【摘要】：研究目的:研究不同強(qiáng)度跑臺(tái)運(yùn)動(dòng)對(duì)學(xué)習(xí)記憶的影響并探討microRNA-483和IGF2在跑臺(tái)運(yùn)動(dòng)后表達(dá)水平的改變及其意義。為此本文通過(guò)三部分闡述研究目的。第一部分:不同強(qiáng)度跑臺(tái)對(duì)學(xué)習(xí)記憶的影響實(shí)驗(yàn)方法:將32只2月齡雄性SD大鼠隨機(jī)分成4組:對(duì)照組(正常進(jìn)食,不進(jìn)行運(yùn)動(dòng)訓(xùn)練);小強(qiáng)度組(18m/min x 30min);中強(qiáng)度組30min(24m/min x30min);大強(qiáng)度組(30m/min x 30min)。6周后進(jìn)行Morris水迷宮測(cè)試,檢測(cè)大鼠的學(xué)習(xí)記憶能力。實(shí)驗(yàn)結(jié)果:經(jīng)過(guò)4天的定向航行,跑臺(tái)訓(xùn)練組的逃避潛伏期相比對(duì)照組明顯縮短,在第一天的定向航行中,中等強(qiáng)度跑臺(tái)訓(xùn)練組與對(duì)照組相比明顯縮短,具有顯著相差異(P0.05)。1天的空間探索發(fā)中,跑臺(tái)訓(xùn)練組在穿臺(tái)次數(shù)中比對(duì)照組明顯增多,但整體的差異顯著性不高,但在路程百分比中,跑臺(tái)訓(xùn)練的中等強(qiáng)度組明顯低于對(duì)照組P0.05),其他各組無(wú)顯著性差異(P0.05)。實(shí)驗(yàn)結(jié)論:跑臺(tái)訓(xùn)練改善認(rèn)知功能,中等強(qiáng)度組效果更為顯著。第二部分:基因芯片分析實(shí)驗(yàn)方法:首先我們把四組基因通過(guò)Venny對(duì)四組基因進(jìn)行交集,找出四組共有基因,再通過(guò)MeV將四組基因的趨勢(shì)圖做出,直觀表達(dá)各組差異基因。本實(shí)驗(yàn)研究的是認(rèn)知功能,通過(guò)基因芯片中四組的GO-memory功能基因進(jìn)行交集,并通過(guò)上調(diào)或下調(diào)差異倍數(shù)最大的基因定位我們的目的基因,再通過(guò)生物信息學(xué)分析,用Target Scan_7.1,miRDB,microRNA.org三個(gè)數(shù)據(jù)對(duì)篩選的目的基因進(jìn)行預(yù)測(cè),利用pubmed數(shù)據(jù)庫(kù)文獻(xiàn)確定最終目的基因及調(diào)控機(jī)制。并將篩選的目的基因及其調(diào)控機(jī)制通過(guò)QPCR,WB,IF進(jìn)行驗(yàn)證。實(shí)驗(yàn)結(jié)果:上調(diào)差異基因共有587個(gè)。下調(diào)的差異基因有604個(gè)。在上調(diào)的memory基因有6個(gè),下調(diào)的memory基因有4個(gè)。通過(guò)找出這10個(gè)對(duì)應(yīng)的fold change值,發(fā)現(xiàn)上調(diào)基因IGF2和下調(diào)基因Htr7作為我們選出重要基因。通過(guò)查閱文獻(xiàn)我們發(fā)現(xiàn)IGF2與學(xué)習(xí)記憶有關(guān),但在跑臺(tái)運(yùn)動(dòng)對(duì)記憶影響相關(guān)的作用研究較少,最終確定IGF2為我們的目的基因。為了更好的找出跑臺(tái)運(yùn)動(dòng)改善學(xué)習(xí)記憶的調(diào)控機(jī)制,我們通過(guò)Target Scan_7.1,miRDB,microRNA.org三個(gè)數(shù)據(jù)庫(kù)共預(yù)測(cè)到microRNA 69個(gè),將三個(gè)數(shù)據(jù)庫(kù)進(jìn)行交集,發(fā)現(xiàn)TargetScan_7.1與miRDB數(shù)據(jù)庫(kù),都存在microRNA-483和microRNA665我們又分別對(duì)IGF2與microRNA-483和IGF2與microRNA665在pubmed里研究進(jìn)展進(jìn)行檢索,最終選定IGF2與microRNA-483作為我們最終的研究對(duì)象,并提出假說(shuō)跑臺(tái)運(yùn)動(dòng)改善學(xué)習(xí)記憶中可能通過(guò)microRNA-483對(duì)IGF2的調(diào)控作用達(dá)到的。QPCR結(jié)果發(fā)現(xiàn)IGF2在跑臺(tái)訓(xùn)練后的表達(dá)水平都較對(duì)照組高,且中強(qiáng)度表達(dá)水平最高,與對(duì)照組(P0.05)和小強(qiáng)度組(P0.05)比具有顯著性差異。而microRNA-483的表達(dá)水平在跑臺(tái)訓(xùn)練后的表達(dá)水平都比對(duì)照組低。WB結(jié)果:IGF2的蛋白表達(dá)量在跑臺(tái)訓(xùn)練組都是升高的,尤其是在中等強(qiáng)度組。IF結(jié)果證明IGF2在海馬神經(jīng)元大量表達(dá)。實(shí)驗(yàn)結(jié)論:跑臺(tái)訓(xùn)練后大量和學(xué)習(xí)記憶相關(guān)的基因表達(dá)改變,其中IGF2與microRNA-483的表達(dá)變化可能是學(xué)習(xí)記憶改善的關(guān)鍵因素。第三部分:IGF2和microRNA-483對(duì)海馬神經(jīng)元的影響實(shí)驗(yàn)方法:用PC12細(xì)胞系進(jìn)行si IGF2片段的篩選。體外培養(yǎng)海馬神經(jīng)元后,將海馬神經(jīng)元細(xì)胞分成8組,分別為Normal組、Reagent組、IGF2 normal(IGF2-NC)組,siIGF2組、mimic negative control CY3(mi-NC)、inhitibor negative control(in-NC)組,microRNA-483 inhitibor(in-483)組、microRNA-483 mimic(mi-483)組,每組3個(gè)孔,Normal組細(xì)胞不做處理,觀察神經(jīng)元存活情況。實(shí)驗(yàn)結(jié)果:通過(guò)PC12細(xì)胞篩選有效片段,發(fā)現(xiàn)Si-IGF2-3干擾效果最好。正常海馬神經(jīng)元細(xì)胞在倒置顯微鏡下8小時(shí)開(kāi)始貼壁并有較小突起,1天后基本貼壁完成,7天后神經(jīng)元胞體飽滿,突起增長(zhǎng)形成網(wǎng)絡(luò)結(jié)構(gòu)。IGF2和microRNA-483轉(zhuǎn)染成功3天后發(fā)現(xiàn)siIGF2,mi-RNA 483組的神經(jīng)元數(shù)目明顯低于其它各組(P0.05,P0.01),但兩者沒(méi)有統(tǒng)計(jì)學(xué)差異(P0.05)實(shí)驗(yàn)結(jié)論:IGF2可以有效促進(jìn)海馬神經(jīng)元的存活,而microRNA-483明顯抑制海馬神經(jīng)元的存活,他們之間可能存在潛在的負(fù)調(diào)控關(guān)系。綜上:中等強(qiáng)度跑臺(tái)運(yùn)動(dòng)通過(guò)上調(diào)IGF2和下調(diào)microRNA-483促進(jìn)神經(jīng)元存活,從而改善學(xué)習(xí)記憶功能。
[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.
【學(xué)位授予單位】：云南師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：G804.2

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9 辛成齊;椰棗microRNA鑒定及其在果實(shí)發(fā)育過(guò)程中的表達(dá)譜研究[D];中國(guó)科學(xué)院北京基因組研究所;2015年

10 李棟;先天性心臟病血漿microRNA表達(dá)譜及與GATA4靶序列單核苷酸多態(tài)性的關(guān)聯(lián)研究[D];山東大學(xué);2015年

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