高氟干扰T淋巴细胞分化定向蛋白质相互作用研究

项目名称： 高氟干扰T淋巴细胞分化定向蛋白质相互作用研究

项目编号： No.31201963

项目类型： 青年科学基金项目

立项/批准年度： 2013

项目学科： 畜牧学与草地科学、兽医学、水产学

项目作者： 王宏伟

作者单位： 河南科技大学

项目金额： 21万元

中文摘要： 针对高氟干扰T淋巴细胞分化定向这一前期研究结果，本项目拟建立小鼠染氟模型，以胸腺为研究对象，采用 2D-DIGE技术筛选加氟组与对照组之间的差异蛋白，探讨氟对胸腺蛋白差异表达的影响；并以筛选出的关键差异蛋白为"诱饵"，构建关键蛋白DNA-BD 载体，用酵母双杂交系统，从小鼠胸腺cDNA文库中筛选与其相互作用的蛋白，采用免疫共沉淀和GST-pull down技术验证酵母双杂交结果，利用Western blotting和免疫荧光技术，检测互作蛋白的表达情况，经生物信息学分析，构建蛋白质相互作用系统图谱。最后，建立体外氟处理胸腺细胞模型，针对关键互作蛋白分子设计小分子siRNA，构建siRNA真核表达载体，干扰胸腺细胞关键蛋白的表达，验证关键蛋白表达对T淋巴细胞分化定向的影响。从蛋白质相互作用的角度，阐明高氟干扰T淋巴细胞分化定向的蛋白质蛋与白质相互作用机制，为地氟病的防治提供理论依据。

中文关键词： 氟；T细胞；分化；蛋白组学；iTRAQ

英文摘要： It is found in our previous study that high fluoride interferes the T lymphocyte differentiation direction. In the present study, mice were used to establish the fluorosis animal model, 2D-DIGE technology was used to analyse the difference proteins expression of thymus treated by fluoride. To explore the protein-protein interaction in the lymphocyte differentiation direction, the thymus cDNA library was subjected to yeast two-hybrid system. Co-immunoprecipiation and GST-pull down technology were associated to validate the protein interaction results. Western blotting and immunofluorescence technique were used to detect the proteins expression. By use the bioinformatics analysis, protein interaction schematic diagram was designed on the basis of the results mentioned above. Furthermore, to explore the effect of key protein in the T lymphocyte differetaition direction,thymus cell culture was used to establish the fluorosis model in vitro, the cell model was subjected to siRNA expression vector targeting at key protein. This study provides a theoretical basis on the protein-protein interaction mechanism of high fluoride in interfering the T cell differentiation direction and an attractive prevention and cure for the control of endemic fluorosis.

英文关键词： fluoride；T-lymphocyte；Differentiation；Proteomics；iTRAQ