不同强度跑台运动对大鼠学习记忆功能的影响及microRNA-483与IGF2的调控机制研究

发布时间：2019-07-01 12:55

【摘要】：研究目的:研究不同强度跑台运动对学习记忆的影响并探讨microRNA-483和IGF2在跑台运动后表达水平的改变及其意义。为此本文通过三部分阐述研究目的。第一部分:不同强度跑台对学习记忆的影响实验方法:将32只2月龄雄性SD大鼠随机分成4组:对照组(正常进食,不进行运动训练);小强度组(18m/min x 30min);中强度组30min(24m/min x30min);大强度组(30m/min x 30min)。6周后进行Morris水迷宫测试,检测大鼠的学习记忆能力。实验结果:经过4天的定向航行,跑台训练组的逃避潜伏期相比对照组明显缩短,在第一天的定向航行中,中等强度跑台训练组与对照组相比明显缩短,具有显著相差异(P0.05)。1天的空间探索发中,跑台训练组在穿台次数中比对照组明显增多,但整体的差异显著性不高,但在路程百分比中,跑台训练的中等强度组明显低于对照组P0.05),其他各组无显著性差异(P0.05)。实验结论:跑台训练改善认知功能,中等强度组效果更为显著。第二部分:基因芯片分析实验方法:首先我们把四组基因通过Venny对四组基因进行交集,找出四组共有基因,再通过MeV将四组基因的趋势图做出,直观表达各组差异基因。本实验研究的是认知功能,通过基因芯片中四组的GO-memory功能基因进行交集,并通过上调或下调差异倍数最大的基因定位我们的目的基因,再通过生物信息学分析,用Target Scan_7.1,miRDB,microRNA.org三个数据对筛选的目的基因进行预测,利用pubmed数据库文献确定最终目的基因及调控机制。并将筛选的目的基因及其调控机制通过QPCR,WB,IF进行验证。实验结果:上调差异基因共有587个。下调的差异基因有604个。在上调的memory基因有6个,下调的memory基因有4个。通过找出这10个对应的fold change值,发现上调基因IGF2和下调基因Htr7作为我们选出重要基因。通过查阅文献我们发现IGF2与学习记忆有关,但在跑台运动对记忆影响相关的作用研究较少,最终确定IGF2为我们的目的基因。为了更好的找出跑台运动改善学习记忆的调控机制,我们通过Target Scan_7.1,miRDB,microRNA.org三个数据库共预测到microRNA 69个,将三个数据库进行交集,发现TargetScan_7.1与miRDB数据库,都存在microRNA-483和microRNA665我们又分别对IGF2与microRNA-483和IGF2与microRNA665在pubmed里研究进展进行检索,最终选定IGF2与microRNA-483作为我们最终的研究对象,并提出假说跑台运动改善学习记忆中可能通过microRNA-483对IGF2的调控作用达到的。QPCR结果发现IGF2在跑台训练后的表达水平都较对照组高,且中强度表达水平最高,与对照组(P0.05)和小强度组(P0.05)比具有显著性差异。而microRNA-483的表达水平在跑台训练后的表达水平都比对照组低。WB结果:IGF2的蛋白表达量在跑台训练组都是升高的,尤其是在中等强度组。IF结果证明IGF2在海马神经元大量表达。实验结论:跑台训练后大量和学习记忆相关的基因表达改变,其中IGF2与microRNA-483的表达变化可能是学习记忆改善的关键因素。第三部分:IGF2和microRNA-483对海马神经元的影响实验方法:用PC12细胞系进行si IGF2片段的筛选。体外培养海马神经元后,将海马神经元细胞分成8组,分别为Normal组、Reagent组、IGF2 normal(IGF2-NC)组,siIGF2组、mimic negative control CY3(mi-NC)、inhitibor negative control(in-NC)组,microRNA-483 inhitibor(in-483)组、microRNA-483 mimic(mi-483)组,每组3个孔,Normal组细胞不做处理,观察神经元存活情况。实验结果:通过PC12细胞筛选有效片段,发现Si-IGF2-3干扰效果最好。正常海马神经元细胞在倒置显微镜下8小时开始贴壁并有较小突起,1天后基本贴壁完成,7天后神经元胞体饱满,突起增长形成网络结构。IGF2和microRNA-483转染成功3天后发现siIGF2,mi-RNA 483组的神经元数目明显低于其它各组(P0.05,P0.01),但两者没有统计学差异(P0.05)实验结论:IGF2可以有效促进海马神经元的存活,而microRNA-483明显抑制海马神经元的存活,他们之间可能存在潜在的负调控关系。综上:中等强度跑台运动通过上调IGF2和下调microRNA-483促进神经元存活,从而改善学习记忆功能。
[Abstract]:Objective: To study the effect of different intensity treadmill exercise on learning and memory and to study the changes of microRNA-483 and IGF2 expression level after treadmill exercise and its significance. For this purpose, the purpose of the study is set forth in three parts. The first part: The effect of different intensity treadmill on learning and memory:32-month-old male SD rats were randomly divided into 4 groups: control group (normal feeding, no exercise training), small-intensity group (18 m/ min x 30 min), medium intensity group 30 min (24 m/ min x 30 min); After 6 weeks, Morris water maze test was performed to test the learning and memory capacity of the rats. The experimental results showed that, after 4 days of directional navigation, the escape latency of the treadmill training group was significantly shorter than that of the control group. In the first day of the directional navigation, the middle-strength treadmill training group was significantly shortened compared with the control group, with a significant phase difference (P0.05). In the first day of space exploration, There was no significant difference between the training group and the control group, but the overall difference was not high, but the middle intensity group of the treadmill training was significantly lower than that of the control group (P0.05). The results of the experiment show that the training of the treadmill improves the cognitive function, and the effect of the medium-intensity group is more significant. The second part: The gene chip analysis experiment method: First, we set four groups of genes to the four groups of genes through Venny to find out four groups of common genes, and then, the trend chart of four groups of genes was made by MeV, and the difference genes of each group were directly expressed. In the experiment, the cognitive function was studied, and the gene of the target gene was located through four groups of the GO-memory functional genes in the gene chip, and the target gene was located by regulating or down-regulating the maximum gene of the difference, and the target gene was analyzed by bioinformatics, using the Target Scan _ 7.1 and the miRDB. The objective gene of the screen was predicted by the three data of the microRNA.org, and the final target gene and the regulatory mechanism were determined by using the pubmed database. And the screening target gene and the control mechanism thereof are verified through the QPCR, the WB and the IF. The results of the experiment were as follows: the up-regulation of the differential gene was 587. The down-cut differential gene has 604. There are 6 down-regulated memory genes and 4 down-regulated memory genes. By finding the 10 corresponding fold change values, the up-regulation gene IGF2 and the down-regulation gene Htr7 were found to be an important gene for us. By consulting the literature we found that IGF2 is related to learning and memory, but the effect of the treadmill exercise on the memory effect is less, and finally, it is determined that IGF2 is the target gene for us. In order to find out the regulation and control mechanism for improving learning and memory, we used the target Scan _ 7.1, the miRDB and the microRNA.org three databases to predict the microRNA 69, and the three databases were intersected to find the TargetScan _ 7.1 and the miRDB database. There are microRNA-483 and microRNAs 665. We also search for the progress of IGF2 and microRNA-483 and IGF2 and microRNAs in pubmed, and finally select IGF2 and microRNA-483 as our final research object, and put forward that the hypothesis running-table exercise can improve the regulation of IGF2 by microRNA-483 in learning and memory. The results showed that the expression level of IGF2 was higher in the control group than in the control group, and the expression level of the medium intensity was the highest in the control group (P0.05) and the small-intensity group (P0.05). The expression level of microRNA-483 was lower than that of control group. WB results: The amount of protein expression of IGF2 was elevated in the treadmill training group, especially in the medium-strength group. IF The results of IF showed that IGF2 was expressed in the hippocampus. It is concluded that the expression of IGF2 and microRNA-483 may be a key factor in the improvement of learning and memory. The third part: the effect of IGF2 and microRNA-483 on the hippocampal neurons: screening of the si-IGF2 fragment by PC12 cell line. In vitro, the hippocampal neurons were divided into 8 groups, namely, the normal group, the Reagent group, the IGF2 normal (IGF2-NC) group, the siIGF2 group, the mimetic control CY3 (mi-NC), the in-NC group, the microRNA-483 inhtibor (in-483) group and the microRNA-483 mic (mi-483) group, and the normal group cells did not process to observe the survival condition of the neurons. The results showed that the effect of Si-IGF2-3 was the best in the screening of effective fragments by PC12 cells. The cells of the normal hippocampal neurons began to adhere to each other for 8 hours under the inverted microscope and had a small protrusion. After 1 day, the cells were basically attached to the cells. After 7 days, the cells of the neurons were full and the growth of the protrusions was increased to form the network structure. After 3 days of successful transfection of IGF2 and microRNA-483, the number of neurons in the siIGF2 and mi-RNA 483 groups was significantly lower than that of the other groups (P0.05, P0.01), but there was no statistical difference between them (P0.05). The results showed that IGF2 could effectively promote the survival of the hippocampal neurons, while microRNA-483 significantly inhibited the survival of the hippocampal neurons. There may be potential negative control relationships between them. In general, medium-intensity treadmill exercise improved learning and memory by up-regulation of IGF2 and down-regulation of microRNA-483 to promote neuronal survival.
【学位授予单位】：云南师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：G804.2

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