【作者】 张展翅；

【导师】 崔慧先；

【作者基本信息】 河北医科大学 ， 人体解剖与组织胚胎学， 2012， 博士

【摘要】 海马是中枢神经系统的重要脑区，与学习、记忆等生理功能有着密切的关系，许多神经系统的退行性疾病如阿尔茨海默病、癫痫等也与该部位的病变有关。重复经颅磁刺激(Repetitivetranscranial magnetic stimulation,rTMS)是在TMS基础上发展起来的新的神经电生理技术，通过脉冲磁场产生感应电流，激活神经元，通过暂时改变被刺激组织的兴奋性而发挥治疗作用。研究表明rTMS已成功用于阿尔茨海默病、癫痫、抑郁症等疾病的治疗。前期研究认为老化相关的认知功能下降可能和神经元的丢失有关，但越来越多的证据证实，在正常老化过程中，神经元的数量并没有发生明显的变化，而是神经元间信息传递的特化结构—突触的数量及完整性发生了改变，出现不同程度的下降。突触在其结构以及功能方面的修饰称为突触可塑性，是学习记忆的基础。研究突触可塑性与神经系统发育、脑缺血损伤后修复以及学习记忆等功能密切相关，已成为近年来研究的热点。研究发现恒磁场作用能促进培养神经元突起的生长，应用磁刺激(magnetic stimulation, MS)干预对数生长期的PC12细胞后使细胞突起生长明显增多，多巴胺分泌增加。动物实验同样证实MS能增加血管痴呆大鼠海马CA1区及MPTP诱导的帕金森大鼠纹状体突触素的表达，提示MS具有促进突触重塑的功能。但近年来关于MS的研究大多集中在它对病理状态下突触重塑与再生的影响，而关于MS对正常脑发育、神经网络构建等影响的研究较少， MS对突触可塑性的调节作用涉及到细胞内众多分子通路，内在机制复杂，目前对MS干预体外培养的神经元的内在机制尚缺乏深入系统的研究。海马神经元原代培养比那些在体外经过多次传代的神经细胞株更能代表体内神经细胞正常的发育状态，是研究神经细胞生长、发育的理想模型。因此我们选取原代培养的海马神经元，施以不同强度的刺激参数，通过观察MS对海马神经元突触超微结构的影响、突触可塑性相关蛋白、胞浆内钙离子浓度、NR2B、pCaMKII、pCREB等的表达变化，探讨磁刺激对正常培养的海马神经元突起生长及突触可塑性相关蛋白的调节作用。并通过对钙信号转导通路干预作用的研究，明确MS发挥作用的确切机制，为临床进一步研究MS对正常脑可塑性的影响提供理论基础。第一部分重复磁刺激对原代海马神经元突起生长和c-fos激活作用的研究目的:探讨磁刺激是否能够引起原代海马神经元c-fos的表达，促进神经元突起生长、并观察磁刺激对海马神经元存活率的影响。方法:将原代培养的海马神经元随机分为对照组（正常培养无任何干预）；假刺激组（置于相同磁场环境但不接受磁刺激）；30%最大刺激强度组（约1.11T刺激强度作用于细胞）；40%最大刺激强度组（约1.48T刺激强度作用于细胞）。培养24h的神经元换液后开始接受磁刺激。刺激频率为1Hz，每天刺激3组，每组20个序列，每个序列间隔1s，每组间隔1min，每天固定时间刺激，连续刺激5d，磁头距离细胞1cm。培养第7d进行MAP2免疫荧光显色，观察磁刺激对海马神经元突起数目和突起生长长度的影响，并通过免疫荧光显色和免疫印迹法检测各组海马神经元c-fos的表达。应用MTT微量比色法观察磁刺激对海马神经元存活率的影响。结果:原代培养的海马神经元经MAP2染色鉴定纯度大于90%，可用于实验。2个刺激强度组多突起神经元(n=2、n≥3)占总神经元的比例以及神经元突起长度明显高于对照组(P<0.05)，且30%强度组的比例高于40%强度组，差异有统计学意义(P<0.05)。假刺激组和对照组间无明显差异(P>0.05)。C-fos免疫荧光显色结果显示两个刺激强度组c-fos阳性神经元比例明显高于对照组、假刺激组(P<0.01)，而对照组、假刺激组间阳性神经元比例无差异(P>0.05)。免疫印迹结果显示c-fos蛋白表达仅30%强度组表达量增高有统计学意义(P<0.05)，而40%强度组的表达量和对照组比虽有一定的增高趋势，但无统计学意义。MTT法检测细胞存活率显示MS对30%强度组神经元细胞存活率无明显影响，40%强度组细胞存活率明显下降。结论：30%、40%两种强度(1.11T、1.48T)低频(1Hz)磁刺激都能促进原代海马神经元的生长，使细胞突起数目增多、突起长度增加，神经元间连接更紧密。其中30%强度(1.11T)组MS可以激活细胞的功能状态，上调海马神经元c-fos蛋白的表达，并且对神经元存活率无有害影响，而40%强度(1.48T)组c-fos蛋白无明显变化，但海马神经元的存活率出现明显下降。第二部分重复磁刺激对海马神经元突触超微结构和NR2B受体表达的影响目的:探讨重复MS对海马神经元突触界面参数及NR2B受体表达的影响，初步明确MS对海马神经元突触可塑性的调节作用。方法:利用细胞透射电镜观察海马神经元突触界面参数的变化。利用免疫荧光染色、RT-PCR和免疫印迹观察MS对海马神经元NR2B mRNA和蛋白表达的影响。结果:30%强度组突触后致密物的厚度、突触界面曲率和对照组比，都出现显著增加(P<0.01)，而突触前膜活性带长度的增加也较对照组有统计学意义(P<0.05)。40%强度组突触后致密物的厚度、突触突触界面曲率和对照组比增加明显(P<0.01, P<0.05)，但突触前膜活性带长度无明显变化(P>0.05)。突触间隙宽度各实验组间的差异无统计学意义(P>0.05)。NR2B免疫荧光定量分析显示30%、40%强度组NR2B阳性神经元显著增多，免疫反应性明显高于对照组、假刺激组，差异有统计学意义(P<0.01)，RT-PCR结果显示与对照组，30%强度组、40%强度组NR2B mRNA的表达明显增高(P<0.01)，而假刺激组、对照组间NR2B mRNA表达水平无统计学差异(P>0.05)。免疫印迹结果显示30%强度组、40%强度组NR2B蛋白表达水平明显高于对照组、假刺激组(P<0.01)。结论:两种强度MS(1.11T、1.48T)都能提高原代海马神经元NR2B受体mRNA和蛋白的表达水平，并且对突触界面参数有调节作用，30%强度MS(1.11T)对海马神经元突触界面参数的调节作用优于40%强度组(1.48T)。海马神经元NR2B受体mRNA和蛋白的表达上调可能是MS改善突触可塑性的机制之一。第三部分重复磁刺激对原代海马神经元钙信号转导通路的影响目的:探讨磁刺激对海马神经元胞浆内[Ca2+]i的影响及对下游信号通路的激活作用，对MS调节突触可塑性的可能机制进行初步探讨。方法:利用流式细胞仪观察磁刺激后海马神经元胞浆内[Ca2+]i的变化。利用免疫印迹法观察磁刺激对神经元CaMKII(pCaMKII)、CREB(pCREB)蛋白表达的影响。并应用Hoechst33258免疫荧光显色检测磁刺激对培养神经元凋亡率的影响。结果:两个刺激强度组[Ca2+]i和对照组比均出现明显增加(P<0.05，P<0.01)，其中40%强度组[Ca2+]i的增加明显高于30%强度组(P<0.01)，假刺激组和对照组间[Ca2+]i无明显差别(P>0.05)。免疫印迹结果表明和对照组比，30%、40%两个刺激强度组CaMKII总蛋白水平无明显变化(P>0.05)，但其磷酸化水平明显提高，分别较对照组提高了28.9%和17.7%。假刺激组CaMKII和pCaMKII蛋白表达和对照组比无统计学意义(P>0.05)。MS能引起海马神经元pCREB蛋白表达水平增高，30%、40%强度组pCREB的蛋白水平较对照组分别提高了59.1%和13.6%(P<0.01, P<0.05)。MS对海马神经元CREB的总蛋白表达水平无影响(P>0.05)，Hoechst33258免疫荧光染色结果显示30%强度组细胞凋亡率和对照组比无明显变化，但40%强度磁刺激造成细胞凋亡率增加。结论:30%强度磁刺激使海马神经元胞浆内Ca2+浓度升高，激活钙信号转导通路，提高神经元pCaMKⅡ和pCREB蛋白表达水平，表现出促进细胞生长的作用，对细胞凋亡率无明显影响。而40%强度磁刺激使海马神经元胞浆内Ca2+浓度显著升高，虽然提高了pCaMKⅡ和pCREB蛋白表达水平，对细胞生长也有促进作用，但由于造成了钙超载，最终导致海马神经元凋亡率明显增加。第四部分：CaMKII-pCREB通路参与磁刺激促进海马神经元突触可塑性相关蛋白的表达目的:研究MS对海马神经元突触可塑性相关蛋白表达的影响，给予CaMKII抑制剂是否能拮抗MS的调节作用，明确MS对海马神经元突触可塑性的调节作用及机制。方法:将原代海马神经元分为对照组，假刺激组，30%强度刺激组，40%强度刺激组，K1组（先应用0.5umol/L的KN93预处理神经元3h后，再开始接受30%强度磁刺激），K2组（先应用0.5umol/L的KN93预处理神经元3h后，再开始接受40%强度磁刺激）。磁刺激干预条件及时间不变。应用免疫荧光染色、RT-PCR和免疫印迹对突触素(SYN)、活性相关的细胞骨架蛋白(Arc)和微管相关蛋白2(MAP2)的表达进行检测。结果:免疫荧光显色结果显示，SYN免疫荧光阳性物质表现为特征性颗粒状或点状，主要分布在神经元的胞膜及突起上，呈串珠状分布，和对照组比30%强度组免疫阳性物质明显增多。Arc主要表达于神经元的胞体、突起和树突棘，部分阳性产物呈点状分布，30%强度组点状阳性物质明显增多。MAP2主要表达于神经元的胞体和突起，可见30%强度组神经元突起间连接较相关对照组更紧密。荧光定量分析显示30%强度组SYN、Arc和MAP2免疫荧光强度较对照组明显增高(P<0.01)，K1组免疫荧光性较30%强度组分别下降了13.5%、29.6%和13.9%。RT-PCR和免疫印迹结果显示，30%强度磁刺激使SYN、Arc和MAP2的mRNA的表达水平较对照组比分别增加了77.8%、58.7%和46.4%，而蛋白水平分别增加了41.8%、89.1%和43.7%。而经KN93预处理后，K1组SYN、Arc和MAP2的mRNA和蛋白表达和30%强度组比均出现明显下降(P<0.01)，mRNA水平分别下降了35.4%、27.4%和22.7%，蛋白水平分别下降了21.7%、41.1%和18.4%。K2组SYN、Arc和MAP2的mRNA和蛋白水平和对照组比无明显变化(P>0.05)。提示KN93能部分拮抗磁刺激引起的突触可塑性相关蛋白表达的增加。明确了Ca2+信号转导通路在MS促进海马神经元突起生长及可塑性调节中的作用。结论：MS通过Ca2+-CaM/CaMKII-pCREB通路提高突触可塑性相关蛋白SYN、Arc和MAP2的表达，加强对突触可塑性的调节作用，KN93预处理能部分拮抗MS的调节作用。但不同刺激参数调节效果不同，30%强度MS(1.11T)对海马神经元突触可塑性的调节效果优于40%强度(1.48T)。更多还原

【Abstract】 As one of the most important brain areas of the central nervous system,hippocampus have a close relationship with learning, memory, and otherphysiological function, many neurodegenerative diseases, such as Alzheimer’sdisease and epilepsy, also accompanied by the lesion of it. Repetitivetranscranial magnetic stimulation (rTMS) is a new neural electrophysiologicaltechnique developed on the basis of transcranial magnetic stimulation and itdelivered magnetic stimulation (MS) to the brain in the repetitive impulseform, through the pulse magnetic field generating induction current, activatingthe neurons and playing a therapeutic role by temporarily changing theexcitability of the stimulated tissue. Studies have shown that rTMS has beenused successfully in the treatment of the diseases such as Alzheimer’s disease,epilepsy, and depression. Early studies suggested that aging related cognitivefunction decline may be related to the loss of neurons, but more and moreevidences showed that, in the normal aging process, the number of neuronshad no significant change, but the specialized structure of informationtransmission between neurons--the number and the integrity of synapses hadchanged, with a different degree of decline. Synaptic plasticity refers to themodification of synaptic connectivity in the structure and function which isclosely related to the nervous system development, injury repair, learning andmemory, and the other important brain functions, has become a researchhotspot recently.Studies had showed that the constant magnetic field could contribute tothe growth of cultured neurons and affect the direction of newborn axons. Theneurite outgrowth and the secretion of DA all increased significantly after theapplication of MS intervention on PC12cells in the logarithmic phase. Additionally, animal experiments also confirmed that MS could increase thesynaptophysin expression in hippocampal CA1area of the vascular dementiarat and in striatum of MPTP-induced the Parkinson rat, which indicated thatMS could promote the ability of synaptic plasticity. In recent years the studiesabout MS mostly concentrated on synaptic remodeling and regeneration in thepathological conditions, while the research on normal brain development andneural network construction was scarce, the regulation function of MS onsynaptic plasticity involved in many intracellular molecular pathways andcomplex mechanism. The primary cultured hippocampal neurons were morerepresentative of the normal developmental status of nerve cells in vivo thanthose cell lines after several passages in vitro, were the ideal model for thestudy of neurons’ growth and development. Therefore, we selected primarycultured hippocampal neurons, to impose different intensity stimulationparameters, by observing the effect of MS on hippocampal neuronal synapticultrastructure, the expression changes of synaptic plasticity related protein,intracytoplasmic calcium concentration, NR2B, pCaMKII and pCREB afterMS, to explore the mechanism of MS on primary cultured hippocampalneurons neurite outgrowth and synaptic plasticity. And throughthe interventionstudy on the calcium signal transduction pathways, we can clear the precisemechanisms of MS playing the role and to provide a theoretical basis forfurther clinical studies on normal brain plasticity.1Research of repetitive magnetic stimulation on neurite growth and c-fosactivation in primary cultured hippocampal neuronsObjective: to investigate whether magnetic stimulation can cause c-fosexpression, promote neurite growth of primary cultured hippocampal neurons,observe the effects of MS on hippocampal neuron survival.Methods: The primary cultured hippocampal neurons were divided intofour groups, control group (remained in the incubator for7days), sham group(exposed to the magnetic stimulation apparatus for the identical length of time,but did not receive stimulation),30%maximum stimulus intensity group(30%intensity,1.1T),40%maximum stimulus intensity group (40%intensity, 1.48T), the neurons were stimulated after24h, at a rate of1Hz, and themaximum output intensity of magnetic field was3.7Tesla. Continuousstimulation5d, the stimulation coil was held paralleled1cm above the dish.Cellular immunofluorescence staining was executed on day7after plating，tocount the multiple neurites neurons and the cell neurite length of MAP2positive neurons, the c-fos expression in each group was detected byimmunofluorescence staining and Western blot. The effect of magneticstimulation on hippocampal neuron survival was detected by MTT.Results: The purity of MAP2positive neurons in primary culturedhippocampal neurons was more than90%, suitable for the experiment. Thepercentage of multiple neurites neurons（n=2、n≥3）and the cell neurite lengthin both stimulation group were significantly higher than that in control group(P<0.05). Meanwhile it was higher in30%intensity group than that in40%intensity group, there was significant difference in the results (P<0.05). Whilethere was no significant difference between the control and sham stimulationgroup was observed in the growth of neurons (P>0.05). C-fosimmunofluorenscence staining showed that after MS the proportion of c-fospositive neurons increased significantly, and the increase in MS group wasstatistically significant compared with control and sham group (P<0.01), andit had no statistical meaning between control and sham group (P>0.05).Western blot analysis showed that30%intensity MS significantly increasedthe c-fos protein expressin (P<0.05), while the increase in40%intensitygroup had no statistical significance (P>0.05). Cell survival tested by MTTshowed that30%intensity MS had no effect on it, while the survivaldecreased obviously in40%intensity group.Conclusion: Both intensities (1.11T,1.48T) low frequency (1Hz) MSpromoted the neurite outgrowth of primary cultured hippocampus neurons,increased the number and length of neurites, and made the connectionsbetween neurons more closely.30%intensity (1Hz,1.11T) low frequency MSactivated the cell function, increased c-fos protein expression in hippocampalneurons and had no harmful effects on neuron survival, while the cell survival rate decreased obviously in40%intensity group.2The influence of repetitive magnetic stimulation on synapticultrastructure of primary cultured hippocampal neurons and NR2Breceptor expressionObjective: to explore the influence of repetitive MS on synapticultrastructure and NR2B receptor expression of primary cultured hippocampalneurons, make clear the role of MS in the synaptic plasticity regulation ofhippocampal neurons.Methods: Cell transmission electron microscope was used to observe thechanges in the synaptic interface parameters of primary cultured hippocampalneurons. We used immunofluorescence staining, RT-PCR and Western-blotanalysis to explore the expression of NR2B mRNA and protein in differentgroup after MS.Results: After MS, the postsynaptic density thickness and curvature ofthe synaptic interface in30%intensity group had a significant increase(P<0.01) compared to control group, and the increase in presynaptic length ofthe active zoneswas also statistically significant (P<0.05). While40%intensity MS only promoted the postsynaptic density thickness and curvatureof the synaptic interface (P<0.01, P<0.05), the change in presynaptic length ofthe active zones had no statistical significance compared to control group. TheMS had no effect on the synaptic cleft width. Immunofluorescencequantitative analysis showed that NR2B-positive neurons immunoreactivity in30%and40%intensity group was significantly higher than that in controlgroup and sham group after MS, and the difference was statisticallysignificant (P<0.01), RT-PCR result showed that the increase of NR2B mRNAin both MS group was statistically significant compared with control andsham group, and Western blot result also indicated significant difference(P<0.01).Conclusion: Both intensities (1.11T,1.48T) low frequency (1Hz) MScould promote the NR2B mRNA and protein expression of primary culturedhippocampal neurons, had a regulatory role on the synaptic interface parameters, and the adjustment effect of30%intensity (1.11T) MS is betterthan that of40%intensity (1.48T). Upregulation of NR2B receptor mRNAand protein expression in hippocampal neurons may be one of the mechanismsof MS to improve synaptic plasticity.3The influence of repetitive magnetic stimulation on calcium signalingtransduction pathway of primary cultured hippocampal neuronsObjective: To investigate the effect of MS on intracellular Ca2+concentration and the activation of downstream signaling pathways, to explorethe possible mechanism of MS regulating the synaptic plasticityMethods: Flow cytometry analytical technique was used to observe thechanges of the intracellular Ca2+concentration and intervention of MS. Weused Western-blot analysis to explore the effect of MS on the proteinexpression of CaMKII(pCaMKII)、CREB(pCREB) in different group. Theinfluence of MS on neurons apoptosis rate was detected by Hoechst33258immunofluorescence staining.Results: Intracellular [Ca2+]i showed a significant increase both in30%and40%intensity group compared to control group (P<0.05, P<0.01), and theincrease in40%intensity group was significantly higher than that in30%intensity group (P<0.01). Compared to control group, the change of [Ca2+]ihad no significant difference in sham group (P>0.05). Western blot analysisshowed that, the total protein level of CaMKII was not different in each group(P>0.05), while the expression of pCaMKII was significantly up-regulated by28.9%and17.7%in30%and40%intensity group respectively. The proteinexpression of pCaMKII had no significant difference in control and shamgroup (P>0.05). The pCREB protein expression also increased by59.1%and13.6%in30%and40%intensity group respectively and MS had nosignificant effect on the total protein level of CREB (P>0.05). Hoechst33258fluorescence staining showed that the neuron apoptosis rate had no obviouschange in30%intensity group compared to control group, but increasedsignificantly in40%intensity group.Conclusion:30%intensity MS elevated intracellular Ca2+concentration, activated calcium signal transduction pathway, improved pCaMKII andpCREB protein expression, had no harmful effect on hippocampal neurons.There was no significant changein apoptosis rate was found in30%intensitygroup compared to control group. While in40%intensity group, MSsignificantly promoted the intracellular Ca2+concentration, althoughimproved the protein expression of pCaMKII and pCREB, calcium overloadcaused an obvious increase in apoptosis rate of the hippocampal neurons.4CaMKII-pCREB pathways involved in the regulatory role of magneticstimulation promoting the synaptic plasticity-related proteins expressionObjective: To research the influence of repetitive MS on the expressionof synaptic plasticity-related proteins, and whether CaMKII inhibitor KN93administration can antagonize the regulatory role, further to make clear therole and mechanism of MS on synaptic plasticity in hippocampal neurons.Methods: The primary cultured hippocampal neurons were divided intosix groups, control group, sham group,30%maximum stimulus intensitygroup, K1group (pretreated with0.5umol/L KN93for3h, and then began toaccept the30%intensity of MS),40%maximum stimulus intensity group, K2group (pretreated with0.5umol/L KN93for3h, and then began to accept the40%intensity of MS). MS intervention condition and time was the same asbefore. Immunofluorescence staining, RT-PCR and Western-blot analysis wereused to detect the expression of synaptophysin (SYN), Activity regulatedcytoskeletal protein (Arc) and microtubule-associated protein2(MAP2).Results: In primary cultured hippocampal neurons, SYNimmunofluorescence-positive material showed beaded distribution along theneuronal processes, granular or point-like, and the cellularimmunofluorescence intensity in30%intensity group was significantly higherthan that in control group. Arc protein expression was mainly located inneuronal cell bodies, processes and dendritic spines, some positive productsshowed punctate distribution. A marked increase of the punctate positivematerial was observed in30%intensity group. MAP2was mainly expressedin the body and dendrites of the neurons and the connection among the neurons in30%intensity group was more closely than that in other groups.Fluorescence quantitative analysis showed that the SYN、Arc and MAP2immunofluorescence intensity in K1group decreased by13.5%,29.6%and13.9%respectively compared to control group. RT-PCR and Western blotanalysis showed that in30%intensity group, SYN、Arc and MAP2mRNAexpression levelswere significantly increased by77.8%,58.7%and46.4%thanthat in control group, and protein levels increased respectively41.8%,89.1%and43.7%. While in K1group, SYN, Arc and MAP2mRNA and proteinexpression showed a significant decrease compared to30%intensity group,mRNA levels were decreased by35.4%,27.4%and22.7%, and protein levelswere decreased by21.7%,41.1%and18.4%respectively. SYN, Arc andMAP2mRNA and protein levels had no significant change in K2groupcompared with control group (P>0.05). These suggested that KN93administration could partly antagonize the regulatory role of MS, reduced thesynaptic plasticity-related protein expression. We further to make clear therole of MS on neurite outgrowthandplasticity regulation in hippocampalneurons.Conclusion: By Ca2+-CaM/CaMKII-pCREB pathways, MS improvedthe expression of synaptic plasticity-related protein SYN、Arc and MAP2,strengthened the regulatory role of MS on synaptic plasticity, KN93pretreatment could partially inhibit the effect of MS. But different stimulatingparameters results in different stimulus effects. The regulation function of30%intensity MS (1.11T) on hippocampal neuronal synaptic plasticity wasbetter than that of the40%intensity (1.48T).更多还原