儿童哮喘病情及预后评估的蛋白质组学研究
摘要
背景：支气管哮喘作为一种常见疾病，在儿童期间发病率在世界范围内有逐渐上升的趋势。儿童哮喘与成人哮喘具有同样的病理生理基础，但由于儿童处于生长发育阶段，因此具有其自身的特点。小儿气道炎症也会随年龄增长而发生变化，最终约2/3患儿疾病自然缓解、症状消失，另外1/3左右患儿病情持续、发展为成人哮喘。儿童哮喘这种随年龄变化而呈现的不同自然病程及预后转归现象目前都还没有合理的解释，其自然病程演变规律及机制还不清楚，然而疾病的自限性和进展性的差异提示它们存在本质上的不同。目前，临床上通常以临床症状、肺功能及治疗效果来衡量哮喘轻重及控制情况，但绝大多数稳定期哮喘患儿肺功能正常。研究表明即使部分患儿肺功能正常，其仍持续存在小气道慢性炎症，而该种慢性炎症的持续存在是导致儿童哮喘反复发作和病情进展导致控制不良的基础。因而肺功能是评估儿童哮喘病情不够敏感的指标。早期明确哮喘患儿病情严重程度及其转归方向对指导儿童哮喘治疗和减轻家长及患儿心理负担有重要意义。但目前临床上缺乏能够准确评估哮喘病情及预后的客观指标，更缺少针对儿童哮喘病情及预后评估的前瞻性的研究。疾病相关的生物靶标（biomarker）可提示疾病相关信息，可用于疾病的辅助诊断、病情进展的监测和预后及疾病的预防。目前关于儿童哮喘的生物靶标研究还很局限，因此，急需新兴的生物靶标来辅助客观地评估儿童哮喘。蛋白质组学的出现，为哮喘生物靶标的筛选提供了新的希望。采用蛋白质组学方法筛选疾病相关的蛋白靶标，可发现疾病相关的系列蛋白靶标及潜在药物靶标，还可发现目前没有同疾病相关联的新蛋白。
目的：筛选潜在用于评估儿童哮喘病情及预后的蛋白靶标。
方法：通过使用二维差异凝胶电泳技术（two dimensional differential in-gelelectrophoresis，2D-DIGE），分离及筛选不同轻重程度稳定期哮喘儿童（根据GINA方案，间歇发作、轻度持续、中度持续、重度持续组）及健康儿童血浆蛋白，进一步经基质辅助激光解吸/电离-飞行时间质谱（matrix-assisted laser desorption/ionization-time offlight-mass spectrometry，MALDITOF/TOF MS）鉴定，然后经ELISA方法进行另一扩大人群临床验证试验，明确各蛋白靶标在病情轻重不同组别的分布规律，筛选评估儿童哮喘病情轻重的蛋白靶标（候选蛋白靶标多组间比较统计分析采用单因素方差分析）；同时对哮喘儿童进行为期3年的队列研究，根据其转归情况（完全缓解、好转、持续、进展），再次行血ELISA靶标检测，分别分析入组初及3年随访后的个体候选靶标表达水平及其随访前后的蛋白靶标水平的差值，分别按照转归分组进行多组间比较的单因素方差分析，筛选评估儿童哮喘预后的蛋白靶标。
结果:通过蛋白质组学分析，组间共筛选出36个差异蛋白（p<0.05），20个蛋白点经质谱鉴定，代表了8种蛋白（同种蛋白由于同型异构体的分子量或等电点的不同可出现在凝胶的不同位置），其中4个蛋白靶标经扩大人群ELISA试验验证，在组间差别有统计学意义，分别为抗凝血酶III （antithrombin-III， AT-III）、 α2巨球蛋白（alpha2-macroglobulin，A2M）、CD5L（CD5antigen-like, CD5L）、补体C3（Complement3,C3）。在稳定期儿童哮喘与对照组间比较，AT-III在哮喘组表达明显高于对照组（p<0.01）；C3、A2M在对照组高于哮喘组（p<0.05）；在稳定期哮喘组内比较差异有显著性，AT-III随病情加重表达有升高趋势（ANOVAp<0.05），CD5L、A2M随病情加重表达有逐渐下降的趋势（ANOVA,p<0.05, p<0.01），其中AT-III与A2M呈负相关（r=-0.259，p<0.05）,与CD5L呈负相关（r=-0.276，p<0.05）,与FEV1%/FVC%呈负相关（-0.362，p<0.05）；CD5L与A2M之间呈正相关（r=0.303, p<0.01），与FEV1%/FVC%呈正相关（r=0.264，p<0.05）。队列研究中（3年随访前后）各转归组间比较，CD5L表达在入组时（CD5L前）各转归组间分布差别有显著性（p<0.05），随转归加重其表达下降。在随访结束时（CD5L后）其表达在各转归组间分布差别有显著性（p<0.01），随转归加重其表达下降。CD5L差值（CD5后－CD5前）在各转归组间分布差别有显著性（p<0.05），在加重及持续组其差值下降高于健康对照；AT-III前及AT-III后在各转归组间分布差别无显著性，但其表达随转归加重由升高趋势。AT-III差值在各转归组间分布差别有显著性（p<0.05），在好转组其差值降低高于持续、加重组及健康对照组；A2M前在各转归组间分布差别有显著性（p<0.05），健康对照组与完全缓解组及加重组间存在差异，A2M后在各转归组间分布差别无显著性，A2M前及A2M后其表达随转归变化无明显趋势。A2M差值在各转归组间分布差别无显著性；补体C3前、补体C3后及补体C3差值在各转归组间分布差别皆无显著性，其随转归变化无明显趋势。
结论:本研究通过蛋白质组学方法发现了儿童哮喘评估病情及预后的相关系列蛋白靶标。AT-III、A2M、补体C3可辅助诊断儿童哮喘；AT-III、A2M、CD5L与儿童哮喘疾病严重相关，AT-III、CD5L与儿童哮喘预后转归相关。血浆蛋白靶标AT-III、A2M、CD5L、补体C3有望作为辅助儿童哮喘诊断及评估疾病严重程度及预后的有力工具。
Background: Bronchial asthma is a common disease in children, due to multiplefactors, the incidence of the disease grow higher than ever. Childhood asthma has a naturalhistory different from that of adult asthma. Symptoms of2/3asthma children will disappearwhen they reach adult age, while another1/3will continue and finally develop adult asthma.This phenomenon implies that there must be some underlying mechanisms in it; Besidesthat, Clinically we evaluate asthma disease severity and control condition by clinicalmanifestations and lung function tests, but most of their lung function tests show normalwhile the bronchial inflammations are still there or progressing. Evaluating the severity andprognosis of childhood asthma is important to the clinical management of the disease andunderstanding the pathogenesis of it, also help to relieve the parents’ anxiety about thefuture of their children’s disease. Currently we lack reliable tests that are sensitive andobjective in evaluating childhood asthma, and prospective studies in this field are needed.Disease related biomarkers can help to diagnose and evaluate diseases objectively. Proteomicmethods can help to find series of disease related biomarkers, even to find novel biomarkerswhich never be connected with the disease.
Objective: We want to use proteomic methods to find childhood asthma relatedbiomarkers.
Methods: Two dimensional differential gel electrophoresis techniques (2D–DIGE)were used upon plasma samples of stable asthmatic children (intermittent, mild persist,moderate persist, severe persist, subgrouped according to GINA) and healthy controls toseparate and screen for differential expression of proteins. The candidate proteins wereidentified by Matrix-Assisted Laser Desorption/Ionization Time of Flight MassSpectrometry (MALDI TOF/TOF MS). Then were validated by ELISA in another largerpediatric population to evaluate candidate protein markers associated with disease severity; These candidate markers then have been further validated by ELISA in a large population. Inthis longitudinal cohort study we conducted a3years follow up trial with various groups inview of clarifying the disease association with childhood asthma prognosis.
Results: By comparing protein levels in the plasma samples of asthmatic and healthycontrols using proteomic techniques,36protein markers were found differentially expressed(p<0.05) between4asthmatic and healthy control groups,20proteins were identified byMass Spectrometry which represent set of8proteins. Further validation of these proteins in aseparate large clinical group was preformed. We found that a panel of four biomarkers(antithrombin–III (AT–III), alpha2-macroglobulin(A2M), CD5antigen-like(CD5L), Com-plement3(C3)) consistently showed significantly differential expressions between differentasthmatic groups and healthy control group. AT-III, C3, A2M were differentially expressedbetween asthmatic and healthy control group (p<0.05); among the different asthmaticgroups, A-TIII expressions showed a trend of gradually raising up with asthmatic diseaseseverity (ANOVA p<0.05), A2M and CD5L expressions showed reversed trends with thedisease severity (ANOVA,p<0.05, p<0.01). it was demonstrated that AT-III had a negativecorrelations with A2M (r=-0.259, p<0.05), CD5L(r=-0.276，p<0.05), and FEV1%/FVC%(r=-0.362, p<0.05), respectively; similarly, CD5L had a positive correlations with A2M(r=0.303, p<0.01) and FEV1%/FVC%(r=0.264，p<0.05), respectively. Among differentprognostic groups, A-TIII expressions showed a trend of raising up with asthmaticprognostic severity, CD5L showed reversed trend with asthmatic prognostic severity. A2Mand C3showed no obvious trend with asthmatic prognostic severity.
Conclusions: Our study demonstrated that proteomics is a useful method in identifyasthma related novel biomarkers. The result showed that a panel of four biomarkers (CD5L,AT-III, A2M, and C3) seems to relate closely with childhood asthma disease severity andprognosis. The combination of the biomarkers can be a powerful tool in evaluating andmonitoring childhood asthma progress. The biomarkers may also underpin the diseasemechanism which warrant further exploration in understanding asthma pathogenesis.Now we lack specific indexes to evaluate childhood asthma, plasma biomarkers are a goodchoice to reflect the disease condition of childhood asthma, especially for the childhood population who are difficult to go through invasive examinations.
目的：筛选潜在用于评估儿童哮喘病情及预后的蛋白靶标。
方法：通过使用二维差异凝胶电泳技术（two dimensional differential in-gelelectrophoresis，2D-DIGE），分离及筛选不同轻重程度稳定期哮喘儿童（根据GINA方案，间歇发作、轻度持续、中度持续、重度持续组）及健康儿童血浆蛋白，进一步经基质辅助激光解吸/电离-飞行时间质谱（matrix-assisted laser desorption/ionization-time offlight-mass spectrometry，MALDITOF/TOF MS）鉴定，然后经ELISA方法进行另一扩大人群临床验证试验，明确各蛋白靶标在病情轻重不同组别的分布规律，筛选评估儿童哮喘病情轻重的蛋白靶标（候选蛋白靶标多组间比较统计分析采用单因素方差分析）；同时对哮喘儿童进行为期3年的队列研究，根据其转归情况（完全缓解、好转、持续、进展），再次行血ELISA靶标检测，分别分析入组初及3年随访后的个体候选靶标表达水平及其随访前后的蛋白靶标水平的差值，分别按照转归分组进行多组间比较的单因素方差分析，筛选评估儿童哮喘预后的蛋白靶标。
结果:通过蛋白质组学分析，组间共筛选出36个差异蛋白（p<0.05），20个蛋白点经质谱鉴定，代表了8种蛋白（同种蛋白由于同型异构体的分子量或等电点的不同可出现在凝胶的不同位置），其中4个蛋白靶标经扩大人群ELISA试验验证，在组间差别有统计学意义，分别为抗凝血酶III （antithrombin-III， AT-III）、 α2巨球蛋白（alpha2-macroglobulin，A2M）、CD5L（CD5antigen-like, CD5L）、补体C3（Complement3,C3）。在稳定期儿童哮喘与对照组间比较，AT-III在哮喘组表达明显高于对照组（p<0.01）；C3、A2M在对照组高于哮喘组（p<0.05）；在稳定期哮喘组内比较差异有显著性，AT-III随病情加重表达有升高趋势（ANOVAp<0.05），CD5L、A2M随病情加重表达有逐渐下降的趋势（ANOVA,p<0.05, p<0.01），其中AT-III与A2M呈负相关（r=-0.259，p<0.05）,与CD5L呈负相关（r=-0.276，p<0.05）,与FEV1%/FVC%呈负相关（-0.362，p<0.05）；CD5L与A2M之间呈正相关（r=0.303, p<0.01），与FEV1%/FVC%呈正相关（r=0.264，p<0.05）。队列研究中（3年随访前后）各转归组间比较，CD5L表达在入组时（CD5L前）各转归组间分布差别有显著性（p<0.05），随转归加重其表达下降。在随访结束时（CD5L后）其表达在各转归组间分布差别有显著性（p<0.01），随转归加重其表达下降。CD5L差值（CD5后－CD5前）在各转归组间分布差别有显著性（p<0.05），在加重及持续组其差值下降高于健康对照；AT-III前及AT-III后在各转归组间分布差别无显著性，但其表达随转归加重由升高趋势。AT-III差值在各转归组间分布差别有显著性（p<0.05），在好转组其差值降低高于持续、加重组及健康对照组；A2M前在各转归组间分布差别有显著性（p<0.05），健康对照组与完全缓解组及加重组间存在差异，A2M后在各转归组间分布差别无显著性，A2M前及A2M后其表达随转归变化无明显趋势。A2M差值在各转归组间分布差别无显著性；补体C3前、补体C3后及补体C3差值在各转归组间分布差别皆无显著性，其随转归变化无明显趋势。
结论:本研究通过蛋白质组学方法发现了儿童哮喘评估病情及预后的相关系列蛋白靶标。AT-III、A2M、补体C3可辅助诊断儿童哮喘；AT-III、A2M、CD5L与儿童哮喘疾病严重相关，AT-III、CD5L与儿童哮喘预后转归相关。血浆蛋白靶标AT-III、A2M、CD5L、补体C3有望作为辅助儿童哮喘诊断及评估疾病严重程度及预后的有力工具。
Background: Bronchial asthma is a common disease in children, due to multiplefactors, the incidence of the disease grow higher than ever. Childhood asthma has a naturalhistory different from that of adult asthma. Symptoms of2/3asthma children will disappearwhen they reach adult age, while another1/3will continue and finally develop adult asthma.This phenomenon implies that there must be some underlying mechanisms in it; Besidesthat, Clinically we evaluate asthma disease severity and control condition by clinicalmanifestations and lung function tests, but most of their lung function tests show normalwhile the bronchial inflammations are still there or progressing. Evaluating the severity andprognosis of childhood asthma is important to the clinical management of the disease andunderstanding the pathogenesis of it, also help to relieve the parents’ anxiety about thefuture of their children’s disease. Currently we lack reliable tests that are sensitive andobjective in evaluating childhood asthma, and prospective studies in this field are needed.Disease related biomarkers can help to diagnose and evaluate diseases objectively. Proteomicmethods can help to find series of disease related biomarkers, even to find novel biomarkerswhich never be connected with the disease.
Objective: We want to use proteomic methods to find childhood asthma relatedbiomarkers.
Methods: Two dimensional differential gel electrophoresis techniques (2D–DIGE)were used upon plasma samples of stable asthmatic children (intermittent, mild persist,moderate persist, severe persist, subgrouped according to GINA) and healthy controls toseparate and screen for differential expression of proteins. The candidate proteins wereidentified by Matrix-Assisted Laser Desorption/Ionization Time of Flight MassSpectrometry (MALDI TOF/TOF MS). Then were validated by ELISA in another largerpediatric population to evaluate candidate protein markers associated with disease severity; These candidate markers then have been further validated by ELISA in a large population. Inthis longitudinal cohort study we conducted a3years follow up trial with various groups inview of clarifying the disease association with childhood asthma prognosis.
Results: By comparing protein levels in the plasma samples of asthmatic and healthycontrols using proteomic techniques,36protein markers were found differentially expressed(p<0.05) between4asthmatic and healthy control groups,20proteins were identified byMass Spectrometry which represent set of8proteins. Further validation of these proteins in aseparate large clinical group was preformed. We found that a panel of four biomarkers(antithrombin–III (AT–III), alpha2-macroglobulin(A2M), CD5antigen-like(CD5L), Com-plement3(C3)) consistently showed significantly differential expressions between differentasthmatic groups and healthy control group. AT-III, C3, A2M were differentially expressedbetween asthmatic and healthy control group (p<0.05); among the different asthmaticgroups, A-TIII expressions showed a trend of gradually raising up with asthmatic diseaseseverity (ANOVA p<0.05), A2M and CD5L expressions showed reversed trends with thedisease severity (ANOVA,p<0.05, p<0.01). it was demonstrated that AT-III had a negativecorrelations with A2M (r=-0.259, p<0.05), CD5L(r=-0.276，p<0.05), and FEV1%/FVC%(r=-0.362, p<0.05), respectively; similarly, CD5L had a positive correlations with A2M(r=0.303, p<0.01) and FEV1%/FVC%(r=0.264，p<0.05), respectively. Among differentprognostic groups, A-TIII expressions showed a trend of raising up with asthmaticprognostic severity, CD5L showed reversed trend with asthmatic prognostic severity. A2Mand C3showed no obvious trend with asthmatic prognostic severity.
Conclusions: Our study demonstrated that proteomics is a useful method in identifyasthma related novel biomarkers. The result showed that a panel of four biomarkers (CD5L,AT-III, A2M, and C3) seems to relate closely with childhood asthma disease severity andprognosis. The combination of the biomarkers can be a powerful tool in evaluating andmonitoring childhood asthma progress. The biomarkers may also underpin the diseasemechanism which warrant further exploration in understanding asthma pathogenesis.Now we lack specific indexes to evaluate childhood asthma, plasma biomarkers are a goodchoice to reflect the disease condition of childhood asthma, especially for the childhood population who are difficult to go through invasive examinations.
引文
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