纳米SiO2暴露对中枢海马影响及GAP-43相关机制研究

With the development of nanometer technology, as a new material nanometer material has been widely used in machinery, textile, war industry, cosmetics, household appliances, medicine, chemical industry and many other fields. With the industrialization of nanotechnology and widely used of nanomaterials, the chances of people to contact nanomaterials are increased, and the chances of the producers, researchers and consumers to contact nanomaterials are increased furtherly. Studies have been found that the nanoparticles can enter into the central nervous system by crossing the blood-brain barrier or via the olfactory nerve pathway, and into the different brain regions to deposit. The hippocampus may be the main target area in the central nervous system of nanometer particles accumulating and effecting. Nanometer silicon dioxide (SiO2) is an important member of nanometer materials. The opportunities of contacting nanometer SiO2 are more and more because of its highest yield and wide application, and this will continue to increase. Therefore, the biological safety and neural biological effect of the nanometer SiO2 will be more important. At present, the domestic focus of nanometer SiO2 on experimental animal is pulmonary toxicity and reproductive toxicity, and abroad focused mainly on nanometer SiO2 cell toxicity study. So far, nanometer SiO2 affects on the central nervous system and its biological effects have been reported never. Growth associated protein (GAP-43) is closely related with the neural plasticity. It is the prefer protein to study hippocampal neurogenesis and injury. It can be inferred that the GAP-43 and its signal transduction pathway may be one of the important mechanisms of nanometer SiO2 effecting hippocampal neural functions. Our study will focus on the influence of nanometer SiO2 on central hippocampal function from the overall level, organization level and cell level by Western blot, real-time fluorescent quantitative RT-PCR, immunohistochemical and electrophysiological techniques, respectively. In this process, the expression changes of GAP-43 and its signal transduction pathway proteins will be also observed. Our aim is to understand the role of GAP-43 in the process of nanometer SiO2 affects hippocampal neuronal function, and make clear its molecular pathways. This will be important in providing a new idea and theory for further study of nanometer SiO2 induced learning and memory impairment and abnormal neurological behavior, and to explore the prevention and control measures.

纳米材料的广泛应用使其生物安全性问题日益突显。研究发现，纳米颗粒可穿越血脑屏障进入中枢神经系统，并转运至不同脑区沉积，中枢海马可能是纳米颗粒蓄积和作用的主要靶区。纳米SiO2 产量高、应用广，其神经生物效应鲜有报道。本研究拟观察纳米SiO2对海马神经功能的影响并探讨其可能机制。神经生长相关蛋白(GAP-43) 是研究中枢海马神经功能和损伤的首选蛋白，与神经可塑性密切相关。本课题采用Western blot、实时荧光定量PCR、免疫组化和神经电生理等技术，分别从整体、组织和细胞水平研究纳米SiO2对中枢海马功能的影响，以及在此过程中GAP-43及其信号转导通路蛋白表达的变化，旨在认识GAP-43表达的变化在纳米SiO2影响海马相关神经功能中的作用，明确其分子途径，为进一步研究纳米SiO2诱导学习记忆功能损害和神经行为异常的机制、探讨防治措施提供新的思路和理论依据。

纳米材料的广泛使用使其生物安全性问题日益凸显。本研究从GAP-43及其信号转导途径入手，探讨GAP-43在纳米二氧化硅（nano-silicon dioxide,nano-SiO2）损害认知功能过程中的变化及其在信号转导途径中是否发挥这重要的调控作用。本实验以大鼠为研究对象，分为整体水平和离体脑片干预两个水平。结果：第一部分：nano-SiO2对大鼠认知功能的影响。经过4周nano-SiO2暴露，结果显示，体重涨幅L组与C组相比无显著性差异，但有减小趋势，随着nano-SiO2浓度的增加，H组体重增长显著低于C组（P<0.05）。HFS后，各组PS峰值增加幅值明显升高，出现LTP。与C组比较，L组的PS峰增幅显著提高（P<0.05）；H组PS峰增幅更高（P<0.01）。HE染色实验结果显示nano-SiO2干预组大鼠海马神经元出现明显的组织细胞损伤的形态学改变，有剂量-效应关系。第二部分： 1.经nano-SiO2干预后，L组和H组较C组GAP-43 mRNA表达量显著降低（P<0.05），PKC和CaMK2 mRNA未发生改变。2.免疫组化结果，L组和H组较C组蛋白阳性神经细胞数目少、体积小、染色较浅， L组PKC较C组有降低趋势，H组PKC显著降低（P<0.01）。3.western blot结果显示，海马组织中干预组GAP-43、P-GAP-43、PKC蛋白表达量降低（P<0.05，P<0.01）； CaMKⅡ蛋白表达量L组无差异，H组较C组显著降低（P<0.01）；大脑皮层中L组GAP-43、P-GAP-43、PKC、CaMKⅡ蛋白表达量较C组低（P<0.05），H组较C组显著降低（P<0.01）。4.脑片L组GAP-43 mRNA表达量较C组低（P<0.05），M组和H组较C组显著降低（P<0.01）；5.L组GAP-43、P-GAP-43、PKC、CaMKⅡ蛋白表达量较C组低（P<0.05），M组和H组较C组显著降低（P<0.01），有剂量-效应关系。结论：nano-SiO2可能通过影响海马和大脑皮层中以GAP-43为调控子的信号转导通路，损害机体的学习记忆功能。因此GAP-43可能作为改善nano-SiO2损害认知功能的药物靶点，对改善毒性作用，保护职业人群健康，并为进一步探寻有效的医学防治措施提供一定理论依据。