WO2015113478A1 - 外周血白细胞介素25水平在哮喘分型中的应用及分型方法 - Google Patents

外周血白细胞介素25水平在哮喘分型中的应用及分型方法 Download PDF

Info

Publication number
WO2015113478A1
WO2015113478A1 PCT/CN2015/071419 CN2015071419W WO2015113478A1 WO 2015113478 A1 WO2015113478 A1 WO 2015113478A1 CN 2015071419 W CN2015071419 W CN 2015071419W WO 2015113478 A1 WO2015113478 A1 WO 2015113478A1
Authority
WO
WIPO (PCT)
Prior art keywords
asthma
peripheral blood
patients
inhaled
level
Prior art date
Application number
PCT/CN2015/071419
Other languages
English (en)
French (fr)
Inventor
甄国华
薛峥
赵建平
徐永健
Original Assignee
华中科技大学同济医学院附属同济医院
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 华中科技大学同济医学院附属同济医院 filed Critical 华中科技大学同济医学院附属同济医院
Publication of WO2015113478A1 publication Critical patent/WO2015113478A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6869—Interleukin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/54—Interleukins [IL]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/12—Pulmonary diseases
    • G01N2800/122—Chronic or obstructive airway disorders, e.g. asthma COPD

Definitions

  • the invention belongs to the medical field, and specifically relates to the prediction of sensitivity to inhaled hormone response in patients with bronchial asthma.
  • Bronchial asthma is a common respiratory disease, and the incidence is gradually rising due to factors such as air pollution.
  • An acute attack of bronchial asthma can endanger the patient's life and is one of the important diseases of critical illness in internal medicine.
  • One of the main drugs for the treatment of bronchial asthma is inhaled hormones, but it has been found in clinical practice that inhaled hormones have only a significant effect on some asthma patients, but have no obvious therapeutic effect on other asthma patients [see: 1. Szefler SJ, Martin RJ, King TS, Boushey HA, Cherniack RM, Chinchilli VM, Craig TJ, et al. Significant variability in response to inhaled corticosteroids for persistent asthma. J Allergy Clin Immunol. 2002 Mar; 109(3): 410-8]. Therefore, it is of great clinical significance to look for biomarkers that predict the sensitivity of bronchial asthma patients to inhaled hormonal responses.
  • the current markers that may predict the sensitivity of asthmatic patients to inhaled hormonal responses are detected by fiberoptic bronchoscopy specimens or by the expression levels of Th2-related genes (CLCAl, POSTN, SERPINB2) in induced sputum samples [see: Woodruff PG] , Modrek B, Choy DF, Jia G, Abbas AR, Ellwanger A, Koth LL, Arron JR, Fahy JV. T-helper type 2-driven inflammation defines major subphenotypes of asthma. Am J Respir Crit Care Med.2009Sep 1;180 (5): 388-95; Peters MC, Mekonnen ZK, Yuan S, Bhakta NR, Woodruff PG, Fahy JV.
  • induced sputum is time-consuming and laborious. It takes about 1 hour for each asthma patient to collect sputum-inducing specimens, then separate the cells in the induced sputum, extract cellular RNA, and detect the expression of related genes by RT-PCR. The use of the method is also greatly limited. Currently an urgent need to predict asthma against inhaled hormones Non-invasive and simple detection of reaction sensitivity, while having high sensitivity and specificity of biomarkers.
  • Another object of the invention is to provide a method of typing asthma. Another object of the present invention is to provide a biomarker for identifying susceptibility to hormonal therapy in asthma patients, i.e., to find novel, biomarkers that are effective, non-invasive, and rapid in predicting susceptibility to inflammatory response in bronchial asthma patients.
  • the method for providing asthma typing according to the present invention is to detect the expression level of interleukin-25 (IL-25) in peripheral blood of asthma patients, and the peripheral blood IL-25 level is greater than 55 pg/ml for hormone sensitive type; peripheral blood IL A -25 level of less than 55 pg/ml is hormone insensitive.
  • Hormone-sensitive patients are sensitive to inhaled hormones, and lung function can be significantly improved by inhaling hormones. Inhalation of hormones in patients with hormone-insensitive patients did not significantly improve lung function.
  • the novel biomarker for predicting susceptibility to bronchial asthma in patients with bronchial asthma and which is non-invasive and rapid is peripheral blood IL-25.
  • IL-25 in peripheral blood of asthma patients We examined the expression of IL-25 in peripheral blood of asthma patients, and systematically correlated the expression of IL-25 with the hormonal reactivity of asthma patients to evaluate whether IL-25 can be used to identify asthma patients for hormone therapy. Sensitive biomarkers. We found that after using inhaled sex hormones, asthmatic patients with high levels of IL-25 improved lung function significantly, while asthmatic patients with low IL-25 expression did not significantly improve lung function. Therefore, detecting the level of IL-25 in peripheral blood of asthma patients is a novel biomarker that can effectively predict the response of asthmatic patients to inhaled hormones. Moreover, the detection of IL-25 in peripheral blood belongs to non-invasive detection, and the detection method by ELISA is simple and rapid. In addition, IL-25 antibodies are being developed as a therapeutic drug for asthma, and detection of IL-25 in peripheral blood will also guide which asthma patients will be applied to IL-25 antibodies in the future.
  • the ELISA assay for peripheral blood IL-25 is as follows:
  • the tube for collecting blood should be a disposable pyrogen-free, endotoxin-free tube.
  • EDTA is recommended for plasma anticoagulants. Avoid using hemolysis, hyperlipidemia specimens.
  • the specimen should be clear and transparent, and the suspended matter should be removed by centrifugation.
  • specimens are not detected in time after collection, they should be packed in one use and frozen in -20 °C, -70 °C refrigerator to avoid repeated freezing and thawing, and test within 3-6 months.
  • Standard Add 1.0 ml of the standard & specimen universal dilution to the lyophilized standard and let stand for 15 minutes. After it is fully dissolved, mix gently (concentration: 2000 pg/ml). Then dilute as needed. (The standard curve uses the following concentrations: 2000, 1000, 500, 250, 125, 62.5, 31.25, 0 pg/ml). The reconstituted standard stock solution (2000 pg/ml) is discarded if not used up.
  • Biotinylated antibody working solution 30 ⁇ concentrated biotinylated antibody was diluted to 1 ⁇ working solution with biotinylated antibody dilution 20 minutes before use, according to the amount required for the current test. Used on the same day.
  • Enzyme conjugate working solution according to the amount required for the current test, 20 minutes before use, using enzyme The 30x concentrated enzyme conjugate was diluted to 1 x working solution with the conjugate dilution. Used on the same day.
  • Blank wells plus standard & specimen universal dilutions add the appropriate standard or different concentration standard (100 ul / well) to the corresponding wells, seal the reaction well with sealing paper, and incubate for 90 minutes at 36 °C.
  • TMB chromogenic substrate
  • IL-25 in peripheral blood of some asthma patients was significantly increased (>55pg/ml), which we call "IL-25 high” asthma patients.
  • the peripheral blood IL-25 level in some patients with asthma is in the same range as the normal control group ( ⁇ 55 pg/ml), which we call "IL-25 low” asthma patients (see Figure 1).
  • Pulmonary function in patients with high peripheral blood IL-25 levels (>55 pg/ml) was significantly improved, with FEV1 increasing by 0.35-0.37 L and PD20 increasing by 0.50-0.52 mg.
  • Peripheral blood IL-25 levels are novel biomarkers for predicting the sensitivity of asthmatic patients to inhaled hormonal responses. If the peripheral blood IL-25 level is greater than 55pg/ml, the patient is sensitive to inhaled hormones, and lung function can be significantly improved by inhaled hormones; if the IL-25 level is less than 55pg/ml, the patient is not sensitive to inhaled hormones, by inhaling hormones. There is no significant improvement in the treatment of lung function, and other treatments other than inhaled hormones, such as leukotriene receptor antagonists, should be taken.
  • peripheral blood IL-25 levels are novel biomarkers for predicting the sensitivity of asthmatic patients to inhaled hormonal responses. If the peripheral blood IL-25 level is greater than 55pg/ml, the patient is sensitive to inhaled hormones, and lung function can be significantly improved by inhaled hormones; if the IL-25 level is less than 55pg/ml, the patient is not sensitive to inhaled hormones, by inhaling hormones. There is no significant improvement in the treatment of lung function, and other treatments other than inhaled hormones, such as leukotriene receptor antagonists, should be taken.
  • the present invention has the following advantages: the present invention proves that peripheral blood IL-25 can be used as a novel biomarker for predicting the sensitivity of bronchial asthma patients to inhaled glucocorticoid, which is a non-invasive and effective detection. The method has significant progress over the prior art.
  • the results of the present invention reveal that if the level of IL-25 in peripheral blood of patients with bronchial asthma is lower than 55 pg/ml, it is considered to be Hormone-insensitive asthma, which is an "IL-25 low" asthma patient, is not suitable for treatment with inhaled glucocorticoids. Other treatments, such as leukotriene receptor antagonists, are recommended.
  • the bronchial asthma typing method of the invention can quickly predict which bronchial asthma patients are suitable for treatment with inhaled glucocorticoid and improve the therapeutic effect of bronchial asthma.
  • a method for treating asthma is obtained by administering an inhaled glucocorticoid to an asthmatic patient whose peripheral blood IL-25 level is greater than 55 pg/ml.
  • budesonide can be used at 200 ⁇ g each time, twice a day for 24 weeks.
  • FIG. 1 Results of peripheral blood IL-25 levels in normal controls and asthmatic patients. Compared with the normal control group, the peripheral blood IL-25 level of some asthma patients was significantly increased (>55pg/ml, IL-25 high asthma patients), and the peripheral blood IL-25 level of some asthma patients was in the same range as the normal control group. ( ⁇ 55pg/ml, IL-25 low asthma patients).
  • FIG. 1 Changes in lung function FEV1 after inhaled corticosteroids in patients with low IL-25 and low IL-25. Changes in lung function FEV1 after 4 weeks and 8 weeks of inhaled corticosteroids by detecting IL-25 high asthma group (>55 pg/ml), IL-25 low asthma group ( ⁇ 55 pg/ml) before inhaled hormone use (0) The results showed that compared with the IL-25-low asthma group, the improvement of lung function FEV1 was more significant in the IL-25-high asthma group after 4 and 8 weeks of inhaled corticosteroid use (P ⁇ 0.05).
  • Figure 3 Changes in lung function PD20 after inhaled corticosteroids in patients with low IL-25 and low IL-25.
  • IL-25 high asthma group IL-25 low asthma group before and after inhaled corticosteroids (0) and using inhaled hormones
  • the results showed that: with IL-25 low asthma group Compared with the IL-25 high asthma group, the improvement of pulmonary function PD20 was more significant after 4 and 8 weeks of inhaled corticosteroid use (P ⁇ 0.05).
  • IL-25 in peripheral blood was taken from patients, and the level of IL-25 in peripheral blood was determined by ELISA.
  • the level of IL-25 in 38 patients was higher than 55pg/ml ("IL -25 high"), designated as hormone-sensitive asthma, inhaled glucocorticoid therapy in these 38 patients with hormone-sensitive asthma, treated with budesonide (200 ⁇ g twice a day) for 8 weeks.
  • Asthma patients with high IL-25 in peripheral blood showed significant improvement in lung function after treatment, with an increase in FEV1 of 0.35-0.37 L and an increase of PD20 of 0.50-0.52 mg (see Figures 2 and 3).
  • IL-25 low peripheral blood IL-25 levels below 55 pg/ml
  • 41 patients with hormone-insensitive asthma were treated with inhaled glucocorticoids.
  • Treatment with budesonide (200 ⁇ g twice daily) for 8 weeks showed no improvement in lung function after treatment, with FEV1 increasing only 0.11-0.12 L and PD20 increasing only 0.03-0.04 mg (see Figures 2 and 3).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Medicinal Chemistry (AREA)
  • Urology & Nephrology (AREA)
  • Cell Biology (AREA)
  • Analytical Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Food Science & Technology (AREA)
  • Public Health (AREA)
  • Epidemiology (AREA)
  • Pulmonology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

一种哮喘分型的方法以及外周血IL-25作为哮喘分型的生物标记物的应用，检测哮喘患者外周血白细胞介素25（IL-25）的表达水平，外周血IL-25大于55pg/ml为激素敏感型哮喘；外周血IL-25水平小于55pg/ml为激素不敏感型哮喘。在使用吸入性激素之后，IL-25水平高的哮喘患者肺功能改善明显，而IL-25表达低的哮喘患者肺功能没有明显的改善。

Description

外周血白细胞介素25水平在哮喘分型中的应用及分型方法 技术领域
本发明属于医学领域，具体涉及支气管哮喘患者对吸入激素反应敏感性的预测。
背景技术
支气管哮喘是常见的呼吸系统疾病，由于空气污染等因素，发病率逐渐上升。支气管哮喘急性发作时可危及到患者的生命，是内科急危重症的重要病种之一。目前治疗支气管哮喘的主要药物之一是吸入激素，但是临床实践中发现，吸入激素仅对一部分哮喘病人有明显疗效，而对其余的哮喘病人没有明显的治疗作用[参见：1.Szefler SJ，Martin RJ，King TS，Boushey HA，Cherniack RM，Chinchilli VM，Craig TJ，et al.Significant variability in response to inhaled corticosteroids for persistent asthma.J Allergy Clin Immunol.2002Mar；109(3)：410-8]。因此，寻找预测支气管哮喘患者对吸入激素反应敏感性的生物标记物有重要的临床意义。
目前已有的可能预测哮喘患者对吸入激素反应敏感性的标记物是通过纤维支气管镜刷片标本或者诱导痰标本中Th2相关基因(CLCAl，POSTN，SERPINB2)的表达水平的检测[参见：Woodruff PG，Modrek B，Choy DF，Jia G，Abbas AR，Ellwanger A，Koth LL，Arron JR，Fahy JV.T-helper type 2-driven inflammation defines major subphenotypes of asthma.Am J Respir Crit Care Med.2009Sep 1；180(5)：388-95；Peters MC，Mekonnen ZK，Yuan S，Bhakta NR，Woodruff PG，Fahy JV.Measures of gene expression in sputum cells can identify T(H)2-high and T(H)2-low subtypes of asthma.J Allergy Clin Immunol.2013Sep 24.pii：S0091-6749(13)01209-8]。但是，纤维支气管镜刷片标本的检测属于有创伤性检查，需要进行局部麻醉，然后在患者气道内插入纤维支气管镜获取刷片标本，再提取刷片标本细胞中的RNA，通过RT-PCR的方法检测相关基因的表达。因此通过纤维支气管镜刷片标本中相关基因的表达水平检测来预测哮喘患者对激素敏感性的方法在哮喘患者中难以推广。诱导痰的检测费时费力，每个哮喘患者收集一次诱导痰标本需要1小时左右的时间，然后再分离诱导痰中的细胞，提取细胞RNA，通过RT-PCR的方法检测相关基因的表达，因此该方法的使用也受到很大的限制。目前亟待一种有效预测哮喘对吸入激素 反应敏感性的无创简便的检测方法，同时又有较高的敏感性和特异性的生物标记物。
发明内容
本发明的任务是提供一种哮喘分型的方法。本发明的另一个任务是提供一种用于鉴别哮喘患者对激素治疗敏感性的生物学标记物，即寻找预测支气管哮喘患者对吸入激素反应敏感性的有效而且无创快捷的新型生物标记物。
本发明提供哮喘分型的方法是：检测哮喘患者外周血白细胞介素25(interleukin-25，IL-25)的表达水平，外周血IL-25水平大于55pg/ml为激素敏感型；外周血IL-25水平小于55pg/ml为激素不敏感型。激素敏感型患者对吸入激素敏感，通过吸入激素治疗肺功能可以明显改善。激素不敏感型患者吸入激素治疗肺功能无明显改善。本发明提供的预测支气管哮喘患者对吸入激素反应敏感性的有效而且无创快捷的新型生物标记物是外周血IL-25。
我们检测哮喘患者外周血IL-25的表达水平，并且把IL-25的表达与哮喘患者对激素的反应性进行了系统的相关性分析，来评价IL-25能否成为鉴别哮喘患者对激素治疗敏感性的生物学标记物。我们发现在使用吸入性激素之后，IL-25水平高的哮喘患者肺功能改善明显，而IL-25表达低的哮喘患者肺功能没有明显的改善。所以，检测哮喘患者外周血IL-25的水平，是一种可以有效预测哮喘患者对吸入激素反应的新型生物标记物。而且，外周血IL-25的检测属于无创检测，通过ELISA检测方法简便快捷。除此之外，IL-25的抗体作为哮喘的治疗药物正在研制过程之中，外周血IL-25的检测同样将指导IL-25抗体将来适用于哪些哮喘患者。
方法：
我们收集了32例健康对照者和79例哮喘患者。在签署知情同意书后，所有对照和哮喘患者进行基础肺功能(FEV1％，PD20等)的测定，并且收集外周血标本。哮喘患者给予吸入性糖皮质激素(ICS)布地奈德吸入(200μg，一天两次)治疗8周，分别在第4周、第8周检测肺功能及收集外周血，通 过吸入性糖皮质激素治疗前后肺功能的改善程度来确定对哮喘患者吸入性糖皮质激素的敏感性，并且对收集的外周血标本通过ELISA方法进行IL-25的检测。然后对外周血IL-25水平与吸入性糖皮质激素治疗前后肺功能的改善程度之间的关系。
外周血IL-25的ELISA测定方法如下：
标本收集：
1.收集血液的试管应为一次性的无热原，无内毒素试管。
2.血浆抗凝剂推荐使用EDTA。避免使用溶血，高血脂标本。
3.标本应清澈透明，悬浮物应离心去除。
4.标本收集后若不及时检测，按一次使用量分装，冻存于-20℃，-70℃电冰箱内，避免反复冻融，3-6月内检测。
检测前准备工作：
1.提前20分钟从冰箱中取出试剂盒，以平衡至室温。
2.用双蒸水将20×浓缩洗涤液稀释成1×工作液。
3.标准品：加入标准品&标本通用稀释液1.0ml至冻干标准品中，静置15分钟，待其充分溶解后，轻轻混匀(浓度为2000pg/ml)。然后根据需要进行稀释。(标准曲线使用以下浓度：2000、1000、500、250、125、62.5、31.25、0pg/ml)。复溶标准品原液(2000pg/ml)若未用完的废弃。
4.生物素化抗体工作液：按当次试验所需要得用量，使用前20分钟，用生物素化抗体稀释液将30×浓缩生物素化抗体稀释成1×工作液。当日使用。
5.酶结合物工作液：按当次试验所需要的用量，使用前20分钟，用酶 结合物稀释液将30×浓缩酶结合物稀释成1×工作液。当日使用。
操作步骤：
1.从已平衡至室温的密封袋中取出试验所需板条。
2.空白孔加标准品&标本通用稀释液，其余相应孔中加标本或不同浓度标准品(100ul/孔)，用封板胶纸封住反应孔，36℃孵箱孵育90分钟。
3.提前20分钟准备生物素化抗体工作液。
4.洗板5次。
5.空白孔加生物素化抗体稀释液，其余孔加入生物素化抗体工作液(100ul/孔)。用新封板胶纸封住反应孔，36℃孵箱孵育60分钟。
6.提前20分钟准备酶结合物工作液。避光室温(22-25℃)放置。
7.洗板5次。
8.空白孔加酶结合物稀释液，其余孔加入酶结合物工作液(100ul/孔)。用新封板胶纸封住反应孔，36℃孵箱，避光孵育30分钟。
9.打开酶标仪电源，预热仪器，设置好检测程序。
10.洗板5次。
11.加入显色底物(TMB)100ul/孔，避光36℃孵箱，避光孵育15分钟。
12.加入终止液100ul/孔，混匀后即刻测量OD450值(3分钟内)。
结果判断：
1.每个标准品和标本的OD值应减去空白孔的OD值。
2.以标准品浓度作横坐标，OD值作纵坐标，手工绘制或用软件绘制标准曲线。通过标本的OD值可在标准曲线上查出其浓度。
3.若标本OD值高于标准曲线上限，应适当稀释后重测，计算浓度时应 乘以稀释倍数。
结果：与正常对照组相比，部分哮喘患者外周血IL-25水平明显升高(＞55pg/ml)，我们称为“IL-25高”哮喘患者。部分哮喘患者外周血IL-25水平与正常对照组在相同范围内(≤55pg/ml)，我们称为“IL-25低”哮喘患者(参见附图1)。外周血IL-25水平高(＞55pg/ml)的哮喘患者肺功能明显改善，FEV1增加0.35-0.37L，PD20增加0.50-0.52mg。而外周血IL-25水平低(≤55pg/ml)的哮喘患者肺功能没有明显改善，FEV1仅增加0.11-0.12L，PD20仅增加0.03-0.04mg(参见附图2和3)。
结论：外周血IL-25水平是预测哮喘患者对吸入激素反应敏感性的新型生物标记物。如果外周血IL-25水平大于55pg/ml，该患者对吸入激素敏感，通过吸入激素治疗肺功能可以明显改善；如果IL-25水平小于55pg/ml，该患者对吸入激素不敏感，通过吸入激素治疗肺功能无明显改善，应当采取除吸入激素外的其他治疗方法，如白三烯受体拮抗剂等。
本发明揭示外周血IL-25水平是预测哮喘患者对吸入激素反应敏感性的新型生物标记物。如果外周血IL-25水平大于55pg/ml，该患者对吸入激素敏感，通过吸入激素治疗肺功能可以明显改善；如果IL-25水平小于55pg/ml，该患者对吸入激素不敏感，通过吸入激素治疗肺功能无明显改善，应当采取除吸入激素外的其他治疗方法，如白三烯受体拮抗剂等。本发明相比现有技术具有如下优点：本发明证实了外周血IL-25可以做为预测支气管哮喘患者对吸入性糖皮质激素敏感性的新型生物标记物，该指标为无创快捷和有效的检测方法，比现有技术具有显著的进步。
本发明结果揭示：如果支气管哮喘患者外周血IL-25的水平低于55pg/ml，则认为是 激素不敏感型哮喘，即“IL-25低”的哮喘患者，不适宜使用吸入性糖皮质激素治疗，建议使用其他治疗方法，如白三烯受体拈抗剂等。本发明支气管哮喘分型方法，可以快速预测哪些支气管哮喘患者适合采用吸入性糖皮质激素进行治疗，提高支气管哮喘的治疗效果。
根据本发明结果，得到了一种治疗哮喘方法，该方法是对外周血IL-25水平大于55pg/ml的哮喘患者施以吸入性糖皮质激素治疗。具体可以使用布地奈德每次200μg，一天两次，疗程为24周。
附图说明
图1.正常对照组和哮喘患者外周血IL-25水平检测结果。与正常对照组相比，部分哮喘患者外周血IL-25水平明显升高(＞55pg/ml，IL-25高哮喘患者)，部分哮喘患者外周血IL-25水平与正常对照组在相同范围内(≤55pg/ml，IL-25低哮喘患者)。
图2.IL-25高和IL-25低的哮喘患者使用吸入激素后肺功能FEV1的变化。通过检测IL-25高哮喘组(＞55pg/ml)、IL-25低哮喘组(≤55pg/ml)在使用吸入激素前(0)和使用吸入激素4周和8周后肺功能FEV1的变化，结果显示：与IL-25-low哮喘组相比，IL-25-high哮喘组在使用吸入激素4周和8周后肺功能FEV1的改善更加显著(P＜0.05)。
图3.IL-25高和IL-25低的哮喘患者使用吸入激素后肺功能PD20的变化。通过检测IL-25高哮喘组、IL-25低哮喘组在使用吸入激素前(0)和使用吸入激素4周和8周后肺功能PD20的变化，结果显示：与IL-25低哮喘组相比，IL-25高哮喘组在使用吸入激素4周和8周后肺功能PD20的改善更加显著(P＜0.05)。
具体实施方式
实施例1
对哮喘专科医生确诊为支气管哮喘的79例患者，抽取患者外周血2ml，通过ELISA方法测定患者外周血IL-25的水平，其中38例外周血IL-25的水平高于55pg/ml(“IL-25高”)，定为激素敏感型哮喘，对这38例激素敏感型哮喘患者进行吸入性糖皮质激素治疗，使用布地奈德(200μg，一天两次)治疗8周。外周血IL-25高的哮喘患者在治疗后肺功能明显改善，FEV1增加0.35-0.37L，PD20增加0.50-0.52mg(参见附图2和3)。
其余41例外周血IL-25的水平低于55pg/ml(“IL-25低”)，定为激素不敏感型哮喘，对这41例激素不敏感型哮喘患者进行吸入性糖皮质激素治疗，使用布地奈德(200μg，一天两次)治疗8周，治疗后肺功能改善不明显，FEV1仅增加0.11-0.12L，PD20仅增加0.03-0.04mg(参见附图2和3)。

Claims (5)

  1. 一种哮喘分型的方法，其特征是：检测哮喘患者外周血白细胞介素25(interleukin-25，IL-25)的表达水平，外周血IL-25水平大于55pg/ml为激素敏感型哮喘；外周血IL-25水平小于55pg/ml为激素不敏感型哮喘。
  2. 外周血IL-25作为哮喘分型的生物标记物的应用。
  3. 一种哮喘的治疗方法，其特征是：对外周血IL-25水平大于55pg/ml的哮喘患者施以吸入性糖皮质激素治疗。
  4. 根据权利要求3所述的治疗方法，其特征在于所述的施以吸入性糖皮质激素治疗的具体方法是：使用布地奈德每次200μg，一天两次。
  5. 根据权利要求4所述的治疗方法，其特征在于用药24周。
PCT/CN2015/071419 2014-01-29 2015-01-23 外周血白细胞介素25水平在哮喘分型中的应用及分型方法 WO2015113478A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201410042817.5A CN103792375B (zh) 2014-01-29 2014-01-29 外周血白细胞介素25水平在哮喘分型中的应用及分型方法
CN201410042817.5 2014-01-29

Publications (1)

Publication Number Publication Date
WO2015113478A1 true WO2015113478A1 (zh) 2015-08-06

Family

ID=50668244

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2015/071419 WO2015113478A1 (zh) 2014-01-29 2015-01-23 外周血白细胞介素25水平在哮喘分型中的应用及分型方法

Country Status (2)

Country Link
CN (1) CN103792375B (zh)
WO (1) WO2015113478A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103792375B (zh) * 2014-01-29 2017-07-07 华中科技大学同济医学院附属同济医院 外周血白细胞介素25水平在哮喘分型中的应用及分型方法

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101711256A (zh) * 2007-04-18 2010-05-19 医疗研究局 针对il-25的抗体
WO2010131009A2 (en) * 2009-05-15 2010-11-18 Medical Research Council Treatment of il-25 mediated diseases with tlr antagonists
CN102245638A (zh) * 2008-09-30 2011-11-16 医疗研究局 针对il-25的抗体
CN103097416A (zh) * 2010-03-30 2013-05-08 詹森生物科技公司 人源化il-25抗体
CN103792375A (zh) * 2014-01-29 2014-05-14 华中科技大学同济医学院附属同济医院 外周血白细胞介素25水平在哮喘分型中的应用及分型方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2009231733B2 (en) * 2008-03-31 2015-12-24 Genentech, Inc. Compositions and methods for treating and diagnosing asthma

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101711256A (zh) * 2007-04-18 2010-05-19 医疗研究局 针对il-25的抗体
CN102245638A (zh) * 2008-09-30 2011-11-16 医疗研究局 针对il-25的抗体
WO2010131009A2 (en) * 2009-05-15 2010-11-18 Medical Research Council Treatment of il-25 mediated diseases with tlr antagonists
CN103097416A (zh) * 2010-03-30 2013-05-08 詹森生物科技公司 人源化il-25抗体
CN103792375A (zh) * 2014-01-29 2014-05-14 华中科技大学同济医学院附属同济医院 外周血白细胞介素25水平在哮喘分型中的应用及分型方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BALLANTYNE, S.J. ET AL.: "Blocking IL -25 prevents airway hyperresponsiveness in allergic asthma", JOURNAL OF ALLERGY CLINICAL IMMUNOLOGY, vol. 6, no. 120, 31 December 2007 (2007-12-31), XP022383425 *
PETERSEN, B.C. ET AL.: "Interleukin-25 induces type 2 cytokine production in a steroid -resistant interleukin-17RB+ myeloid population that exacerbates asthmatic pathology", NATURE MEDICINE, vol. 5, no. 18, 31 May 2012 (2012-05-31), XP055216543 *

Also Published As

Publication number Publication date
CN103792375B (zh) 2017-07-07
CN103792375A (zh) 2014-05-14

Similar Documents

Publication Publication Date Title
Ambrosino et al. Gender differences in patients with COVID-19: a narrative review
US10132809B2 (en) Differential expression of protein markers for the diagnosis and treatment of eosinophilic esophagitis
Gessner et al. Angiogenic markers in breath condensate identify non-small cell lung cancer
Begum Breast cancer in Pakistan: a looming epidemic
Meng et al. Endotypes of chronic rhinitis: a cluster analysis study
Peng et al. Correlation of Krebs von den Lungen-6 and fibronectin with pulmonary fibrosis in coronavirus disease 2019
Li et al. Glutathione S-transferase P1 correlated with oxidative stress in hepatocellular carcinoma
Stanojkovic et al. Relationship between bone resorption, oxidative stress and inflammation in severe COPD exacerbation
Mahmood et al. Severity of coronavirus disease 19: Profile of inflammatory markers and ACE (rs4646994) and ACE2 (rs2285666) gene polymorphisms in Iraqi patients
Puliani et al. NETest: a systematic review focusing on the prognostic and predictive role
Huang et al. Serum pepsinogen levels are correlated with age, sex and the level of Helicobacter pylori infection in healthy individuals
Zhao et al. Sublingual vein parameters, AFP, AFP-L3, and GP73 in patients with hepatocellular carcinoma
Wismol et al. The values of nasal provocation test and basophil activation test in the different patterns of ASA/NSAID hypersensitivity
WO2015113478A1 (zh) 外周血白细胞介素25水平在哮喘分型中的应用及分型方法
Negrut et al. The Multisystem Impact of Long COVID: A Comprehensive Review
Trueba et al. Effects of academic exam stress on nasal leukotriene B4 and vascular endothelial growth factor in asthma and health
Wasilewska et al. Urinary OPN excretion in children with glomerular proteinuria
Mermutluoğlu et al. Evulation of prolidase enzyme, and galectin levels as a marker for fibrosis in patients with chronic hepatitis B.
Trivedi et al. Effect of Smoking and Its Cessation on the Transcript Profile of Peripheral Monocytes in COPD Patients
Qian et al. Podoplanin neutralization reduces thrombo-inflammation in experimental ischemic stroke by inhibiting interferon/caspase-1/GSDMD in microglia
Garcia-Marcos et al. The utility of sputum eosinophils and exhaled nitric oxide for monitoring asthma control with special attention to childhood asthma
Zuo et al. The persistence of SARS-CoV-2 in tissues and its association with long COVID symptoms: a cross-sectional cohort study in China
WO2010103585A1 (ja) メタボリック症候群又はその構成疾患の評価法
JP7328950B2 (ja) 乳幼児アトピー性皮膚炎の検出方法
Zhang et al. Reduced cortisol in the absence of bacterial infection in patients with hepatitis B virus cirrhosis

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15743858

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15743858

Country of ref document: EP

Kind code of ref document: A1