSS 17.0统计软件进行统计学分析。各组研究对象性别构成比以百分率表示,组间比较采用χ2检验。各组研究对象年龄、FEV1%、FEV1/FVC和外周血单个核细胞中NLRP3 mRNA、ASC mRNA、Caspase-1 mRNA表达水平和血清中IL-1β及IL-18水平均以x±s表示,2组间样本均数比较采用两独立样本t检验,多组间样本均数比较采用单因素方差分析。各检测指标之间的相关性采用Pearson相关分析。以P < 0.05为差异有统计学意义。

2 结果 2.1 各组研究对象一般临床资料

各组研究对象性别构成比和年龄比较差异均无统计学意义(P>0.05)。与对照组比较,急性发作期、慢性持续期和缓解期组哮喘患儿FEV1%和FEV1/FVC均降低(P < 0.05),且急性发作期组 < 慢性持续期组 < 缓解期组,组间比较差异有统计学意义(P < 0.05)。见表 2

表 2 各组研究对象一般临床资料 Tab. 2 General clinical materials of subjects in various groups
Group n Gender[n(η/%)] Age(year) FEV1%(η/%) FEV1/FVC(η/%)
Male Female
Control 60 38(63.33) 22(36.67) 6.71±3.14 92.09±5.03 96.40±8.92
Acute exacerbation 91 54(59.34) 37(40.66) 6.44±2.54 69.67±12.55*△# 66.74±9.10*△#
Chronic persistent 49 27(55.10) 22(44.90) 6.18±2.90 78.82±7.65*△ 76.63±10.09*△
Clinical remission 36 19(52.78) 17(47.22) 6.73±3.05 85.47±8.39* 83.36±7.87*
χ2/F 1.322 0.395 72.437 132.011
P 0.724 0.757 < 0.01 < 0.01
* P < 0.05 compared with control group; P < 0.05 compared with clinical remission group;# P < 0.05 compared with chronic persistent group.
2.2 各组研究对象外周血单个核细胞中NLRP3、ASC和Caspase-1 mRNA表达水平及血清中IL-1β和IL-18水平

与对照组比较,哮喘组患儿外周血单个核细胞中NLRP3、ASC和Caspase-1 mRNA表达水平及血清中IL-1β和IL-18水平明显升高(P < 0.01)。见表 3

表 3 各组研究对象外周血单个核细胞中NLRP3、ASC和Caspase-1 mRNA表达水平及血清中IL-1β和IL-18水平 Tab. 3 Expression levels of NLRP3, ASC and Caspase-1 mRNA in peripheral blood mononuclear cells and levels of IL-1β and IL-18 in serum of subjects in various groups
(x±s)
Group n NLRP3 mRNA ASC mRNA Caspase-1 mRNA IL-1β[ρB/(ng·L-1)] IL-18[ρB/(ng·L-1)]
Control 60 1.14±0.08 1.10±0.08 1.10±0.11 2.55±1.12 247.92±76.53
Asthma 176 1.98±0.30 1.72±0.25 1.84±0.29 7.26±2.35 382.97±81.77
t 21.400 19.038 19.486 14.978 11.225
P < 0.01 < 0.01 < 0.01 < 0.01 < 0.01
2.3 不同分期组哮喘患儿外周血单个核细胞中NLRP3、ASC和Caspase-1 mRNA表达水平及血清中IL-1β和IL-18水平

哮喘急性发作期患儿外周血单个核细胞中NLRP3、ASC和Caspase-1 mRNA表达水平及血清IL-1β和IL-18水平高于慢性持续期和缓解期患儿,且慢性持续期患儿高于缓解期患儿(P < 0.05)。见表 4

表 4 不同分期哮喘患儿外周血单个核细胞中NLRP3、ASC和caspase-1 mRNA表达水平及血清中IL-1β和IL-18水平 Tab. 4 Expressionlevels of NLRP3, ASC and caspase-1 mRNA in peripheral blood mononuclear cells and levels of IL-1β and IL-18 in serum of children with different stages of asthma
(x±s)
Group n NLRP3 mRNA ASC mRNA Caspase-1 mRNA IL-1β[ρB/(ng·L-1)] IL-18[ρB/(ng·L-1)]
Acute exacerbation 91 2.22±0.16*△ 1.91±0.09*△ 2.06±0.17*△ 8.91±1.55*△ 427.85±61.34*△
Chronic persistent 49 1.86±0.12* 1.64±0.14* 1.73±0.13* 6.47±1.34* 365.81±62.85*
Clinical remission 36 1.55±0.10 1.35±0.11 1.44±0.10 4.18±1.10 292.86±65.83
F 325.988 354.143 238.586 155.077 62.351
P < 0.01 < 0.01 < 0.01 < 0.01 < 0.01
* P < 0.05 compared with clinical remission group; P < 0.05 compared with chronic persistent group.
2.4 哮喘患儿各项指标相关性分析

Pearson相关分析结果显示:哮喘患儿外周血单个核细胞中NLRP3 mRNA表达水平与ASC、Caspase-1 mRNA表达水平和血清中IL-1β、IL-18水平呈正相关关系(P < 0.05),而与FEV1%和FEV1/FVC呈负相关关系(P < 0.05);ASC mRNA表达水平与Caspase-1 mRNA表达水平和血清中IL-1β、IL-18水平呈正相关关系(P < 0.05),而与FEV1%和FEV1/FVC呈负相关关系(P < 0.05);Caspase-1 mRNA表达水平与血清中IL-1β和IL-18水平呈正相关关系(P < 0.05),而与FEV1%和FEV1/FVC呈负相关关系(P < 0.05);血清中IL-1β和IL-18水平与FEV1%和FEV1/FVC呈负相关关系(P < 0.05)。见表 5

表 5 哮喘患儿各项指标相关性(相关系数r) Tab. 5 Correlations of various indexes in children with asthma (correlation coefficient r)
Index NLRP3 ASC Caspase-1 IL-1β IL-18 FEV1% FEV1/FVC
NLRP3 - 0.814* 0.739* 0.695* 0.547* -0.511* -0.888*
ASC 0.814* - 0.775* 0.706* 0.570* -0.476* -0.497*
Caspase-1 0.739* 0.775* - 0.669* 0.516* -0.494* -0.475*
IL-1β 0.695* 0.706* 0.669* - 0.509* -0.414* -0.471*
IL-18 0.547* 0.570* 0.516* 0.509* - -0.299* -0.370*
“-”:No data; * P < 0.05.
3 讨论

研究[7-8]表明:早期、规范化干预管理对促进儿童哮喘康复和改善预后具有重要意义。目前,临床上就诊的多数哮喘患儿症状较为轻微,临床医师可以根据典型症状进行诊治,但对于一些症状不典型或发病早期患儿诊断相对比较困难,且一些并发症状亦会影响哮喘诊治[9]。哮喘作为一种慢性气道炎症性疾病,对于生长发育期的儿童,即使表现出相同的临床症状,也不能真实反映其呼吸道炎症水平[10],因此积极探索反映哮喘患儿病情的生物学标志物,对指导临床开展个体化治疗及改善哮喘患儿预后具有重要意义。

NLRP3炎症小体是固有免疫和应激系统的重要防御成分,在炎症性疾病和自身免疫性疾病和肿瘤等多种疾病发生发展中发挥重要作用[11-12]。正常情况下,NLRP3处于非活性的抑制状态,一旦被侵入机体的病原微生物或自身产生的危险信号激活,可与ASC和Caspase-1相互作用形成炎症小体,并促进Caspase-1成熟活化,而活化的Caspase-1又可进一步促进IL-1β成熟分泌,从而引发炎症性疾病[13-14]。相关动物实验[15]结果显示:NLRP3可影响巨噬细胞极化,卵清蛋白诱导的过敏性哮喘小鼠支气管肺泡灌洗液中M2细胞数量增加,NLRP3炎症体被激活。研究[16]显示:卵清蛋白致敏小鼠气道反应性升高,炎症增加,纳米颗粒暴露会增加卵清蛋白致敏小鼠肺组织中NLRP3和Caspase-1的表达,导致活性Caspase-1的产生。TSAI等[17]通过siRNA转染沉默人类支气管上皮细胞中NLRP3基因可降低过敏原粉尘螨介导的细胞凋亡及IL-1β分泌,提示NLRP3可能在粉尘螨介导的哮喘发病中发挥重要作用。有研究[18]通过检测哮喘患儿及其家人炎性体基因功能多态性显示:功能获得性NLRP1变体rs11651270、rs12150220和rs2670660与哮喘有关联,rs11651270和rs2670660与哮喘患儿哮喘严重程度及IgE水平有关。本研究结果显示:哮喘患儿外周血中NLRP3炎症小体组成成分及下游效应因子IL-1β、IL-18水平较对照组明显升高,提示NLRP3炎症小体通路可能参与了哮喘患儿发病过程。ROSSIOS等[19]指出:NLRP3炎性体在重症哮喘患者痰标本中表达水平最高。本研究结果显示:急性发作期患儿外周血单个核细胞中NLRP3、ASC和Caspase-1 mRNA表达水平和血清IL-1β和IL-18水平高于哮喘慢性持续期和缓解期患儿,且慢性持续期高于缓解期,说明外周血中NLRP3炎症小体组成成分及下游效应因子IL-1β和IL-18水平与哮喘患儿临床分期有关,可能参与了哮喘患儿疾病进展过程。

FEV1%是目前诊断哮喘及治疗效果评估的主要指标[20],本研究结果显示:与对照组比较,急性发作期、慢性持续期和缓解期哮喘患儿FEV1%和FEV1/FVC均降低,且急性发作期 < 慢性持续期 < 缓解期,进一步说明哮喘患儿出现了肺功能降低,特别是急性发作期患儿肺功能明显降低。Pearson相关分析显示:哮喘患儿外周血单个核细胞中NLRP3、ASC和Caspase-1 mRNA表达水平及血清中IL-1β和IL-18水平互相之间均呈正相关关系,且均与FEV1%和FEV1/FVC呈负相关关系,进一步说明NLRP3炎症小体通路参与了哮喘患儿发病及进展过程,可作为反映哮喘患儿病情分期及气道炎症水平的指标。

综上所述,哮喘患儿外周血NLRP3炎症小体及下游因子IL-1β和IL-18表达水平升高,且与临床分期有关,NLRP3炎症小体通路可能参与了哮喘患儿发病过程,有望成为哮喘患儿诊断及病情监测的潜在指标。

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