孤独症谱系障碍诊断和预测蛋白标志物的筛选验证及相关机制研究

The autism spectrum disorder (ASD) is characterized by impairments in reciprocal social interaction and communication along with restricted and repetitive behaviors. The epidemiology of ASD is continuously increasing all over the world with huge social and economical burdens. Although the exact causes of ASD are still unknown, previous studies suggest that autism can be considered as a multi-factorial disease in which both genital and environmental factors are involved. As the etiology of autism is not completely understood, there is still no medication available for the treatment of this disorder. However, some behavioral treatments are available to improve core and associated symptoms of autism, particularly when initiated at an early stage. Thus, there is an increasing demand for finding biomarkers of autism which could help to identify children with ASD at an early stage. In our previous study, we have obtained the differentially expressed proteins between the autistic subjects and controls. Here, we will further obtain the candidate proteins for diagnosis of ASD by different ways, including animal models, literature surveys and bioinformatics analysis. These proteins would further be validated by using a multiple reaction monitor (MRM) assay to confirm them as important biomarkers. In addition, the plasma and PBMCs cell samples of 6 month old high-risk infants (brother and sister were diagnosed as ASD) were obtained by prospective epidemiological study. At the age of 2 years, they were diagnosed and the samples of 6 months of age were used to screen and validate for early prediction markers. Next, these differentially expressed proteins will be further analyzed with bioinformatics approaches and verified for their possible roles in the pathophysiology of autism. In particular, we will isolate rat brain microglia and PBMCs cell from model mice to investigate whether they have “parallel activation response”. This project is of utmost significance as it will contribute practically for the welfare of human being by exploring the molecular mechanism of autism, especially in the aspects of finding diagnostic and prediction biomarkers for the disease.

孤独症谱系障碍(ASD)是一组以交流、语言障碍和行为异常为特征的发育障碍性疾病。由于病因及发病机制不明，无特效药物和特异性标志物。但早发现、早干预可明显改善预后。本项目拟在前期血浆及外周血单核细胞(PBMCs)蛋白质组学研究基础上，扩大范围(如生物信息预测脑“入血”相关蛋白等)筛选候选诊断标志物。应用靶标蛋白质组学技术从血浆、PBMCs细胞及二者共同变化蛋白的角度，进行不同样本量人群验证，获得ASD诊断标志物。通过流行病学研究，获得高风险婴儿(兄姐为ASD的婴儿)6月龄时的血浆及PBMCs细胞，在他们2岁时对他们进行诊断，取病例和对照6月龄时的样品，采取与诊断标志物研究相似的策略，获得早期预测标志物。分析蛋白功能和信号转导通路，从动物和人群水平对关键蛋白进行验证。分离模型鼠脑小胶质细胞和PBMCs细胞，探讨它们是否存在“平行性激活反应”。研究将为揭示疾病机制、实现早期诊断和预测奠定基础。

孤独症谱系障碍（ASD）是一组以交流、语言障碍和行为异常为特征的神经系统发育障碍性疾病。给个人、家庭和社会带来沉重的精神、经济负担。由于发病机制尚未阐明，缺乏有效防治药物，治疗以教育和行为干预为主。但早发现、早干预可明显改善预后。目前，临床上诊断主要依靠量表，有一定的主观性。因此，寻找用于早期客观诊断的生物标志物，探讨与之关联的疾病机制，具有重要意义。.本项目在前期研究基础上，应用iTRAQ（同位素标记相对和绝对定量）定量蛋白质组学技术分析了ASD 患儿与健康对照外周血单核细胞（PBMCs）差异蛋白，数据库生物信息分析预测脑“入血”ASD 相关蛋白及实验室验证，以及应用多组学方法与质谱多反应监测技术（MRM）开展ASD诊断标志物的研究。同时，建立了ASD动物模型展开疾病机制的研究。开展了ASD与微量元素关系的研究。 此外，应用项目建立的方法，展开了其他疾病诊断标志物和疾病机制的研究。.项目按计划执行，超额完成了任务。通过项目的实施，获得的与ASD研究相关的结果如下：.（1）获得了系列外周血和外周血单核细胞（PBMCs 细胞）ASD 诊断蛋白标志物，以及外周血和PBMCs 细胞共同差异蛋白诊断标志物。这些标志物与ASD的发病机制相关。结果表明免疫在ASD 的发病中扮演关键角色，ASD 患儿外周血PBMCs 细胞与补体和凝血级联可能在ASD患儿的外周血处于“激活状态。”.（2）尽管ASD 患儿在基因水平可能存在异质性，但仍能从外周血或者PBMCs 细胞寻找到区别病例与对照的诊断生物标志物。.（3）从蛋白质组学的角度探讨并获得了孤独症VPA大鼠模型疾病发生相关机制。.（4）从基因组学、转录组学、蛋白质组学、代谢组学、氧化应激等角度全面综述了国内外ASD 诊断标志物研究的最新进展。.（5）开展了基于病例对照研究的ASD微量元素meta分析，以及儿童血浆微量元素的检测，扩展了当前对微量元素与ASD关系的理解。.我们将在此基础上，对ASD诊断标志物进行凝练验证，开展临床转化研究以及相关发病机制的深入研究。