大骨节病儿童生物标志物及其与软骨细胞坏死和真菌毒素的关系

For the key scientific issues on the biomarker shortage of Kashin-Beck disease (KBD), which has been seriously damaging the public health of the endemic areas in China, biomarkers for KBD children and its relationship with cartilage necrosis and mycotoxins will be mainly studied by using the unique disease resource of KBD in China applying population survey combined with the modern molecular bio-techniques. The main contents including: (1) Being integrated the high-throughput results of the abnormal changes of differentially genes, proteins and metabolic phenotypes from KBD recently, the candidate biomarkers for KBD children will be identified by real-time quantitative polymerase chain reaction (qRT-PCR) and etc. (2) Using the immunohistologic methods as biotin-strept-avidin system，the biomarkers related to chondrocyte necrosis in cartilage of KBD children will be identified. (3) Via the technologies of the cell culture in vitro and the cell biotechnology, biomarkers related to chondrocyte necrosis induced by mycotoxins will be identified by comparing the damage effects of deoxynivalenol (DON) and T-2 toxin on chondrocytes and fibroblasts. (4) The high sensitivity and specificity of biomarkers will be established for the early damages in KBD children through epidemiologic investigation and qRT-PCR. The integration of all the research contents will provide the scientific basis to establish the new molecular techniques for early diagnosis of KBD and to promote the development of cell and animal models, and to explore the originating factor of KBD on a cell and molecular level.

针对严重危害我国病区人口健康的大骨节病缺乏疾病和环境生物标志物的重要问题，利用我国独有大骨节病疾病资源，采用人群调查和现代分子生物学技术相结合的方法，集中研究大骨节病儿童生物标志物及其与软骨细胞坏死和真菌毒素的关系，主要内容有：(1) 整合前期所获大骨节病差异基因、蛋白和代谢物的高通量检测结果，采用实时荧光定量聚合酶链式反应等技术确定大骨节病儿童生物标志物；（2）采用抗生物素蛋白-生物素-碱性磷酸酶等技术确定大骨节病儿童软骨坏死相关的早期损害标志物；（3）采用体外细胞培养和细胞生物学技术比较雪腐镰刀菌烯醇和T-2毒素对软骨细胞和成纤维细胞的损伤作用，确定真菌毒素致软骨细胞坏死的生物标志物；(4）采用大骨节病儿童调查、实时荧光定量聚合酶链式反应等技术验证大骨节病儿童早期损害生物标志物的敏感性和特异性，为建立大骨节病分子早期诊断技术,推进细胞与动物模型研究和探寻始动致病因素，提供可靠的科学依据

本课题主要针对大骨节病缺乏疾病和环境生物标志物及其病因发病机制不明的重要科学问题，经2015-2018年采用大骨节病儿童X线诊断、转录组学、代谢组学和细胞培养等生物学技术，集中研究大骨节病儿童生物标志物及其与软骨细胞坏死和真菌毒素的关系，获得的主要创新性结果有：① 通过检测大骨节病儿童和成人外周血和软骨差异表达基因、血清代谢物，发现20个大骨节病外周血差异表达基因标志物，其判定大骨节病的准确性为90%，灵敏度为85%，特异性为95%；大骨节病儿童血清代谢表型以糖代谢紊乱和支链氨基酸（亮氨酸、异亮氨酸、缬氨酸）代谢异常为特点，提出大骨节病分子诊断的标志物。② 通过人群、组织和细胞、蛋白、基因不同水平层面研究，发现T-2毒素、DON诱导人软骨细胞差异表达基因中三个基因、蛋白（GDF5、COL9A1和MMP-3）与大骨节病软骨细胞差异基因表达存在一致性，提出T-2 毒素和/或脱氧雪腐镰刀菌烯醇损伤软骨细胞且与大骨节病相关的生物标志物。③ 发现T-2 毒素、DON单一或联合均可诱导人软骨细胞系（C28/I2）、肝细胞系（L-02）和肾小管上皮细胞系（HK-2）损伤及其相关基因、信号通路变化；结合动物实验，证实T-2毒素及HT-2毒素、DON可在软骨及其它器官中蓄积，对软骨组织和软骨细胞的损害不具有特异性。④发现早于指骨X线干骺端和骨端轻度异常征象的病区儿童出现Glutamine, N-(2,3-Dihydroxybenzoyl)-L-serine, N6-(L-1,3-Dicarbo xypropyl)-L-lysi-neL-4- Hydroxyphenylglycyl-L-arginine等17个代谢物。这些研究结果为大骨节病生物标志物及其与软骨细胞坏死和真菌毒素关系的深入研究，提供了新的科学依据。