雷帕霉素对大鼠婴儿期遗忘作用的研究

发布时间：2018-01-01 22:15

本文关键词：雷帕霉素对大鼠婴儿期遗忘作用的研究　出处：《南方医科大学》2016年硕士论文　论文类型：学位论文

【摘要】：研究背景早期生活经历对成年后人格和心理社会功能的养成起着非常重要的作用,然而成人仍很少能回忆起幼年时期发生的事件。婴儿期遗忘(infantile amnesia)是指成人对特定年龄前发生的生活事件无法进行回忆的现象,部分研究则定义为与成年期相比,婴幼儿时期/儿童期对记忆自发性遗忘的加速现象(在儿童期为童年期遗忘)。目前人类婴儿期遗忘的边界年龄尚未明确,人类方面的研究主要集中在神经心理学方面。1962年Campbell利用行为学实验(主动回避反应训练)验证了婴儿期遗忘现象同样存在于大鼠身上,此后,使用不同种属的实验动物(如灵长类)和行为学训练方法(如被动回避反应训练、条件性恐惧训练)的实验均验证了婴儿期遗忘现象的存在,为更好地研究其发生机制奠定了基础。目前尚未明确婴儿期遗忘发生的神经生物学机制,但明确的是其发生机制并非单一,可能涉及众多与记忆相关的脑区、神经递质系统和神经内分泌系统等。海马、内侧前额叶皮质均是晚成熟结构,N-甲基-D-天冬氨酸受体(N-methyl-D-aspartic acid, NMD A)、γ-氨基丁酸(gamma-aminobutyric acid, GABA)等受体存在亚单位发育的时空差异等,均可能影响婴幼儿时期的学习记忆功能。海马属于边缘系统结构,也是晚成熟结构,出生后海马仍需经历神经发生、突触发生、神经递质受体发育、髓鞘形成等解剖结构和生理功能的改变。情景式的学习记忆如穿梭箱被动回避反应、条件性恐惧训练均是海马依赖性的学习记忆过程,均需完整的海马功能的参与。Lavenex P等通过测试不同年龄儿童的空间学习记忆,并对比灵长类动物随年龄增长而改变的海马结构,发现婴儿期遗忘时间与海马结构功能的发育差异时间基本一致,提出两者可能存在因果关系。Josselyn SA等也认为海马的发育差异在婴儿期遗忘中起着重要作用,猜测婴幼儿时期海马高水平的神经发生可能与婴儿期的加速遗忘现象相关。海马神经发生与学习记忆相关,在特定情况下,成年海马神经发生可促进依赖海马的新记忆的形成和保留,但对于已形成的旧记忆,过多新生神经元对已有神经环路的持续整合却可能加速原有记忆的遗忘,新生的突触连接也可能对原有环路造成干扰。海马齿状回是大脑神经发生的主要区域之一,齿状回神经发生贯穿于生命体的整个生命过程并随年龄增长而水平逐渐降低,婴幼儿时期神经发生水平较高而成年后水平降低。婴幼儿期高水平的神经发生与记忆保留呈负相关关系在Akers等的研究中同样得到证实：使用17日龄和60日龄小鼠进行条件性恐惧记忆训练,免疫荧光染色检测神经发生水平,结果显示17日龄小鼠比60日龄小鼠有更高水平的海马神经发生,且17日龄小鼠对恐惧记忆的遗忘速度比60日龄快；通过基因和药理学途径减少17日龄小鼠的神经发生可增加其记忆保留；而通过自发轮转运动增加60日龄小鼠的神经发生却加速其记忆遗忘,提示婴幼儿期过高水平的神经发生可能是婴儿期遗忘的发生机制之一。哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin, mTOR)是由2549个氨基酸组成的进化上相对保守的丝氨酸/苏氨酸激酶。mTOR信号通路可接受激素、生长因子、营养因子和能量等多种信号输入,通过PI3K/Akt/mTOR或AMPK等途径来发挥控制基因转录和蛋白质翻译起始、调控细胞周期、调节凋亡和影响代谢等多重作用。雷帕霉素是mTOR的抑制剂,可通过阻止mTOR通路下游效应器P70S6K和4EBPs的磷酸化,抑制相关蛋白的转录和翻译,起着影响免疫、调控细胞增殖、调节凋亡与自噬等作用。据国外的相关研究,雷帕霉素腹腔注射可影响幼鼠海马齿状回新生神经元的增殖。雷帕霉素亦可影响突触可塑性,影响学习记忆等相关过程。动物实验表明,局部脑区内(如海马、杏仁核、皮层)注射或腹腔注射雷帕霉素可损害依赖海马的空间记忆、依赖前额叶皮层或杏仁核的条件恐惧记忆、依赖味觉皮层的条件味觉厌恶记忆等学习记忆过程。而另一方面,雷帕霉素长期低剂量口服则能增强成年小鼠的空间学习记忆和老年鼠被动回避反应训练的记忆保留,改善阿尔茨海默病模型小鼠的记忆损害,增加老年鼠的平均寿命。综合以上研究,提示雷帕霉素对学习记忆的影响可能与身体状态、年龄、用药途径等有关。目前甚少研究关注于探讨雷帕霉素对幼龄大鼠学习记忆的影响。由于雷帕霉素可影响幼龄大鼠海马齿状回新生神经元的增殖,而海马神经发生又与记忆保留相关,推测雷帕霉素或通过影响神经发生作用于婴儿期遗忘。因此,本实验通过研究雷帕霉素腹腔注射对幼龄大鼠被动回避反应训练即学习记忆的影响,同时探讨雷帕霉素腹腔注射对幼龄大鼠海马细胞增殖、神经发生和细胞凋亡的作用,初步探讨雷帕霉素影响婴儿期遗忘的可能机制。具体研究包括以下两部分：第一部分：雷帕霉素对幼龄大鼠被动回避反应训练的影响目的穿梭箱被动回避反应训练17日龄(post-natal day 17, P17)和60日龄(post-natal day 60, P60)大鼠,验证婴儿期遗忘现象。研究雷帕霉素腹腔注射对17日龄大鼠被动回避反应训练记忆保留的影响。方法(1)17日龄和60日龄SD雄性大鼠各42只,各年龄组根据被动回避反应训练时是否给予电击又分为电击组与非电击组,测试各组大鼠被动回避反应训练后O d(即训练刚结束后)、2d和7d的记忆保留。(2)P17 SD雄性大鼠84只,分为溶媒组、雷帕霉素20mg/kg(Rap20组)和雷帕霉素40mg/kg组(Rap40组),各组分别在被动回避反应训练后腹腔注射等体积的溶媒或雷帕霉素溶液。各组根据被动回避反应训练时是否给予电击又分电击组与非电击组,测试训练后2 d(P19)和7 d(P24)的记忆保留。结果(1)17和60日龄大鼠被动回避反应训练结果的差异研究适应阶段反应潜时P17和P60大鼠比较差异无统计学意义(P0.05)。测试时电击组2×3析因分析提示不同年龄组差异有统计学意义(P0.05),测试时不同时间点差异有统计学意义(P0.05)。电击组训练后0d(即训练结束后)P17和P60大鼠反应潜时比较差异无统计学意义(P0.05),训练后2d和7d两年龄组反应潜时比较差异均有统计学意义(P0.05)。P17大鼠训练后2d记忆保留有所减弱,与训练后0d反应潜时比较差异有统计学意义(P0.05),到训练后7d差异更明显,反应潜时下降至接近电击训练前水平。P60大鼠在训练后2d和7d仍保留稳定的记忆水平,与训练后Od反应潜时比较差异均无统计学意义(P0.05)。非电击组P17和P60大鼠均在测试各时间点(训练后0d、2d和7 d)显示出与适应阶段无明显差异的反应潜时(P0.05)。(2)雷帕霉素对P17大鼠被动回避反应训练的影响适应阶段反应潜时溶媒组、Rap20组和Rap40组比较差异无统计学意义(P0.05)。测试时电击组3×2析因分析提示不同剂量组差异有统计学意义(P0.05),测试时不同时间点差异有统计学意义(P0.05)。电击组训练后2d三组反应潜时比较差异无统计学意义(P0.05)；训练后7d反应潜时Rap20组、Rap40组与溶媒组比较差异均有统计学意义(P0.05),即雷帕霉素用药组增加了P17大鼠被动回避反应训练的反应潜时,而溶媒组在训练后7d反应潜时已下降至接近电击训练前水平。Rap20组和Rap40组在训练后2d和7d反应潜时比较差异均无统计学意义(P0.05)。非电击组各组各时间点反应潜时比较差异均无统计学意义(P0.05)。结论17日龄大鼠存在快速遗忘,显示了婴儿期遗忘现象。雷帕霉素腹腔注射可增加P17大鼠对电击训练的记忆保留,减缓婴儿期遗忘现象。第二部分：雷帕霉素对幼龄大鼠海马齿状回细胞增殖、神经发生和细胞凋亡的影响目的探讨雷帕霉素腹腔注射对17日龄大鼠海马齿状回细胞增殖、神经发生和细胞凋亡的影响,初步探讨雷帕霉素影响幼龄大鼠记忆保留的可能机制。方法(1)P17 SD雄性大鼠24只,分为正常组、溶媒组、雷帕霉素20mg/kg组(Rap20组)和40mg/kg组(Rap40组),于17日龄同时间点分别腹腔注射等体积的溶媒或雷帕霉素溶液(正常组不予处理),注射44 h后所有动物腹腔注射BrdU溶液标记增殖细胞,剂量为100mg/kg,于BrdU注射4h后(即P19)每组6只大鼠灌注取脑,行海马齿状回BrdU免疫荧光单标染色。(2)P17 SD雄性大鼠48只,分为正常组、溶媒组、Rap20组和Rap40组,于17日龄同时间点分别腹腔注射等体积的溶媒或雷帕霉素溶液(正常组不予处理),注射12h后所有动物腹腔注射BrdU溶液标记增殖细胞,剂量为50mg/kg,每6h注射一次,共5次,于BrdU末次注射12h和132 h后(即P19和P24),每组各时间点取6只大鼠灌注取脑,行海马齿状回BrdU/DCX免疫荧光双标染色。(3)P17 SD雄性大鼠24只,分为正常组、溶媒组、Rap20组和Rap40组,于17日龄同时间点分别腹腔注射等体积的溶媒或雷帕霉素溶液(正常组不予处理),于注射药物48 h后(即P19)每组6只大鼠灌注取脑,石蜡切片行海马齿状回caspase3免疫组化染色。结果(1)溶媒组与Rap20组、Rap40组BrdU阳性细胞计数比较差异均有统计学意义(P0.05),雷帕霉素能减少海马齿状回的细胞增殖。BrdU阳性细胞计数正常组与溶媒组比较差异无统计学意义(P0.05), Rap20组与Rap40组比较差异有统计学意义(P0.05), Rap40组比Rap20组BrdU阳性细胞计数少。(2)溶媒组与Rap20组、Rap40组BrdU、BrdU/DCX阳性细胞计数在各时间点(P19和P24)比较差异均有统计学意义(P0.05),雷帕霉素能减少海马齿状回的神经发生。BrdU、BrdU/DCX阳性细胞计数正常组与溶媒组各时间点比较差异均无统计学意义5), Rap20组与Rap40组各时间点比较差异均有统计学意义(P0.05),增加雷帕霉素的剂量阳性细胞计数更少。P24与P19相比,各组BrdU、BrdU/DCX阳性细胞计数均有不同程度减少。(3)溶媒组与Rap20组、Rap40组caspase3阳性细胞计数比较差异均有统计学意义(P0.05),雷帕霉素能增加海马齿状回的细胞凋亡。Caspase3阳性细胞计数正常组与溶媒组比较差异无统计学意义(P0.05), Rap20组与Rap40组比较差异有统计学意义(P0.05),Rap40组caspase3阳性细胞计数比Rap20组多。结论雷帕霉素能减少P17幼龄大鼠海马齿状回的细胞增殖和神经发生,增加海马齿状回的细胞凋亡。
[Abstract]:On the background of early life experience to develop personality and psychosocial functioning in adulthood plays a very important role, but adults are still rarely recalled childhood events. Infantile amnesia (infantile amnesia) refers to the adult age specific life before the event can not recall phenomenon, part of the research is defined as compared with adults, children of spontaneous forgetting to accelerate the phenomenon of memory in infancy (/ forgotten childhood in childhood). The human infants forgetting the boundary age is not yet clear, the human aspects of the research mainly concentrated in the aspects of neuropsychological.1962 Campbell using behavioral tests (active avoidance reaction) verification the infantile amnesia phenomenon also exists in the rat body, then use different species of animal (such as primates) and training methods (such as passive avoidance behavior Response training, conditioned fear training) experiments have verified the existence of the phenomenon of infantile amnesia, laid the foundation for better research on its mechanism is not yet clear. Infantile amnesia neurobiological mechanisms occur, but its mechanism is not clear is single, may involve a number of memory related brain regions, neurotransmitter system and neuroendocrine system. The hippocampus and medial prefrontal cortex are late maturity structure, N- methyl -D- aspartate receptor (N-methyl-D-aspartic acid, NMD A), gamma aminobutyric acid (gamma-aminobutyric, acid, GABA) are the development of subunit space-time difference etc. receptor, can affect the function of learning and memory in infancy. The hippocampus belongs to the limbic system, and late maturity structure, still need to experience after the birth of hippocampal neurogenesis, synaptogenesis, neurotransmitter receptor development, myelin formation anatomy The structure and physiological function. The change of learning and memory of scenes such as passive avoidance test, conditioned fear training is the process of learning and memory in hippocampus dependent, are required to complete the.Lavenex P in the hippocampal function through the test of learning and memory of children of different ages in space, hippocampus and contrast of primate animal with age change, found time differential development of infantile amnesia and hippocampal structure and function of the basic agreement, put forward the possible causal relationship between the.Josselyn SA also believes that the developmental differences in infant hippocampus plays an important role in the forgetfulness, speculation during high levels of hippocampal neurogenesis may be associated with infant hippocampus accelerated forgetting phenomenon. Neurogenesis is associated with learning and memory, in certain circumstances, the formation of new adult hippocampal neurogenesis in the hippocampus dependent memory can promote and retain, But for the formation of the old memory, too many new neurons continued integration of existing neural circuits may accelerate the original forgotten memory, connect new synapses can also cause interference to the original loop. The dentate gyrus is one of the main areas of the brain neurogenesis, neurogenesis in the dentate gyrus throughout the whole process of life and gradually decreased with age, infants higher levels of adult neurogenesis and neural levels. High levels of infant and memory retention was negatively related in the research of Akers in the same confirmed: the use of 17 day old and 60 day old mice were conditioned fear memory training, immunofluorescence staining neurogenesis level, the results showed that 17 day old mice had higher levels of hippocampal neurogenesis than 60 day old mice, and 17 day old mice on fear of forgetting At the age of 60 days faster than fast; by genetic and pharmacological approaches to reduce 17 day old mice neurogenesis can increase the memory retention; and through the self rotation rotary motion increase of 60 day old mice have accelerated the neural amnesia, suggesting that infants high levels of neurogenesis may be the pathogenesis of infantile amnesia. The mammalian target of rapamycin (mammalian target of rapamycin, mTOR) is a relatively conservative consisting of 2549 amino acids on the evolution of serine / threonine kinase.MTOR signaling pathway can receive hormone, growth factor, nutrition factor and energy and other inputs, through PI3K/Akt/mTOR or AMPK and other ways to exert control of gene transcription and protein translation initiation. The regulation of cell cycle regulation, apoptosis and metabolism of multiple roles. Rapamycin is an inhibitor of mTOR, by blocking mTOR pathway downstream. Is P70S6K and the phosphorylation of 4EBPs, inhibition of transcription and translation related protein, plays influence immune, regulation of cell proliferation, apoptosis and autophagy. According to the related research abroad, rapamycin intraperitoneal injection can affect neurogenesis in the dentate gyrus of rats. The proliferation effect of synaptic plasticity may influence the learning and memory of rapamycin. Other related process. Animal experiments show that local brain regions (such as the hippocampus, amygdala, cortex) spatial memory injection or intraperitoneal injection of rapamycin can damage the hippocampus dependent fear conditioning, dependent prefrontal cortex or amygdala dependent, gustatory cortex conditioned taste aversion memory learning and memory process. On the other hand, long-term low rapamycin oral dose of reinforcement learning can passive avoidance response training and memory of aged rats memory retention in adult mice, improve Alzheimer's disease model The memory impairment of mice, increase the life expectancy of aged rats. Based on the above study, suggesting that effects of rapamycin on learning and memory and physical condition, age, route of administration and so on. There are few studies focus on the effects of rapamycin on learning and memory in young rats. The effects of rapamycin in young rats neurogenesis in the dentate gyrus the proliferation and hippocampal neurogenesis and memory retention, presumably through the influence of rapamycin or neurogenesis in infantile amnesia. Therefore, through the study of rapamycin intraperitoneal injection on rat passive avoidance response training effects of learning and memory, and discusses on the proliferation of hippocampal cells in young rats by intraperitoneal injection of rapamycin, neurogenesis and the role of apoptosis, to investigate the possible mechanisms of the effects of rapamycin in infancy forgotten. Specific studies include in The two part: the first part: the influence of rapamycin on young rats of passive avoidance response training to passive avoidance test in training at the age of 17 days (post-natal day 17, P17) and 60 days (post-natal day 60, P60) rats, verification of infantile amnesia phenomenon. Research on ray rapamycin intraperitoneal injection of 17 day old rats passive avoidance reaction training memory retention. Methods (1) 17 day old and 60 day old male SD rats 42 rats in each age group, the passive avoidance according to whether or not to give electric shocks and divided into shock group and shock group response training, all rats were tested after training passive avoidance response O D (i.e. just after the end of training), 2D and 7d (2) P17 memory retention. 84 male SD rats, divided into solvent group, rapamycin 20mg/kg (Rap20 group) and rapamycin group 40mg/kg (Rap40 group), each group were in passive avoidance of solvent was injected into the reaction volume after training Or rapamycin solution. Each group based on passive avoidance reaction to shocks and whether the training is divided into non shock group and shock group, test after training 2 D (P19) and 7 d (P24) memory retention. Results (1) 17 and the difference between the 60 day old rats of passive avoidance response training results Research on the adaptive phase of the reaction potential when P17 and P60 rats showed no significant difference (P0.05). The test shock group 2 * 3 factorial analysis showed that the differences between different age groups was statistically significant (P0.05), when tested at different time points were statistically significant (P0.05). 0d shock group after training (i.e. after training) and P60 P17 rat response latency had no significant difference (P0.05), 2D and 7d after training two age group formation. There were significant differences (P0.05) in.P17 rats after training 2D memory retention weakened, and potential 0d response after the training when the difference was statistically significant (P0.05), The difference is more obvious after 7d training, the reaction latency dropped to near the level before the shock training.P60 rats after training in 2D and 7d remained stable memory level, and potential Od reaction after training showed no significant difference (P0.05). The non shock group P17 and P60 rats were tested in different time point (after training 0d 2D and 7 d) reaction shows no significant difference between the latent phase of adaptation (P0.05). (2) the effect of rapamycin on P17 rat passive avoidance reaction influence training adaptation stage of the reaction potential when the solvent group, there was no significant difference between Rap20 group and Rap40 group (P0.05) test. When the electric group of 3 * 2 factorial analysis showed that different dose groups were statistically significant (P0.05), when tested at different time points were statistically significant (P0.05). The shock training group 2D three group reaction latency had no significant difference (P0.05); potential 7d after training in Rap20 group, Rap40 Group and solvent group were statistically significant (P0.05), which increases the reaction potential rapamycin treatment group training passive avoidance response in P17 rats, and solvent group in the latent 7d reaction after training has dropped to near the level before the shock training group.Rap20 and Rap40 group after training 2D and 7d response latency there were no significant differences (P0.05). The non shock group at each time point response latency showed no significant difference (P0.05). The conclusion of the 17 day old rats are quickly forgotten, shows the phenomenon of infantile amnesia. Rapamycin intraperitoneal injection can increase the memory of the shock training P17 rats retained, slow down baby during the period of forgetting phenomenon. The second part: rapamycin on young rat dentate gyrus cell proliferation, neurogenesis and apoptosis effect study of rapamycin intraperitoneal injection of 17 day old rat dentate gyrus cell proliferation, Effect of neurogenesis and apoptosis, to investigate the possible mechanisms of memory in young rats. Methods the retention effect of rapamycin (1) P17 24 male SD rats were divided into normal group, solvent group, rapamycin group 20mg/kg (Rap20 group) and 40mg/kg group (Rap40 group), at the age of 17 at the same time. Intraperitoneal injection of equal volume of solvent or rapamycin solution (the normal group without treatment), 44 h after injection of all animal injected BrdU labeled cells, the dose of 100mg/kg, BrdU after injection of 4H (P19) 6 rats in each group underwent brain perfusion, dentate gyrus BrdU immunofluorescence single stained. (2) P17 48 male SD rats were divided into normal group, vehicle group, Rap20 group and Rap40 group, at the age of 17 at the same time were injected equal volume of solvent or rapamycin solution (the normal group without treatment), 12h after injection of all animal injected BrdU labeling. Cell dose is 50mg/kg, each 6h injection time, a total of 5 times, in the end of BrdU and 132 h after injection of 12h (P19 and P24), each group of each time point, 6 rats were perfused and brains, for dentate gyrus BrdU/DCX immunofluorescence staining. (3) P17 SD male 24 rats were divided into normal group, vehicle group, Rap20 group and Rap40 group, at the age of 17 at the same time were injected equal volume of solvent or rapamycin solution (the normal group without treatment), 48 h after drug injection (P19) with 6 rats in each group perfused brain, paraffin sections. The dentate gyrus Caspase3 immunohistochemical staining. Results (1) the solvent group and Rap20 group, Rap40 group, BrdU positive cells were statistically significant (P0.05), rapamycin can reduce dentate cell proliferation of.BrdU positive cells in normal group and solvent group was not statistically significance (P0.05). Rap20 group and Rap40 group difference There was a significant correlation (P0.05), Rap40 group than in the Rap20 group BrdU positive cells. (2) the solvent group and Rap20 group, Rap40 group, BrdU, BrdU/DCX positive cells at different time points (P19 and P24) showed no significant differences (P0.05), rapamycin can reduce dentate gyrus neurogenesis.BrdU, the number of BrdU/DCX positive cells in normal group and solvent group at each time point, there were no significant differences in 5), Rap20 group and Rap40 group at different time points were statistically significant (P0.05), increase the dose of rapamycin positive cells count less.P24 compared with P19, BrdU group, BrdU/DCX positive cell counts were not

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R965

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