母体期慢性铝暴露对子代大鼠海马LTP、PKC活性和Ng含量的影响

发布时间：2018-07-09 17:21

本文选题：母体期慢性铝暴露 + 海马　；参考：《中国医科大学》2007年硕士论文

【摘要】： 母体期慢性铝暴露对子代大鼠海马LTP、PKC活性和Ng含量的影响前言铝大量蓄积体内可产生毒性作用。国内外许多流行病学调查和研究表明，铝易致神经元损伤，引起智力和认知能力下降等学习和记忆方面的缺欠，且这是一个长期的慢性的神经病理过程，涉及到脑发育的各个阶段。海马是学习记忆的关键脑区，海马长时程增强(long-term potentiation，LTP)是NMDA(N-甲基-D-门冬氨酸)受体依赖性突触传递效能的持续性增强，是公认的脑学习记忆功能在突触水平的研究模型和神经基础。因此，研究铝暴露对LTP及与其突触机制有关的各项生化指标的影响，有助于从突触和蛋白分子水平阐明铝损害脑学习记忆功能的作用机制。目前虽然铝对LTP损害作用的观察很多，但铝对LTP损害作用的突触机制尚未完全阐明。已明确，蛋白激酶C(protein kinase C，PKC)是LTP产生的必需酶，而神经颗粒素(Neurogranin，Ng)是一种突触后脑特异性蛋白质，是PKC的天然作用底物，与LTP的诱导与维持关系密切。同时，母体期是子代脑发育的一个重要时期，母体期铝暴露会不会感染子代，是一个关系到子代健康的大问题。因此，本实验拟采用母体慢性铝暴露大鼠模型，观察母体期铝暴露对子代大鼠海马LTP诱导和维持、PKC活性及Ng含量的影响，以期从PKC及Ng角度为阐明此发育阶段铝影响LTP的机制提供一个新视点。材料与方法一、实验材料 1、实验动物 Wistar大鼠，♀：♂为3：1(体重150-200g)及其所繁殖的子代鼠 2、主要试剂三氯化铝(分析纯)；抗大鼠Ng单克隆抗体：碱性磷酸酶标记的兔抗山羊二次抗体；PepTag非放射性PKC活性测定试剂盒。 3、主要仪器大鼠跳台反应箱；通用电泳仪；凝胶电泳成像系统；低温冷冻离心机；Spectronic 21型的紫外-可见分光光度计：电生理相关仪器；琼脂糖凝胶电泳仪。二、实验方法 1、分组及动物模型建立雌性Wistar大鼠45只随机分为3组，从其与雄性大鼠合笼始至子代断乳止，在饲料相同条件下，分别饮用蒸馏水、0.2％及0.4％氯化铝(AlCl_3)溶液。当其子代鼠断乳(生后21d)后，每窝取出2-3只子代鼠用于实验，雌雄各半。将饮蒸馏水雌鼠的子代鼠作为对照组(control)，断乳后继续饮用蒸馏水。将饮铝溶液雌鼠的子代鼠对应分为0.2％和0.4％两个暴露组(记为0.2％-Al和0.4％-Al组)，断乳后改饮蒸馏水。每组饮水条件从断乳至电生理实验止(时间3-4个月)保持不变，三组动物交替用于实验。饲养动物的室内温度18-23℃，相对湿度45-55％。 2、行为学测定采用跳台法检测铝对学习记忆行为的影响，记录大鼠第1次跳下平台的时间(潜伏期)和5 min内受到电击(即跳下)次数(错误次数)。 3、电生理LTP测定 20％乌拉坦(urethane，6.5ml·kg~(-1)，ip)麻醉后，将动物头部固定于脑立体定位仪上，在Schaffer侧枝部位(坐标：前囟后3.3mm；旁开3.8mm：皮层下3.8mm)插入刺激电极。将记录玻璃微电极插入海马CA_1区部位(坐标：前囟后3.3mm；旁开1.5mm；电极尖端先抵皮层表面，然后逐步推进)行细胞外记录。找到稳定的群体锋电位(population spike，PS)后，首先记录30min的每分钟单脉冲刺激所诱发的PS。并将所得的30次的PS幅值的平均值作为基线值。然后给予同样强度及波宽的短串高频刺激(higll frequency stimuli，HFS)后，观察每分钟单脉冲检验刺激所诱发的PS的幅值变化及此变化所持续的时间。HSF后PS的幅值变化值以相对于基线值的百分数表示(又称PS的相对幅值，示PS增强率)。 4、海马PKC活性的测定采用琼脂糖凝胶电泳方法测定海马PKC的活性。参照PepTag非放射性PKC活性测定试剂盒说明书。 5、Ng含量的测定采用Western blot方法测定海马Ng的含量。 6、脑组织及血液中铝含量测定采用原子吸收石墨炉法测定脑铝和血铝含量。 7、统计学处理采用SPSS13.0(statistical package for the social science，SPSS)统计分析处理软件对数据进行处理，P＜0.05视为有统计学意义。实验所得数据资料用(?)±S表示。实验结果一、记忆行为学指标与对照组大鼠第1次跳下平台的潜伏期(300±0.00秒)相比，0.2％-Al(202.71±81.99秒)和0.4％-Al组(19.67±8.44秒)差异均非常显著，P＜0.01。两暴露组5min内错误次数(1.43±0.85次，，2.47±0.99次)与对照组(0.00±0.00次)相比也均存在显著差异，P＜0.05。两暴露组之间差异也具有统计学意义，P＜0.01。二、电生理LTP测试结果 HS后，与对照组PS幅值增强率(143.94±29.93％)相比，0.2％-Al(115.29±14.80％)和0.4％(103.62±13.70％)，差异均非常显著，P＜0.01。但两暴露组之间差异不显著，P＞0.05。三、海马PKC活性测定结果(PKC活性单位为pmol／min·mg) 与对照组海马组织PKC的全细胞(827.60±42.52)、胞膜(340.99±24.50)、胞浆活性(481.32±22.52)及胞膜与胞浆PKC活性比值(0.7114±0.0686)相比，0.2％-Al组(506.41±20.56、155.69±12.63、55.79±25.98、0.4411±0.0623)和0.4％-Al组(494.65±16.81、140.11±4.44、353.44±8.28、0.3966±0.0163)均降低，差异显著，P＜0.01。但两暴露组的各项指标差异不显著，P＞0.05。四、海马Ng含量测定结果与对照组海马Ng表达的灰度值(62.56±8.74)相比，0.2％-Al组(52.33±4.01)和0.4％-Al组(50.75±5.99)差异显著，P＜0.01。但两暴露组之间差异不显著，P＞0.05。五、脑组织与血中的铝含量测定结果与对照组脑铝含量(6.21±2.37μg／g)、血铝含量(33.9±38.74μg／L)相比，0.2％-Al组(15.68±4.48μg/g、47.43±14.38μg/L)和0.4％-Al组(18.79±5.18μg／g、55.56±19.07μg/L)差异均显著，P＜0.01。但两暴露组之间差异不显著，P＞0.05。讨论母体期是子代脑发育重要阶段，此期的母体铝暴露对子代脑的智力发育是否有影响，是一个值得关注的方面。因此，研究铝及其化合物在母体期的潜在发育毒性，对铝导致的儿童智力和认知能力低下性神经疾病的早期治疗与预防具有重要意义。由实验结果可见，母体慢性铝暴露可损害子代海马LTP的诱导与维持，其损害的程度有随暴露剂量增加而加重的趋势，行为学测试的结果也从侧面反映了这种关系。脑铝与血铝的变化趋势也基本与之一致。本实验结果显示，母体铝暴露的子代大鼠的海马PKC全细胞活性、胞膜、胞浆活性及胞膜与胞浆活性比值，与对照组相比，都分别呈显著性降低，我们分析认为，铝通过抑制PKC活性而使LTP产生的必需物质—B-50及NMDA受体的磷酸化调节发生障碍，从而损害大鼠LTP及导致其学习记忆能力下降。本实验对子代大鼠进行海马Ng的含量测定结果显示，与对照组相比，母体铝暴露的子代大鼠海马Ng含量下降，有统计学意义。推测Ng含量下降的原因之一可能是树突棘数量的减少和细胞缺失，因为有研究表明，铝负荷过量大鼠海马CA_1区神经元核固缩和神经元减少现象明显。总之，对母体实施铝暴露会影响到子代，且铝对在LTP机制中起重要作用的PKC和Ng的影响是多方面的，需要进一步的探讨。结论 1、母体期慢性铝暴露可使子代大鼠第1次跳下平台的潜伏期缩短，5min内错误次数增加，提示该暴露损害了子代大鼠的记忆能力； 2、母体期慢性铝暴露可使子代大鼠PS幅值增强率减小，提示该暴露损害了子代大鼠LTP的诱导与维持； 3、母体期慢性铝损害子代海马LTP的可能机制之一是铝造成了其海马PKC活性和Ng含量的降低。
[Abstract]:Effects of maternal chronic aluminum exposure on LTP, PKC activity and Ng content in hippocampus of offspring rats
Preface
Many epidemiological investigations and studies at home and abroad have shown that aluminum is vulnerable to neuronal damage, causing a lack of learning and memory, such as intellectual and cognitive decline, and this is a chronic, chronic neuropathological process, involving various stages of brain development.
Hippocampus is the key brain area for learning and memory. Long-term potentiation (LTP) is a continuous enhancement of NMDA (N- methy aspartic acid) receptor dependent synaptic transmission efficiency. It is a recognized model of brain learning and memory function at the synaptic level and the basis of the apex. Therefore, the study of aluminum exposure to LTP and its synaptic mechanism The effects of various biochemical indexes are helpful to elucidate the mechanism of aluminum damage to brain learning and memory function from the synapse and protein molecular level. Although there are many observations on the damage effect of aluminum to LTP, the synaptic mechanism of aluminum damage to LTP has not been fully elucidated. It is clear that the egg white kinase C (protein kinase C, PKC) is a necessity for LTP production. Neurogranin (Ng) is a specific protein of the postsynaptic brain, which is a natural protein of the postsynaptic brain. It is a natural substrate for PKC. It is closely related to the induction and maintenance of LTP. At the same time, the parent phase is an important period for the development of the progeny of the offspring. The model of maternal chronic aluminum exposure was used to observe the effect of aluminum exposure on LTP induction and maintenance, PKC activity and the content of Ng in the hippocampus of the parent rat, in order to provide a new view from the angle of PKC and Ng to elucidate the mechanism of aluminum affecting LTP at this stage.
Materials and methods
First, experimental materials
1, experimental animals
Wistar rats were: 3:1 (body weight 150-200g) and their offspring offspring.
2, the main reagent
Aluminum chloride (analytical purity); anti rat Ng monoclonal antibody: alkaline phosphatase labeled Rabbit anti goat two antibody; PepTag non radioactive PKC activity assay kit.
3, the main instrument
Rat platform reaction box; general electrophoretic apparatus; gel electrophoresis imaging system; cryogenic centrifuge; Spectronic 21 ultraviolet visible spectrophotometer: electrophysiological related instrument; agarose gel electrophoresis apparatus.
Two, experimental method
1, group and animal model establishment
45 female Wistar rats were randomly divided into 3 groups, from the cages of the male rats to the offspring of the offspring. Under the same feed conditions, drinking distilled water, 0.2% and 0.4% aluminum chloride (AlCl_3) solution respectively. When their offspring were weaned (after birth 21d), 2-3 offspring were taken out of each nest. As a control group (control), drinking distilled water was continued after weaning. The offspring of female rats were divided into 0.2% and 0.4% exposed groups (group 0.2%-Al and 0.4%-Al), and distilled water was changed after weaning. Each group of drinking water remained unchanged from weaning to electrophysiological experiment (3-4 months), and three groups were used alternately for experiment. The indoor temperature of raising animals is 18-23 degrees, and the relative humidity is 45-55%.
2, behavioural determination
The effect of aluminum on learning and memory behavior was detected by platform method. The time (incubation period) and the number of times (the number of errors) were recorded in the time (incubation period) of the first jumping platform and the 5 min in the rats.
3, electrophysiological LTP determination
After 20% uran (urethane, 6.5ml. Kg~ (-1), IP), the head of the animal was fixed on the brain stereotaxic. The stimulation electrode was inserted into the Schaffer lateral branch (coordinates: 3.3mm in the anterior fontanelle, 3.8mm: subcortical 3.8mm), and the glass microelectrodes were inserted into the hippocampus CA_1 region (coordinates: the fontanelle, 3.3mm; the side opening 1.5mm; the tip tip). When the stable group front potential (population spike, PS) was found, the PS. was first recorded by the single pulse of 30min and the mean value of the 30 PS amplitude was taken as the baseline value. Then the short series of high frequency stimulation (higll frequenc) with the same intensity and wave width (higll frequenc) was given. After y stimuli, HFS), the amplitude changes of the PS induced by the monopulse test per minute and the duration of this change are observed after.HSF, and the amplitude of PS is expressed as a percentage relative to the baseline value (also known as the relative amplitude of the PS, showing the PS enhancement rate).
4, determination of the activity of PKC in the hippocampus
The activity of hippocampal PKC was determined by agarose gel electrophoresis. Reference to PepTag non radioactive PKC activity assay kit was provided.
5, determination of Ng content
The Western blot method was used to determine the content of Ng in the hippocampus.
6, determination of aluminum content in brain tissue and blood
Atomic absorption graphite furnace method was used to determine the content of aluminum and aluminum in brain.
7, statistical treatment
The data are processed with SPSS13.0 (statistical package for the social science, SPSS) statistical analysis and analysis processing software. P < 0.05 is statistically significant. The data obtained from the experiment are expressed in (?) + S.
experimental result
First, the index of memory behavior
Compared with the incubation period of first times (300 + 0 seconds), 0.2%-Al (202.71 + 81.99 seconds) and 0.4%-Al group (19.67 + 8.44 seconds) were significantly different from the control group. The number of errors in 5min in the P < 0.01. two exposure group (1.43 + 0.85, 2.47 + 0.99) was also significantly different from the control group (0 + 0), P < 0.05. exposure group. The difference was also statistically significant, P < 0.01.
Two, electrophysiological LTP test results
After HS, compared with the contrast group PS amplitude enhancement rate (143.94 + 29.93%), 0.2%-Al (115.29 + 14.80%) and 0.4% (103.62 + 13.70%), the difference was very significant, P < 0.01., but there was no significant difference between the two exposed groups, P > 0.05.
Three, the results of hippocampal PKC activity assay (PKC activity unit was pmol / min mg).
The whole cell (827.60 + 42.52) of PKC in the hippocampus of the control group, the membrane (340.99 + 24.50), cytoplasm activity (481.32 + 22.52) and the ratio of cytoplasmic PKC activity (0.7114 + 0.0686), 0.2%-Al group (506.41 + 20.56155.69 + 12.63,55.79 + 25.98,0.4411 + 0.0623) and 0.4%-Al group (494.65 + 16.81140.11 + 4.44353.44 + 8.28,0.3966 + 0.0163) The difference was significant, P < 0.01., but the difference between the two exposure groups was not significant, P > 0.05.
Four, the results of the determination of Ng content in the hippocampus
Compared with the gray value of Ng expression in the hippocampus of the control group (62.56 + 8.74), the difference between the 0.2%-Al group (52.33 + 4.01) and the 0.4%-Al group (50.75 + 5.99) was significant, P < 0.01., but there was no significant difference between the two exposed groups, P > 0.05.
Five, determination of aluminum content in brain tissue and blood
Compared with the control group, the content of Al (6.21 + 2.37 g / g) and the content of blood aluminum (33.9 + 38.74 mu g / L), 0.2%-Al group (15.68 + 4.48 g/g, 47.43 + 14.38 mu g/L) and 0.4%-Al group (18.79 + 5.18 mu / g, 55.56 + 19.07 mu g/L) were all significant differences.
discuss
The maternal stage is an important stage of the subgeneration brain development, and the maternal aluminum exposure in this period is an important aspect of the intellectual development of the offspring brain. Therefore, the study of the potential developmental toxicity of aluminum and its compounds in the parent phase is important for the early treatment and prevention of mental and cognitive impairment of the aluminum induced children. The experimental results showed that the maternal chronic aluminum exposure could damage the induction and maintenance of the hippocampal LTP in the offspring, and the degree of damage increased with the increase of exposure dose. The results of the behavioral test also reflected the relationship. The trend of the change of brain aluminum and blood aluminum was also basically consistent.
The results showed that the PKC whole cell activity, the cell membrane, the cytoplasm activity and the ratio of the cytoplasm and cytoplasm activity of the offspring of the maternal aluminum exposed rats were significantly lower than those of the control group. In our analysis, we believe that the aluminum by inhibiting the activity of PKC makes the phosphorylation of the necessary substance produced by LTP produced by LTP and the phosphorylation of the NMDA receptor to occur. It can damage LTP and decrease learning and memory ability in rats.
In this experiment, the content determination of hippocampal Ng in the offspring of the offspring showed that compared with the control group, the content of Ng in the hippocampus of the offspring of the maternal aluminum exposed rats decreased, which was statistically significant. One of the reasons for the decrease of Ng content may be the decrease of the number of dendritic spines and the loss of cells. The phenomenon of pyknosis and neuron reduction in the nucleus was obvious.
In conclusion, the exposure to maternal aluminum will affect the offspring, and the effects of aluminum on PKC and Ng, which play an important role in the LTP mechanism, are multifaceted and need further exploration.
conclusion
1, chronic aluminum exposure in the mother stage could shorten the incubation period of the first descending platform of the offspring and increase the number of errors in 5min, suggesting that the exposure damage the memory ability of the offspring of the offspring.
2, maternal exposure to chronic aluminum exposure reduced the amplitude of PS in offspring rats, suggesting that the exposure impaired the induction and maintenance of LTP in offspring rats.
3, one of the possible mechanisms of maternal chronic aluminum damage in offspring hippocampus LTP is that aluminum causes the decrease of PKC activity and Ng content in hippocampus.
【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R363

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