NO/NOS在地方性氟中毒神经系统损伤及智力改变分子发病机制中的作用研究

Currently, endemic fluorosis has been listed as a disease of main prevention and cure among existing endemic diseases in our country. Although the neural system damage caused by endemic fluorosis and child amentia situation in endemic fluorosis areas have been highly focused by international fluoride research field, the molecular mechanism of its reaction is still unclear yet, which brings some trouble in preventing and curing endemic fluorosis. This project intends to research on the molecular mechanism of ultrastructural injury on neurons, as well as NO/NOS's intervention effects on the change of protein and gene level, the molecular mechanism of intelligence change and the NOS inhibitor (L-NAME). On the basis of preliminary work, this project plans to choose both adult and child population of patients in endemic fluorosis areas to copy adult and newborn rat models of burning-coal type and drinking-water type fluorosis, and copy fluorosis cell models in vitro culture. Using advanced technologies such as western blot and immunohistochemical method, Real-time PCR, animal behavior detecting and neuropathology examination, this project is discussing NOS/NO and NOS inhibitor's effect on the pathogenesis of neuro injured by fluorosis at protein and gene level, which aims at providing theoretical and experimental basis for preventing and curing researches on pathogenesis of neuro injured by fluorosis, providing experimental basis at protein and gene level for the effects of oxidative stress on the pathogenesis of fluorosis, and at carrying out preliminary research on clinical treatment and drug development.

当前，地方性氟中毒已列为我国现有地方病中重点防治的疾病，地方性氟中毒造成的神经系统损伤和地氟病区儿童智力低下现状已得到国际氟研究领域的高度关注，但其发生的分子机制仍不十分清楚，给地氟病的防治带来一定困难。本项目拟在前期研究工作基础上，选用地氟病区成年患者和儿童群体、复制燃煤型和饮水型氟中毒大鼠及仔鼠模型，复制体外培养氟中毒细胞模型，采用蛋白印迹和免疫组化方法、Real-time PCR、动物行为学检测、神经病理形态学检查等先进手段，来研究神经细胞超微结构损伤的分子机制、NO/NOS在蛋白和基因水平改变，智力改变分子机制，NOS抑制剂（L-NAME）的干预作用，从蛋白，基因水平探讨NOS/NO及NOS抑制剂在氟中毒神经损害发病机制中的作用，为氟中毒神经损伤发病机制和防治研究提供理论和实验依据，为氧化应激在氟中毒发病机制中的作用，从蛋白和基因水平上提供实验依据，为临床治疗和药物开发进行前期研究

地方性氟中毒是我国现有地方病中重点防治的疾病，地方性氟中毒造成的神经系统损伤和地氟病区儿童智力低下现状已得到国际氟研究领域的高度关注，但其发生的分子机制仍不十分清楚，给地氟病的防治带来一定困难。本研究以地氟病区成年患者和儿童群体、氟中毒大鼠及仔鼠模型，体外培养氟中毒细胞模型为研究对象，采用蛋白印迹和免疫组化方法、Real-time PCR、动物行为学检测、神经病理形态学检查等先进手段，来研究神经细胞超微结构损伤的分子机制；NO/NOS 在氟中毒神经损伤发病机制中作用；NOS特异性抑制剂（7-NI及L-NIO）对神经细胞损伤的保护作用及相关关系。我们对地氟病区成年患者和儿童群体血清检测NOS活性及NO含量，发现其NOS活性降低，NO含量增加，差异无显著性；在慢性氟中毒动物成年组及仔代模型实验部分，发现过量氟引起成年及仔代大鼠尿氟、骨氟、脑氟含量均显著性升高；形态学、超微结构及动物行为学改变：尼氏小体显著性下降；神经细胞超微结构损伤；学习能力及空间记忆能力下降；对nNOS蛋白与mRNA水平的影响在染氟早期及低浓度氟时即已发生。过量氟可引起大鼠脑组织NO含量改变，表现为：染氟3个月高氟组大鼠脑组织NO含量显著性下降，染氟6个之低氟组和高氟组大鼠脑组织NO含量显著性升高。相关性分析显示过量氟引起成年及仔代大鼠学习记忆能力降低与脑组织nNOS蛋白表达增加、NO含量增加有关，其间有量效关系。氟中毒细胞模型显示：过量氟可导致大鼠海马神经细胞超微结构损伤和细胞凋亡率升高，这可能和过量氟导致神经细胞nNOS、eNOS蛋白和mRNA表达水平升高、NO含量增多、NOS活力升高有关。NOS特异性抑制剂：7-NI、L-NIO可以拮抗过量氟引起的神经细胞超微结构损伤、eNOS蛋白和mRNA表达及细胞凋亡率升高，起到一定的氟中毒性神经保护作用。这为氟中毒所致神经系统损伤治疗药物的研究提供了实验基础。