高氟干扰T淋巴细胞分化定向蛋白质相互作用研究

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.

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。

本项目建立了小鼠氟暴露模型，以胸腺为研究对象，采用iTRAQ技术筛选了加氟组与对照组之间的差异表达蛋白，探讨氟对胸腺蛋白差异表达的影响。结果显示，高氟可诱导350多个蛋白发生差异表达。进一步的生物信息学分析表明，这些蛋白主要分布于110多个信号通路，其中参与细胞代谢的有30多条，涉及70多个差异蛋白；参与信号调控通路的有50多，涉及160多个差异蛋白，参与疾病发生的有19条通路，涉及60多个差异蛋白。这些差异表达蛋白可以影响胸腺细胞的生物学过程，进而干扰T细胞的分化。进一步利用Western blotting和real-time PCR技术，检测关键互作蛋白的表达情况。基于上述研究结果，经生物信息学分析，构建了蛋白质相互作用系统图谱。同时，本研究还采用体外氟处理胸腺细胞模型，对关键互作蛋白分子进行了研究，采用流式细胞术，Western blotting和real-time PCR技术，验证了关键蛋白表达对T淋巴细胞分化定向的影响。项目从蛋白质相互作用的角度，阐明了高氟干扰T淋巴细胞分化定向的蛋白质相互作用机制，为地氟病的防治提供了理论依据。