本文選題：miRNAs + 基因芯片�。� 參考：《北京體育大學(xué)》2017年碩士論文

【摘要】：研究目的:miRNA是真核生物中廣泛存在的一種長(zhǎng)度約為21-23nt的RNA分子,可通過(guò)與mRNA的結(jié)合抑制其轉(zhuǎn)錄,在眾多生物學(xué)過(guò)程中發(fā)揮重要作用。研究顯示miRNA在肌萎縮過(guò)程中發(fā)揮重要調(diào)控作用,已有研究發(fā)現(xiàn)miRNAs在多種類(lèi)型的肌萎縮中存在差異表達(dá),但在失重性肌萎縮及恢復(fù)過(guò)程中miRNAs表達(dá)譜的變化未見(jiàn)報(bào)道。本實(shí)驗(yàn)擬通過(guò)尾懸吊誘導(dǎo)小鼠失重性肌萎縮研究miRNAs表達(dá)譜的變化,尋找失重性肌萎縮過(guò)程中具有重要調(diào)控作用的miRNAs。研究方法:以C57BL/6小鼠為實(shí)驗(yàn)對(duì)象,尾懸吊14天誘導(dǎo)失重性肌萎縮,動(dòng)態(tài)分析尾懸吊及恢復(fù)過(guò)程中小鼠后肢背側(cè)骨骼肌肌纖維橫截面積及肌萎縮標(biāo)志蛋白和基因表達(dá)的變化;在此基礎(chǔ)上對(duì)小鼠比目魚(yú)肌miRNAs進(jìn)行基因芯片檢測(cè),尋找差異表達(dá)的基因,并通過(guò)qPCR對(duì)部分未見(jiàn)報(bào)道的miRNAs進(jìn)行驗(yàn)證。研究結(jié)果:(1)14天尾懸吊過(guò)程中,小鼠后肢背側(cè)肌重顯著下降,恢復(fù)過(guò)程中逐漸升高;其中比目魚(yú)肌重量丟失程度明顯高于腓腸肌和跖肌,肌纖維橫截面積顯著下降。(2)小鼠比目魚(yú)肌中肌萎縮標(biāo)志基因FoxO3a、MuRF1和Atrogin-1的蛋白和mRNA表達(dá)隨尾懸吊時(shí)間逐漸升高。14天尾懸吊后,FoxO3a的蛋白含量顯著升高(P0.01),Atrogin-1的蛋白含量也顯著升高(P0.01),MuRF1的蛋白含量在尾懸吊7天升高最明顯(P0.05)。(3)FoxO3a、Atrogin-1和MuRF1的基因表達(dá)在尾懸吊過(guò)程中有升高趨勢(shì)。FoxO3a mRNA在尾懸吊3天最高(P0.01),Atrogin-1 mRNA在尾懸吊7天水平最高(P0.01),MuRF1 mRNA在尾懸吊7天表達(dá)最高(P0.01)。(4)芯片結(jié)果顯示,尾懸吊過(guò)程中14個(gè)miRNAs上調(diào),2個(gè)miRNAs在尾懸吊3天和7天上調(diào),4個(gè)miRNAs先下調(diào)后上調(diào),22個(gè)miRNAs下調(diào)。(5)qRT-PCR對(duì)肌肉萎縮過(guò)程中差異表達(dá)的miRNAs進(jìn)行驗(yàn)證,篩選出8個(gè)符合芯片結(jié)果的miRNAs。研究結(jié)論:本研究通過(guò)尾懸吊成功構(gòu)建了小鼠后肢骨骼肌萎縮模型,具有廢用性肌萎縮的特征,包括肌肉重量下降、肌纖維橫截面積的減小和肌萎縮相關(guān)基因的升高。通過(guò)基因芯片對(duì)失重性肌萎縮形成及恢復(fù)過(guò)程中差異表達(dá)的miRNAs進(jìn)行了篩選,并進(jìn)一步利用qRT-PCR對(duì)差異表達(dá)的miRNAs進(jìn)行了驗(yàn)證和分析,篩選到了在失重性肌萎縮中具有潛在調(diào)控作用的miRNAs。
[Abstract]:Objective\ 21-23nt is a kind of RNA molecule widely existing in eukary@@ Studies have shown that miRNA plays an important regulatory role in the process of muscle atrophy. It has been found that there is a differential expression of miRNAs in various types of muscle atrophy, but the changes of miRNAs expression profile during the course of weightlessness and recovery have not been reported. The purpose of this study was to investigate the changes of miRNAs expression profile in mice with weightlessness muscle atrophy induced by tail suspension, and to search for miRNAs that play an important role in the process of weightlessness muscle atrophy. Methods: C57BL/6 mice were used to induce weightlessness muscle atrophy after 14 days of tail suspension. The changes of muscle fiber cross-sectional area, muscle atrophy marker protein and gene expression during tail suspension and recovery were analyzed. On the basis of this, the miRNAs of mouse soleus muscle was detected by gene chip, and the differentially expressed genes were found, and some unreported miRNAs were verified by qPCR. Results the weight loss of soleus muscle was significantly higher than that of gastrocnemius muscle and metatarsal muscle during 14 days of tail suspension. The expression of protein and mRNA of muscle atrophy marker genes FoxO3a and MuRF1 and Atrogin-1 in soleus muscle of mice increased with the tail suspension time. 14 days after tail suspension, the protein content of FoxO3a increased significantly, and the protein content of P0.01 and Atrogin-1 also showed significant increase in mice soleus muscle atrophy marker gene FoxO3a and Atrogin-1. The protein content of P0.01Atrogin-1 increased most obviously during the 7th day of tail suspension. The gene expression of FoxO3a Atrogin-1 and MuRF1 increased during tail suspension. The highest level of P0.01Atrogin-1 mRNA in tail suspension was P0.01Atrogin-1 mRNA at 7 days of tail suspension. The highest level of P0.01Atrogin-1 mRNA was found at 7 days after tail suspension. The gene expression of FoxO3a and MuRF1 was increased during tail suspension. The highest level of P0.01Atrogin-1 mRNA in tail suspension was P0.01Atrogin-1 mRNA during 7 days of tail suspension. The result of the chip showed that, During tail suspension, 14 miRNAs were up-regulated, 2 miRNAs were up-regulated 3 and 7 days after tail suspension, 4 miRNAs were down-regulated and then up-regulated, 22 miRNAs down-regulated .5 miRNAs RT-PCR was used to verify the differential expression of miRNAs during muscle atrophy, and 8 miRNAs were screened out according to the results of microarray. Conclusion: the model of skeletal muscle atrophy in hind limb of mice was successfully constructed by tail suspension. It has the characteristics of disused muscle atrophy, including the decrease of muscle weight, the decrease of cross sectional area of muscle fiber and the increase of gene related to muscle atrophy. The differentially expressed miRNAs during the formation and recovery of weightlessness muscle atrophy were screened by gene microarray, and the differentially expressed miRNAs was verified and analyzed by qRT-PCR. MiRNAs with potential regulatory role in weightlessness muscle atrophy were screened.
【學(xué)位授予單位】：北京體育大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R85

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