【作者】 杨慧；

【导师】 李云生；

【作者基本信息】 天津医科大学 ， 人体解剖与组织胚胎学， 2010， 博士

【摘要】 甲醛为较高毒性物质,研究显示其对大鼠的神经系统有明显的损害作用,在人群中亦发现甲醛具有神经毒性。可见研究甲醛对学习记忆的影响十分必要。海马是脑边缘系统中最重要的结构之一,是学习记忆等高级神经活动的重要部位。CaMKⅡ和Ng是神经系统特异表达的蛋白,尤其高表达于海马和大脑皮层的突触后致密物(post-synaptic density,PSD)中,CaMKⅡ是Ca2+-CaM通路的下游分子,在学习时通过Ca2+内流引起磷酸化而被激活,活化的激酶分子在催化自身磷酸化,从而使激酶分子在学习结束后能持久保持活化状态。实验表明CaMKⅡ是空间学习和记忆的分子基础；大量研究报道,Ng是参与调控学习记忆过程的一个重要蛋白,参与了学习记忆过程中起核心作用的信号转导以及突触可塑性的主要调控环节,有研究进一步证实Ng作为记忆形成机制的上游调控子,通过调控游离的Ca2+和CaM,在PKC介导的信号转导途径中起关键作用。目的本研究旨在以大鼠为模型,通过腹腔注射染毒,观察甲醛对大鼠学习记忆能力、对大鼠脑组织内Ng及CaMKⅡ表达水平的影响,将有助于进一步揭示甲醛损害学习记忆功能的机制,为深入开展防治甲醛神经毒性的研究提供科学的理论依据。方法1.选用健康SPF级SD雄性大鼠88只,体重180-200g,在标准SPF级动物房内饲养。每天上午腹腔注射给药,对照组：1ml生理盐水；低剂量组：5mg/kg甲醛溶液；中剂量组：10mg/kg甲醛溶液；高剂量组：20mg/kg甲醛溶液。染毒时间分别为7天和14天。用Morris水迷宫对大鼠进行行为学检测。实验终期,大鼠经10%水合氯醛腹腔麻醉,一部分断头分离脑组织,将脑组织在冰冷生理盐水中漂洗,用滤纸吸干,置于-80℃冰箱保存；另一部分10%中性甲醛灌注后,将脑组织固定。2.用Morris水迷宫检测大鼠的空间学习记忆能力改变,测试包括定位航行实验和空间探索实验。3.收集14天染毒组大鼠血清和脑组织,采用柱前衍生HPLC法测定不同时相血中(0、7、14天和处死时)及处死后大脑组织中甲醛浓度。4.14天染毒组大鼠海马和前额皮层,采用免疫组化染色并观察,对CaMKⅡNg免疫反应产物在前额皮层、海马DG区和CA3区平均光密度值进行测量分析；应用Western Blotting方法检测海马CaMKⅡ、Ng蛋白水平的变化。结果1.HPLC法测定,脑组织中甲醛值中、高剂量组与对照组和低剂量组间差异有统计学意义(P<0.05)；在7、14天两个时间点,中、高剂量组血甲醛值均显著高于对照组和低剂量组(P<0.05)。结果表明,甲醛在大鼠血液和脑组织中有不同程度的蓄积,并存在时间、剂量效应关系。2.空间探索实验：7天染毒组中剂量组和高剂量组在第一象限的游泳点数及百分比高于对照组及低剂量组且差异具有统计学意义(P<0.05)；14天组空间探索试验中低、中和高剂量组在第一象限的游泳点数及百分比高于对照组且差异具有统计学意义(P<0.05),对照组游泳速度与低、中和高剂量组比较差异具有统计学意义(P<0.05),提示甲醛染毒对大鼠的空间记忆能力有损害。3.甲醛腹腔注射染毒对海马、前额皮层的Ng和CaMKⅡ蛋白表达都有影响,免疫组化结果显示高剂量组平均光密度低于其它三组,差异具有统计学意义(P<0.05)。结论1.建立了甲醛致空间学习记忆能力损害模型,大鼠的空间学习记忆能力受到损害,其中记忆能力的损伤更为严重。2.高剂量甲醛染毒抑制了Ng和CaMKⅡ在海马、前额皮层的表达。3.甲醛染毒后,甲醛在大鼠血液浓度表现为剂量和时间的效应关系；脑组织中甲醛浓度表现为剂量效应关系。更多还原

【Abstract】 Formaldehyde is a toxic substance. Studies have shown that it can be harmful obviously for the nervous system of rats, and it also has been found its neurotoxicity for the people. So the research that the effects of formaldehyde on learning and memory is very essential.Hippocampus is one of the most important structures in the limbic system of brain, and is an important part of advanced nervous activity including of learning and memory. CaMKII and Ng are the specific proteins of nervous system, especially in the post-synaptic density (PSD) of hippocampus and pallium, it has a higher expression. CaMKII is a downstream molecule of Ca2+-CaM pathway, during the study, induced phosphorylation by Ca2+influx and then is activated, activated kinase molecules catalyze autophosphorylation again, and thereby kinase molecules can maintain activated state after learning. Experiments show that CaMKII is the molecular basis of spatial learning and memory, many research report that Ng is an important protein involved in the regulation of learning and memory, which participates the signal transduction played central role during learning and memory, and it is the main regulatory aspect of synaptic plasticity. Studies have further confirmed Ng as an upstream regulatory element on the formation mechanism of memory, which plays a key role in the PKC-mediated signal transduction pathway by regulating free Ca2+and CaM. ObjectiveIn this study,exposing the rats to toxin by intraperitoneal injection, we research the effects of formaldehyde on learning and memory of rats, Ng and CaMKII expression level in brain tissue of rats, which is helpful to further reveal the mechanism of formaldehyde damaging learning and memory, and provide a theoretical scientific basis for the research of preventing the neurotoxicity of formaldehyde. MethodsWe choosed healthy (SPF grade) male rats as subjects; they were randomly divided into4groups, a normal control group given the rats normal saline by intraperitoneal injection every morning, the others groups given the rats formaldehyde solution by intraperitoneal injection every morning, dose respectively5mg/kg,10mg/kg,20mg/kg, the time of exposure separately were7days and14days. After evaluating the learning and memory of rats by the Morris water maze, we anesthetized rats by use of10%chloral hydrate, then separated the serum and brain tissue, the serum and a part of tissue were used to determined the formaldehyde concentration by the method of pre-column derivatization HPLC. Other part of tissue were used for Immunohistochemistry and Western Blotting, and observed the changes of CaMKII, Ng protein expression in hippocampus and prefrontal cortex. ResultsHPLC:The formaldehyde concentration of serum between the middle-dose group, high-dose group are higher than normal control group and low-dose group (P<0.05). The formaldehyde concentration of brain are same as that of serum.In the spatial probe test, middle-dose and high-dose groups swimming time and the percentage of swimming path in the first quadrant compared with the normal and low-dose group’s, the difference has statistically significance(P<0.05) in7days of intraperitoneal injection. In that of14days the three groups of intraperitoneal injection are longer than the normal control group on swimming time and the percentage of swimming path in the first quadrant, the swimming speed of the normal control group compared with other groups, there are statistically significance(P<0.05)On the high-dose group the expression of Ng and CaMKII in hippocampus and prefrontal cortex were significantly lowest on IHC. The expressions of Ng and CaMKII protein in hippocampus of high-dose groups were significantly lower than that of control, low-dose and middle-dose groups but there was no significant difference on WB. Conclusion1. To set up the model of injurying on spatial learning and memory by formaldehyde, there are some damage about the ability of rats’ spatial learning and memory, the ability of rats’ spatial memory were more serious2. After intraperitoneal injection formaldehyde, the expression of Ng and CaMKⅡ in hippocampus and forehead of cortex are inhibited. 3. After intraperitoneal injection formaldehyde, formaldehyde exists different level of accumulation in blood and brain tissue of rats, the extent of accumulation has a dose-response relationship.更多还原