基于稳定同位素标记定量蛋白组学的儿童哮喘中医证候血清标志物筛选与鉴定

In China, the incidence of childhood asthma was increased by 50% in recent ten years. Because of the side effects of hormone, hormone therapy may lead to inadequate treatment or over treatment in the treatment of stable asthmatic children. Traditional Chinese medicine (TCM) can enhance apparently the effect of anti-asthmatic drug and reduce the toxicity of hormone. In TCM, the determinations of treatment based in pathogenesis obtained through differentiation of symptoms and signs of childhood asthma. Determinations of treatment based in pathogenesis obtained through differentiation of symptoms and signs means deducing the causes of a disease and nature of disease according to the outer signs of a disease. Syndrome proteomics guided by the theory of syndrome, is applying the method of the formation of syndrome and to interpret the nature of syndrome in the level of integer proteins expression. So, it will help to reveal the biochemistry basis and pathogenesis of syndromes in childhood asthma. During the pilot study, a number of discriminating protein peaks have found in stable childhood asthma syndrome using SELDI-TOF-MS methods and established protein spectrum. However, the SELDI-TOF-MS technology can only detect one characteristic proteins, and unable to identify and quantify discriminating expressed proteins directly. Therefore, in the present study we used isobaric tags for relative and absolute quantification (iTRAQ) method coupled with two dimensional liquid chromatography/tandem mass spectrometric (2DLC-MS/MS) method for detecting biomarkers of serum biomarkers in stable childhood asthma syndrome. Using Biomarker Pattern Software, serum protein pattern model was established for distinguishing different stable childhood asthma syndromes. The present study, using quantitative proteomics technology, it will help to provide assistance for the evaluation and treatment of stable childhood asthma, and also will help to provide a biological basis for the determinations of treatment based different stable childhood asthma syndromes.

我国儿童哮喘发病率近十年上升了50.6%，西医的激素治疗因其副作用易使其在哮喘稳定期治疗中导致过治疗和治疗不足。中医遵循“急则治其标，缓则治其本”原则，辨清证候确定治法治则，对儿童稳定期哮喘具有一定的疗效。然而目前哮喘中医辨证标准缺乏科学量化而影响中医对儿童哮喘的治疗效果。课题组曾用SELDI-TOF MS法研究儿童哮喘证候，获得多个差异蛋白峰并建立了相应的证候血清蛋白图谱。但该技术只能捕获某一特性蛋白，且无法直接鉴定差异蛋白。为此本课题应用稳定同位素标记定量（iTRAQ）法结合二维液相色谱串联质谱（2DLC-MS/MS）技术平台研究儿童哮喘稳定期证候，同位素标记血清蛋白后经2DLC-MS/MS分离，鉴定并验证证候标志蛋白，构建儿童哮喘稳定期证候蛋白模型。本课题应用定量蛋白质组学技术研究儿童哮喘证候分型，有助于规范儿童哮喘治疗的辨证施治，提高中医疗效，并为阐明证候的生物学本质提供科学依据。

哮喘影响儿童正常的生活及其生长发育，其发病率呈上升趋势。中医遵循“急则治其标，缓则治其本”原则，辨清证候确定治法治则，对儿童稳定期哮喘具有一定的疗效。然而目前哮喘中医辨证标准缺乏科学量化而影响中医对儿童哮喘的治疗效果。本研究采用稳定同位素标记定量（iTRAQ）法结合二维液相色谱串联质谱（2DLC-MS/MS）技术筛选归纳了儿童哮喘各证候特有的差异蛋白峰，获得脾肾阳虚证组特征性蛋白51个，肺肾阴虚证组特征性蛋白29个，肺脾气虚证组特征性蛋白34个，这些蛋白可能是证候外在表现的内在病理机制和物质基础，为整体性评价儿童哮喘中医证候的实质提供证据；筛选获得60个蛋白在儿童哮喘三种中医证候组之间两两比较有差异性，差异的标志蛋白可能体现了儿童哮喘证候间一定的相互关系及演变规律。采用生物信息学分析（GO、KEGG、STRING）分析筛选的儿童哮喘中医证候相关的差异蛋白，主要参与生物调节、代谢过程、生物过程调节、响应刺激等生物过程；最主要参与补体和凝血级联反应通路；且差异蛋白间具有紧密的功能联系。采用ELISA法验证儿童证候差异蛋白ApoCⅢ、CD5L和SAA在儿童哮喘血清中的含量，脾肾阳虚证组ApoCⅢ含量明显低于肺肾阴虚证组和肺脾气虚证组；脾肾阳虚证组CD5L含量明显低于肺脾气虚证组和肺肾阴虚证组；肺肾阴虚证SAA含量明显高于肺脾气虚证和脾肾阳虚证，且SAA含量在三证之间比较均有显著差异。ApoCⅢ、CD5L和SAA在儿童哮喘中医证候间的差异表达可能为儿童哮喘中医证候分型的血清潜在标志物，可在临床和功能方面作进一步的验证。采用决策树建模，构建了由ApoCⅢ、CD5L和SAA这3个蛋白组成的儿童哮喘中医证候蛋白辨证模型，该辨证模型对于肺肾阴虚证的准确率为82.5%，对于肺脾气虚证的准确率为70.0%，对于脾肾阳虚证的准确率为80.0%。构建的儿童哮喘中医证候蛋白辨证模型，可为儿童哮喘证候诊断的客观化提供蛋白质标准，有助于规范儿童哮喘中医治疗的辨证施治。