MAP2在氟致小鼠大脑损伤机制中的作用研究

It has been found that fluoride induced the damaged structure of brain and different expression of MAP2 in cerebral cortex of brain in our study. But the role of MAP2 in mouse brain damaged by fluoride was unclear. So, the animal model (mouse) was established in this study, which simulated natural environment of high fluoride area. The change of MAP2 in brain in different developing period of offspring mouse exposed to fluoride were studied with real-time PCR, Western blotting and immunofluorescence techniques; At the same time, MAP2 gene knockdown in mouse brain were regulated separately by RNA interference technique, and MAP2 gene knockdown mouse model was established in this study. The structures of brain and cell skeleton morphological changes in mouse model exposed to fluoride were observed, and the ability of learning and memory of model mouse were studied with step-down test in order to analyze the correlation of intelligence and MAP2 gene knockdown. And then, the protein interacted with MAP2 in brain were explored with yeast two-gybrid technique and these results were verified with co-immunoprecipiation and GST-pull down technique. Western blotting and immunofluorescence technique were used to detect these protein expression. Schematic diagram of protein interacted with MAP2 were designed on the basis of the above results with bioinformatics analysis. This study will provide abundant evidences to reveal the role of MAP2 in mouse brain damaged by fluoride, to show the action mechanism of nerve cell structure damaged by fluoride and to further provide the rationale of controlling fluorosis effectually.

针对高氟损伤大脑形态结构，引起大脑皮质微管相关蛋白2（MAP2）差异表达这一前期研究结果，本项目模拟高氟区自然环境，建立氟暴露小鼠模型，利用Real-time PCR，Western blot和免疫荧光技术研究MAP2在不同发育时期子代小鼠大脑中的变化规律；利用RNA干扰技术，调控小鼠大脑MAP2基因沉默，建立MAP2基因沉默小鼠模型，观察氟致MAP2基因沉默小鼠大脑组织及细胞超微结构变化，采用跳台法测定小鼠学习记忆能力，分析MAP2基因沉默与智力相关性；利用酵母双杂交技术，筛选MAP2互作蛋白，采用免疫共沉淀和GST-pulldown技术验证酵母双杂交结果，利用Western blot和免疫荧光技术，检测互作蛋白的表达情况，构建蛋白质互作网络图谱，揭示MAP2在氟损伤大脑机制的作用，阐明高氟损伤神经细胞结构的作用机制，为防治动物（包括人）氟中毒提供理论基础。

课题组前期研究发现氟中毒可以降低子代大鼠学习记忆能力，大脑皮质和海马结构损伤，出现病理学改变，神经细胞凋亡率升高，DNA损伤严重等现象。细胞骨架是否也受到损伤?本研究以此为契机，以ICR小鼠、Neuro-2A为研究对象，研究细胞骨架蛋白MAP2在氟中毒发生机理中的作用。.(1)通过自由饮用含氟水（0, 50 or 100 mg/L）建立氟中毒ICR小鼠子代模型。在0 d，10 d，20 d，30 d，60 d和90 d，分别取子代小鼠大脑皮层进行研究。采用水迷宫测定30 d子代小鼠智力水平，结果显示氟处理组的学习记忆能力受到显著影响，潜伏期显著延长，并且穿过目标象限次数减少。HE染色观察到氟处理组大脑皮层锥体细胞数量减少，胶质细胞和皱缩和碎裂。氟暴露组小鼠大脑皮质MAP2、SYP和Dbn的mRNA和蛋白表达水平均不同程度受到抑制，谷氨酸受体蛋白表达也出现下降的趋势（P>0.05）。酵母双杂交成功筛选出与MAP2互作的蛋白-Myosin。推测，氟暴露模型子代小鼠学习记忆能力下降可能是氟抑制骨架蛋白和突触相关蛋白表达，继而影响NMDAR蛋白功能所致。.(2)以Neuro-2A细胞为试验对象，分别外源添加0，1，2，4，6 mM NaF 孵育24 h，探讨氟对神经细胞骨架的影响。结果发现氟暴露能降低细胞的存活率，并且细胞上清中LDH的含量逐渐升高，说明随着NaF浓度的升高，细胞膜的完整性受到破坏，并且呈现剂量依赖性。光学显微镜观察发现氟处理后细胞开始变圆，贴壁性差，神经细胞的正常形态和轴突消失。F-actin染色结果也证实了这一结果。Western blot检测结果发现，NaF处理组细胞骨架蛋白MAP2和突触小泡蛋白SYP的表达量显著下降。ELISA检测结果发现谷氨酸及其受体蛋白的含量显著受到抑制。透射电镜观察细胞超微结构，随着氟浓度增加，神经细胞出现线粒体凝集，空泡化，细胞水肿。RT-PCR方法证实了氟暴露能使细胞骨架的经典调控通路RhoA/ROCK/MLC激活。