时间:2024-03-28 15:06 来源:当代医学 作者:毛黎明,苏保宁*,徐珊珊,董必浩,江丽红
论著栏目
(上海市嘉定区中医医院儿科,上海 201822)
资助项目:上海市中医药管理局专科培育项目[ZY〔2019-2020〕-ZYBZ-34];上海市中医药管理局优势病种项目[ZY〔2018-2020〕-ZYBZ-40];上海市嘉定区卫健委科研课题(2018-KY-ZYY-11)
*通信作者:苏保宁,E-mail:252269819@qq.com
摘要: 目的 探讨白细胞介素-4(IL-4)-589和IL-4受体(IL-4R)rs1801275基因多态性与哮喘患儿IL-4和总免疫球蛋白E(TIgE)含量变化的相关性。方法 选取2017年9月至2020年12月于上海市嘉定中医医院儿科收治的90例哮喘发作患儿作为实验组,另入取同期90名健康体检儿童作为空白组,两组均应用聚合酶链反应(PCR)和DNA测序法进行基因分型。同时采用酶联免疫吸附试验法(ELISA)法检测两组血清IL-4、TIgE水平。结果 两组IL-4R基因rs1801275位点基因型频率和等位基因频率、IL-4基因-589位点基因型频率比较差异无统计学意义;实验组C等位基因频率低于空白组,T等位基因频率高于空白组,差异有统计学意义(P<0.05)。两组TT血清IL-4含量均高于CT、CC,CT均高于CC,且实验组CC、CT、TT血清IL-4含量均高于空白组,差异有统计学意义(P<0.05)。组GG血清IL-4含量高于AA、AG,AG高于AA,且实验组AA、AG、GG血清IL-4含量均高于空白组,差异有统计学意义(P<0.05)。两组TT血清TIgE含量高于CT、CC,CT高于CC,且实验组CC、CT、TT血清TIgE含量均高于空白组,差异有统计学意义(P<0.05)。两组GG血清TIgE含量均高于AA、AG,AG均高于AA,且实验组AA、AG、GG血清TIgE含量均高于空白组,差异有统计学意义(P<0.05)。结论 哮喘患儿 IL-4 基因-589 位点和IL-4Rrs1801275基因多态性与健康未显示出明显差异,TT和GG基因型哮喘患儿IL-4和TIgE水平较其他对应基因型升高,机制需进一步研究。
关键词: 儿童哮喘;IL-4;TIgE;基因启动子;IL-R;多态性
Clinical study on the correlation between interleukin-4 and interleukin-4 receptor gene polymorphisms and serum interleukin-4 and serum total immunoglobulin E content in children with asthma
MAO Liming, SU Baoning, XU Shanshan, DONG Bihao, JIANG Lihong
(Department of Pediatrics, Shanghai Jiading District Chinese Medicine Hospital, Shanghai, 201822, China)
Abstract: Objective To investigate the relationship between interleukin-4 (IL-4) -589 and IL-4 receptor (IL-4R) rs1801275 gene polymorphisms and the levels of IL-4 and total immunoglobulin E (TIgE) in children with asthma. Methods 90 children with asthma attack admitted to the Department of Pediatrics of Shanghai Jiading Hospital of Traditional Chinese Medicine from September 2017 to December 2020 were selected as the experimental group, and 90 healthy children in the same period were enrolled as the blank group. The polymerase chain reaction(PCR) and DNA sequencing were used for genotyping between thetwo groups. The serum levels of IL-4 and TIgE were detected by enzyme-linked immunosorbent assay (ELISA). Results There were no significant differences in genotype frequency and allele frequency of rs1801275 and genotype frequency of IL-4 gene -589 between the two groups. The frequency of C allele in the experimental group was lower than that in the blank group, and the frequency of T allele was higher than that in the blank group, the differences were statistically significant (P<0.05). The serum IL-4 content of TT of the two groups was higher than that of CT and CC, and the serum IL-4 content of CT and TT in experimental group was higher than thosein blank group, the differenceswere statistically significant (P<0.05). The serum IL-4 content of the group GG was higher than that of AA and AG, and the serum IL-4 content of the group AA, AG and GG was higher than thosein the blank group, and the differenceswere statistically significant (P<0.05). The serum TIgE content of TT in two groups was higher than that of CT and CC, and that of CT was higher than that of CC, CT and TT in experimental group were higher than thosein the blank group, the differenceswere statistically significant (P<0.05). The serum TIgE content of GG of the two groups were higher than those of AA and AG, and AG was higher than that of AA, AG and GG in experimental group, and the serum TIgE content of AA, AG and GG were higher than thosein the blank group, the differenceswere statistically significant (P<0.05). Conclusion IL-4 gene-589 and IL-4Rrs1801275 polymorphisms in children with asthma do not show significant differences from those in healthy children. The levels of IL-4 and TIgE in children with TT and GG genotype are higher than those in other genotypes, and the mechanism needs further study.
Keywords: Childhood asthma; Interleukin-4; Total immunoglobulin E; Gene promoter; Interleukin-4 receptor; Polymorphism
哮喘是小儿门急诊高发病种之一,发病机制复杂,世界各地发病率存在一定差异[1]。上海地区儿童哮喘发病率约占7%左右[2],高于全国水平。哮喘作为异质性疾病,表现形式不一,尤其3~6岁的幼儿,反复咳嗽喘息为主要症状,严重影响患儿生命健康[3]。白细胞介素-4(interleukin-4 receptor,IL-4)和总免疫球蛋白E(total immunoglobulin E,TIgE)在哮喘发作机制中发挥核心作用,有研究表明,哮喘发作时IL-4水平增加,但涉及基因启动子区相关性,结论常不一致[4-5]。基于此,本研究旨在探究哮喘急性发作患儿血清TIgE和IL-4含量变化与IL-4启动子区-589和IL-4受体(IL-4 receptor,IL-4R)1801275位点多态性相关性分析,并初步分析儿童哮喘发作机制,现报道如下。
1 资料与方法
1.1 临床资料 选取2017年9月至2020年12月于上海市嘉定中医医院儿科收治的90例哮喘急性期发作患儿为实验组,另选取同期90名健康体检儿童作为空白组。实验组男48例,女42例;年龄3~9岁,平均(4.21±1.49)岁;空白组男46名,女44名;年龄3~9岁,平均(5.16±1.82)岁。两组临床资料比较差异无统计学意义,具有可比性。本研究经嘉定中医院医学伦理委员会审核批准(审批号:2018-007),患儿家属对本研究知情同意并签署知情同意书。
纳入标准:①年龄3~14岁,性别不限;②符合西医小儿哮喘诊断标准[6],同时属于急性发作期;③病情属于中度发作。排除标准:①有严重脏器疾病或先天性疾病;②病情为轻度发作;③哮喘发作持续状态者。
1.2 方法 ①DNA提取及聚合酶链式反应(polymerase chain reaction,PCR)扩增。采集受检者外周静脉血2.5 ml,1 ml用乙二胺四乙酸(ethylene diamine tetraacetic acid,EDTA)
二钠抗凝放置液氮罐备用,用人类基因组DNA提取试剂盒(上海生物工程有限公司)提取基因组DNA,另1.5 ml采用型号为KS50R的离心机(盐城市凯特实验仪器有限公司)离心,取血清于﹣20℃冰箱保存备用。使用Primers5.0引物设计软件,以分段重复、覆盖的原则设计,设计的引物最终与美国国家生物技术信息中心(National Center of Biotechnology Information,NCBI)的生物大分子序列比对搜索工具(basic local alignment search tool,BLAST)核酸数据库、人类全基因组序列进行BLAST比对,验证引物的特异性。验证后的引物由上海生物工程公司进行验证。IL-4-589 P5 TGGGTAAGGACCTTATGGACC,P3 GGTGGCATCTTGGAAACTGT(约 198 bp);Rs1801275 5F AGATCCTCCGCCGAAATGTCCT,5R ACCCTGCTCCACCGCATGTA (约100 bp)。采用30 μl反应体系:15 μl扩增混合物,2 μl镁离子,1 μl引物,2 μl脱氧核糖核酸聚合酶,10 μl超纯水。95 ℃预变性5 min,94 ℃变性30 s,55.6 ℃退火30 s,72 ℃延伸45 s,循环35次,最后72 ℃延伸10 min,扩增后产物经纯化后再回收,测序。②指标检测。采用酶联免疫吸附试验法(enzyme linked immuno sorbent assay,ELISA)检测血清IL-4与TIgE,试剂盒购自联科生物,产品号:EK104-01。
1.3 观察指标 ①比较分析两组患儿IL-4基因-589C/T和IL-4Rrs1801275A/G基因多态性检测结果;②对比分析两组血清IL-4在IL-4基因-589C/T和IL-4Rrs1801275A/G不同基因型含量变化;③对比分析两组血清TIgE在IL-4基因-589C/T和IL-4Rrs1801275A/G不同基因型含量变化。
1.4 统计学方法 采用SPSS 23.0统计学软件进行数据分析,符合正态分布的计量资料以“x±s”表示,采用t检验,多组比较采用F检验,计数资料以[n(%)]表示,采用χ2检验,当min(T)>5,采用Fisher确切概率法检验,以P<0.05为差异有统计学意义。
2 结果
2.1 两组基因检测结果比较 两组IL-4Rrs1801275位点基因型频率和等位基因频率、IL-4基因-589位点基因型频率比较差异无统计学意义;实验组C等位基因频率低于空白组,T等位基因频率高于空白组,差异有统计学意义(P<0.05)。见表1。
表1 两组基因检测结果比较[n(%)]
Table 1 Comparison of gene detection results between two groups [n(%)]
组别 | 例数 | 基因型频率 | 等位基因频率 | ||||||||
IL-4基因-589位点 | IL-4Rrs1801275位点 | IL-4基因-589位点 | IL-4Rrs1801275 | ||||||||
CC | CT | TT | AA | AG | GG | C等位基因 | T等位基因 | A等位基因 | G等位基因 | ||
空白组 | 90 | 34(37.78) | 16(17.78) | 40(44.44) | 53(58.89) | 15(16.67) | 22(24.44) | 53(58.89) | 37(41.11) | 71(78.89) | 19(21.11) |
实验组 | 90 | 31(34.44) | 14(15.56) | 45(50.00) | 59(65.56) | 11(12.22) | 20(22.22) | 39(43.33) | 51(63.33) | 66(73.33) | 24(26.67) |
X2值 | 0.566 0.754 | 1.032 0.597 | 4.358 0.037 | 0.764 0.382 | |||||||
P值 |
注:IL-4,白细胞介素-4
2.2 两组IL-4基因-589位点不同基因型的血清IL-4含量比较 两组TT基因型血清IL-4含量均高于CT、CC,CT均高于CC,且实验组CC、CT、TT血清IL-4含量均高于空白组,差异有统计学意义(P<0.05)。见表2。
表2 两组IL-4基因-589位点的血清IL-4含量比较(x±S,U/ml)
Table 2 Comparison of serum IL-4 level at IL-4-589 locus between the two groups (x±S, U/ml)
组别 | CC | CT | TT | F值 | P值 | |||
例数 | 血清IL-4含量 | 例数 | 血清IL-4含量 | 例数 | 血清IL-4含量 | |||
空白组 | 34 | 21.17±7.89 | 16 | 31.46±5.98a | 40 | 43.39±5.22ab | 108.504 | 0.001 |
实验组 | 31 | 32.83±11.86 | 14 | 42.39±5.62a | 45 | 52.39±4.52ab | 23.761 | 0.021 |
t值 | 4.705 | 5.136 | 8.520 | |||||
P值 | <0.001 | <0.001 | <0.001 |
注:IL-4,白细胞介素-4。与本组CC比较,aP<0.05;与本组TT比较,bP<0.05
2.3 两组IL-4Rrs1801275位点不同基因型的血清IL-4含量比较 两组GG血清IL-4含量高于AA、AG,AG高于AA,且实验组AA、AG、GG血清IL-4含量均高于空白组,差异有统计学意义(P<0.05)。见表3
表3 两组不同IL-4Rrs1801275位点的血清IL-4含量比较(x±S,U/ml)
Table 3 Comparison of serum IL-4 level at IL-4Rrs1801275 locus between the two groups (x±s, U/ml)
组别 | AA | AG | GG | F值 | P值 | |||
例数 | 血清IL-4含量 | 例数 | 血清IL-4含量 | 例数 | 血清IL-4含量 | |||
空白组 | 53 | 23.49±3.08 | 15 | 32.23±2.37a | 22 | 40.79±3.92ab | 14.153 | 0.001 |
实验组 | 59 | 32.17±5.11 | 11 | 46.46±5.98a | 20 | 51.39±5.48ab | 18.352 | 0.016 |
t值 | 41.552 | 39.621 | 40.564 | |||||
P值 | 0.032 | 0.033 | 0.035 |
注:IL-4,白细胞介素-4。与本组AA比较,aP<0.05;与本组AG比较,bP<0.05
2.4 两组IL-4-589位点不同基因型的血清TIgE含量比较 两组GG血清IL-4含量高于AA、AG,AG高于AA,且实验组AA、AG、GG血清IL-4含量均高于空白组,差异有统计学意义(P<0.05),见表4。
表4 两组不同IL-4-589位点的血清TIgE含量比较(x±S,ug/L)
Table 4 Comparison of serumTIgE level at IL-4-589 locus between the two groups (x±S, ug/L)
组别 | CC | CT | TT | F值 | P值 | |||
例数 | 血清TIgE含量 | 例数 | 血清TIgE含量 | 例数 | 血清TIgE含量 | |||
空白组 | 34 | 97.17±32.45 | 16 | 227.46±58.46a | 40 | 473.39±62.15ab | 41.391 | <0.001 |
实验组 | 31 | 156.83±39.86 | 14 | 326.39±63.62a | 45 | 522.39±75.34ab | 61.682 | <0.001 |
t值 | 17.224 | 21.334 | 27.693 | |||||
P值 | <0.001 | <0.001 | <0.001 |
注:TIgE,总免疫球蛋白E。与本组CC比较,aP<0.05;与本组CT比较,bP<0.05
2.5 两组IL-4Rrs1801275位点不同基因型血清TIgE含量比较 两组GG血清TIgE含量均高于AA、AG,AG均高于AA,且实验组AA、AG、GG血清TIgE含量均高于空白组,差异有统计学意义(P<0.05),见表5
表5 两组不同IL-4Rrs1801275位点血清TIgE含量比较(x±S,μg/L)
Table 5 Comparison of serumTIgE level atIL-4Rrs1801275 locus between two groups (x±S, μg/L)
组别 | AA | AG | GG | F值 | P值 | |||
例数 | 血清TIgE含量 | 例数 | 血清TIgE含量 | 例数 | 血清TIgE含量 | |||
空白组 | 53 | 115.36±28.68 | 15 | 279.23±52.39a | 22 | 324.79±63.92ab | 49.722 | <0.001 |
实验组 | 59 | 163.52±52.53 | 11 | 346.46±71.98a | 20 | 551.39±75.48ab | 59.173 | <0.001 |
t值 | 11.551 | 20.624 | 11.563 | |||||
P值 | <0.001 | <0.001 | <0.001 |
注:TIgE,总免疫球蛋白E。与本组AA比较,aP<0.05;与本组AG比较,bP<0.05
3 讨论
哮喘是儿童期最常见的慢性呼吸道疾病之一。多种细胞和细胞组分共同参与的气道慢性炎症性疾病,这种慢性炎症导致气道反应性的增加,通常出现广泛多变的可逆性气流受限,并引起反复发作性的喘。其中炎症因子堆积是导致慢性气道炎症主要机制,细胞因子IL-4和IgE起主要作用。IL-4作为哮喘发作中重要因子之一[8],作用于IL-4R后启动一系列反应起作用。IL-4-33位点和-589位点基因多态性的突变可能与功能变化相关[9];IL-4R功能区更加复杂,其中,有六个位点多态性变化与IL-4含量具有相关性[10]。哮喘发作时,Th1和辅助型T细胞2(T helper 2 cell,Th2)细胞功能失调,Thl类细胞因子分泌不足,Th2类细胞因子增加,Th1/Th2比例失衡,IL-4介导B细胞合成IgE增加,高水平IgE与肥大细胞表面受体结合,肥大细胞活化脱颗粒,具有弱吞噬功能[11]。存在于血液中的肥大细胞活化的颗粒含有肝素、组织胺、5-羟色胺,由细胞崩解后释放出以及颗粒中的其他物质,产生过敏反应及炎症变化[12]。本研究选择IL-4基因-589和IL-4RRs1801275作为观察位点,根据遗传学原理,Hardy-Weillberg平衡定律,入院后首次检测,将IL-4做了基因型实际值与预测值的比对,对比结果提示检测符合遗传学规律。与相关文献[13]相似,提示此次检测符合要求。有文献报道,IgE和IL-4在哮喘发作时升高,有过敏史者,血清IgE升高的发生率在IL-4Rα基因型AA和AG高于对照组[14-15]。
本研究结果显示,两组IL-4Rrs1801275位点基因型频率和等位基因频率、IL-4基因-589位点基因型频率比较差异无统计学意义;实验组C等位基因频率低于空白组,T等位基因频率高于空白组,差异有统计学意义(P<0.05),可能C、T等位基因与哮喘易感有关。
本研究结果还显示,两组TT基因型血清IL-4含量均高于CT、CC,CT均高于CC,且实验组CC、CT、TT血清IL-4含量均高于空白组(P<0.05);两组GG血清IL-4含量高于AA、AG,AG高于AA,且实验组AA、AG、GG血清IL-4含量均高于空白组(P<0.05);提示哮喘急性发作时两个因子均升高。两组GG血清IL-4含量高于AA、AG,AG高于AA,且实验组AA、AG、GG血清IL-4含量均高于空白组,差异有统计学意义(P<0.05),提示TT基因型突变可能与哮喘有关联;两组GG血清TIgE含量均高于AA、AG,AG均高于AA,且实验组AA、AG、GG血清TIgE含量均高于空白组,差异有统计学意义(P<0.05),提示该位点突变与血清IL-4、TIgE有关联性,是否影响哮喘严重程度尚需进一步研究。
综上所述,哮喘患儿IL-4 基因多态性与健康儿童无明显差异,可能与儿童Th2均占优势从而导致过敏体质高发有关。哮喘急性发作时,血清IL-4和TIgE在TT 和GG基因型中含量最高,需进一步深入研究其机制。
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