【作者】 张尤新；

【导师】 孙黎光；

【作者基本信息】 中国医科大学 ， 细胞生物学， 2007， 博士

【摘要】 目的重金属铅是一种普遍存在的环境神经毒物，铅暴露导致儿童学习记忆认知能力异常日益严重。1943年Byers和Lord首次报道铅对儿童认知行为和智力发育的远期危害。儿童和成人对铅的神经毒作用敏感性和反应性不同，发育中的中枢神经系统对铅的神经毒性尤为敏感。铅对胚胎期和幼年期的毒害主要表现为记忆力和注意力的变化，而且一经损害不可逆转。目前研究认为突触部位有一些蛋白激酶分子在学习记忆时被磷酸化而激活参与学习记忆过程。现发现海马空间记忆形成过程中至少有三种蛋白激酶被激活，即Ca²⁺／钙调蛋白依赖的蛋白激酶Ⅱ(Ca²⁺／Calmodulin dependent proteinkinaseⅡ，CaMKⅡ)、蛋白激酶A（protein kinase A，PKA）、细胞外信号调节激酶（extracellular signal regulated kinase，ERIC），活化的激酶分子再催化自身磷酸化，使激酶分子在学习记忆中保持长期活化状态。其中CaMKⅡ是一种具有8～12个亚单位的酶，它是脑内含量最丰富的蛋白激酶，在海马和新皮层的突触后神经元致密层（Postsynaptic Density，PSD）密度最高。海马和大脑皮层是学习和记忆的结构基础。由于CaMKⅡ的特点是亚基上Thr-286磷酸化可使Ca²⁺-依赖性CaMKⅡ变成Ca²⁺-非依赖性CaMKⅡ，这是CaMKⅡ活性可以在Ca²⁺浓度下降后仍能保持较长时间的原因，因此认为CaMKⅡ的自身磷酸化是记忆的一种分子机制。现已证明丝裂原活化蛋白激酶（Mitogen activated protein kinase，MAPK）参与到海马依赖的长时记忆-空间记忆。发现用ERK抑制剂SL327可以损伤小鼠的空间学习记忆。在大鼠海马LTP诱导中ERK2的激活是必需的。大鼠和小鼠的联想和空间记忆的形成过程中也与ERK2的激活密切相关。LTP可由cAMP诱导产生，同时还伴有新蛋白的合成，这些新合成的蛋白在长期记忆中起关键作用。突触激活产生的钙离子内流激活Ca²⁺／CAM敏感的腺苷酸环化酶（adenylate cyclate，AC），催化ATP合成cAMP。最终通过磷酸化激活cAMP反应元件结合蛋白（cAMP response element binding protein，CREB）导致合成新的AMPA受体，在LTP维持和长期记忆中起重要作用。综上所述，这些蛋白激酶（CAMKⅡ、PKA、PKC、ERK1／2等）均通过调节基因表达，新蛋白质的合成来影响长期学习记忆。CaMKⅡ酶活下降和铅中毒均可导致空间学习下降，但这两种学习记忆损伤是否具有某种联系至今还不清楚。本课题组自1995年起开始研究铅对神经系统信号转导的影响，运用神经生理，神经行为及分子生物学等方法已证明不同浓度铅可使神经元内Ca²⁺浓度升高，PKC、ERK1／2表达及活性改变，并使c-fos、c-jun表达改变，NOS活性改变；并测定了PKC与小鼠记忆行为间的关系，证明PKC在LTP的诱发和维持中起重要作用。基于CAMKⅡ在学习记忆中的重要位置，本研究在前期实验结果的基础上拟观察铅对CaMKⅡ在学习记忆中的影响，证明其与ERK1／2、CREB等在长时程增强（Long Term Potentiation，LTP）中的关系，为学习记忆机理的研究提供有力依据，将铅中毒作用机制的研究进一步深入，为优生优育，提高国民素质提供理论依据。方法1、急性铅暴露的方法如下：利用颈部脱臼法处死大鼠，迅速取出脑海马，放到预冷并通以95％O₂+5％CO₂的人工脑脊液（Artificial cerebrospinal fluid，ACSF）中，将脑海马切成350μm厚的脑片。在稳定培养2 h后，对其进行醋酸铅或谷氨酸及KN-93处理，在0，3，7.5，15，30，60，120min收集脑片。利用磷酸化及非磷酸化抗体，Western blots方法测定ERK2和CREB活性及表达；利用RT-PCR方法测定CaMKⅡmRNA表达水平。观察铅对脑片ERK2和CREB活性及表达的影响。2、体内慢性铅暴露的方法如下：Wistar大鼠购自中国医科大学实验动物中心，大鼠受孕后随机分为染铅组和对照组。染铅组饮用0.2％醋酸铅水溶液，对照组饮用自来水。各自持续整个妊娠期和哺乳期。为确保仔鼠具有相同的营养条件，每只母鼠最多哺育8只仔鼠。于20 d仔鼠断乳后，染铅组仔鼠直接饮用与母鼠相同铅浓度的水溶液。对照组仔鼠直接饮用自来水。于仔鼠断乳第一天（即20 d）起始，再分别于40 d、60 d、80 d，将仔鼠颈部脱臼法处死，迅速取出海马，放入液氮中，此为慢性铅暴露组及对照组海马标本。利用非磷酸化抗体，Western blots方法测定ERK2及CREB表达量，观察铅对海马总量ERK2及总量CREB表达的影响。结果1、急性铅暴露对脑片ERK2活性（即pERK2）的影响急性铅暴露的脑片培养过程中，在ACSF中醋酸铅浓度为20μmol／L，于0，3，7.5，15，30，60，120 min时间点收集脑片，利用磷酸化抗体，Western blots结果显示早期ERK2活性升高，30 min处降至最低，以后向正常恢复。2、急性铅暴露对脑片总量ERK2表达的影响在醋酸铅浓度为20μmol／L的ASCF培养中，于前述相同时间点收集脑片，利用非磷酸化抗体，Western blots结果显示总量ERK2表达未受影响。3、慢性铅暴露对总量ERK2表达的影响慢性铅暴露状态下的新生仔鼠分别于20 d，40 d，60 d，80 d颈脱臼法处死，利用非磷酸化抗体，Western blots结果显示总量ERK2表达降低。4、急性铅暴露对脑片CREB活性（即pCREB）及总量CREB表达的影响急性铅暴露脑片培养过程中，在ACSF中醋酸铅浓度为20μmol／L，于0，3，7.5，15，30，60，120min时间处收集脑片，利用磷酸化抗体，Western blots结果显示CREB活性呈时间依赖性降低。与测定CREB活性的相同时间点收集脑片，利用非磷酸化抗体，Westernblots结果显示总量CREB表达未受影响。5、慢性铅暴露对脑片总量CREB表达的影响慢性铅暴露状态下的新生仔鼠分别于20 d，40 d，60 d，80 d颈脱臼法处死，利用非磷酸化抗体，Western blots结果显示总量CREB表达未受影响。6、急性铅暴露对CaMKⅡmRNA水平影响脑片培养过程中的铅暴露，对CaMKⅡ的mRNA水平没有明显影响。7、CaMKⅡ抑制剂KN-93处理脑片，对ERK2活性及表达的影响在脑片培养过程中，经CaMKⅡ抑制剂KN-93处理，利用磷酸化及非磷酸化抗体，Western blots结果显示KN-93能降低谷氨酸引起的ERK2活性的升高，总量ERK2表达未受影响。8、CaMKⅡ抑制剂KN-93处理脑片，对CREB活性及表达的影响在脑片培养工程中，经CaMKⅡ抑制剂KN-93处理，利用磷酸化及非磷酸化抗体，Western blots结果显示KN-93能降低谷氨酸引起的CREB活性的升高，总量CREB表达未受影响。结论1、急性铅暴露导致的空间学习记忆损害和LTP形成抑制是通过影响ERK2的磷酸化水平实现的，对总量ERK2表达水平没有显著性影响。慢性铅暴露同时影响到总量ERK的基因表达。2、急性铅暴露引起CREB的磷酸化水平下降，对总量CREB的表达没有显著性影响。3、急、慢性铅中毒可能是主要通过抑制CaMKⅡ活性来影响下游信号分子造成学习记忆功能损伤。更多还原

【Abstract】 ObjectivePb is the common environmental neural toxicant.It’s well known that exposureto Pb results in learning and memory disorder in children. In 1943,Byers and Lordfirstly reported that Pb could endanger Children cognition behavior and intelligencedevelopment in long term. Their sensibility and reactivity to neural poisonous effectof Pb isn’t equal between the adults and children and the central nervous system ismore sensitive to Pb at developing stage. Pb toxicity brings about the nonreversibleimpairment of memory and attention in embryonic and infant period.At the present time, research has demonstrated that some protein kinases insynapte were activated by phosphorylation involved in learning and memory. Thereare three protein kinases at least to be activated in the form of hippocampal spatiallearning process, including Ca²⁺/Calmodulin dependent protein kinaseⅡ（CaMKⅡ）, protein kinase A （PKA） and extracellular signal regulated kinase（ERK1/2） which maintain in long-playing activation state by self-phosphorylation.CaMKⅡhas 8～12 subunits and is the richest kinase in the post synapticdensity of hippocampal and new cortex where are the basic structures of learningand memory. As the result of its phosphorylated Thr-286, Ca²⁺-dependent CaMKⅡbecomes Ca²⁺-independently which explains why CaMKⅡcan keep longtimeactivity following Ca²⁺ reduction.Mitogen activated protein kinase （MAPK） is also implicated inhippocampus-dependent long-term potentiation （LTP） and spatial learning. As anevidence, ERK inhibitor SL327 could impair spatial learning in mouse, andactivation of ERK2 was necessary in the form of LTP in rat hippocampus as well asthe formation process of association and spatial memory.Along with cAMP-induced LTP, some new proteins were synthesized which playcrucial roles in long-term memory.Ca²⁺-influx elicited by synaptic activation could activate Ca²⁺/CaM- sensitive adenylate cyclate （AC） and eventually phosphorylatedcAMP response element binding proten （CREB） leading to synthesize AMPAreceptor participating in LTP and long-term memory.In a word, these kinases including CaMKⅡand PICA,PKC and ERK1/2 all canaffect long-term learning and memory by regulating genes expression andsynthesizing new proteinsAlthough both CaMKⅡdecreasing activation and Pb toxicity lead to spatiallearning disorder, their relationship is not well defined, especially, whether Pb canaffect CaMKⅡor not.Our team have researched the effect of Pb on nervous system signaltransduction since 1995.It was proved that Pb could increase Ca²⁺ in neuron andalter activation of PKC and ERK1/2, NOS,c-fos and c-jun, and PKC played animportant role in the form and maintenance of LTP. Based on CaMKⅡis alsoinvolved in learning and memory, our aim is to explore the impact of Pb on CaMKⅡin learning and memory and testify a certain relationship between it and ERK1/2or CREB in LTP to provide forceful evidence for the mechanism study of learningand memory, which will contribute to a good birth and care and improve citizenquality.MethodsTreatment with acute Pb exposure as follows:The healthy Wistar rats were provided by the experiment animal centre ofChinese medical university and put to death by neck dislocation. Then, thehippocampus were take out quickly and cut into 350μm slices in cold ACSFventilated with 95% O₂ and 5% CO₂. After stabilizing culture for 2 hours, thehippocampal slices were treated by different concentrations of Pb and KN 93（CaMKⅡspecial inhibitor） and cultured for desired times（0, 3, 7.5, 15, 30, 60,120min）. To confirm the effects of Pb and KN93 on ERK2 and CERB, Westernblots was used to determine the active ERK2 and CERB and their total proteinexpression,and CaMKⅡmRNA expression was detected by RT-PCR.Treatment with chronic Pb exposure in vivo as follows:The pregnant rats were divided randomly into Pb group drinking 0.2% acetic acid lead and the control drinking Running water through to gestation and lactationperiod. Be to ensure that the child mice had identical nutrition condition, eachmother feeded 8 children at most. After ablactation at 21 d, child mice of Pb groupdrank directly 0.2% acetic acid lead as same as mothers and the child mice ofcontrol group drank Running water. To obtain chronic Pb-exposed and controlhippocampal slice, neck dislocation was uesed to put rats to death at 40 d, 60 d and80 d respectively after ablactation initiation. Western blots was to determine thetotal proein exprssion of ERK2 and the active CREB to observe the effect chronicPb exposure on ERK2 and CREB in b hippocampal slicesResults1. The effect of actue Pb exposure on active ERK2Actut exposing hippocampal slices to 20mmol/L Pb increased active ERK2 atfirst and decreased it later（minimum at 30 min） and resumed normal levelgradually.2. The effect of actue Pb exposure on total protein expression ofERK2Actut exposing hippocampal slices to 20mmol/L Pb had no effect on the totalprotein expression of ERK2.3. The effect of chronic Pb exposure on total protein expression ofERK2Chronic exposure to 0.2% Pb could reduce the total protein expression of ERK2on 20 d and 40 d, 60 and,80 d.4. The effect of actue Pb exposure on the active CREB and its totalprotein expressionActut exposure to 20mmol/L Pb could inhibit CREB activity and had no effecton the total protein expression of CREB.5. The effect of chronic Pb exposure on total CREBChronic exposure to 0.2% Pb had no effect on the total protein expression ofCREB on 20 d and 40 d, 60 and,80 d. 6. The effect of actue Pb exposure on CaMKⅡmRNA expressionActut exposure to Pb had no effect on CaMKⅡmRNA expression.7. The effect of CaMKⅡinhibitor KN 93 on ERK2 activityThe hippocampal slices treated with CaMKⅡinhibitor KN-93 could inhibit theincreasing activity of ERK2 induced by glutamic acid.8. The effect of CaMKⅡinhibitor KN 93 on active CREBKN-93 treatment could prevent the increasing activity of CREB induced byglutamic acid.Conclusion1. Actue Pb exposure-induced spacial learning and memory disorder wascorelative closely to its effect on the ERK2 activity.Chronic Pb exposure couldaffect the total protein expression of CREB rather than actue Pb exposure.2. Actue Pb exposure could block CREB activity.3. Actue and chronic Pb toxicosis were likely to inhibit CaMKⅡactivity andaffect its downstream signal resulting in nervous system impairment.更多还原