本文選題：大鼠 + 睪丸��； 參考：《揚(yáng)州大學(xué)》2017年碩士論文

【摘要】：實(shí)驗(yàn)?zāi)康?探討mi RNA對(duì)高脂膳食誘導(dǎo)的肥胖大鼠生精細(xì)胞凋亡的調(diào)控,并初步揭示有氧運(yùn)動(dòng)對(duì)雄性高脂膳食大鼠生精細(xì)胞凋亡的mi RNA干預(yù)作用。實(shí)驗(yàn)方法:將48只雄性健康SD大鼠隨機(jī)分為4組:普通膳食對(duì)照組(A)、高脂膳食組(B)、普通膳食+運(yùn)動(dòng)組(C)、高脂膳食+運(yùn)動(dòng)組(D),每組各12只;A組和C組,普通飼料喂養(yǎng);B組和D組,高脂、高熱量飼料喂養(yǎng)。C組及D組大鼠進(jìn)行10周60min無負(fù)重游泳運(yùn)動(dòng),每周6天。實(shí)驗(yàn)結(jié)束后,各組大鼠數(shù)量為A組8只,B組11只,C組10只,D組11,測(cè)量大鼠體重及睪周脂重。將A組大鼠分為A01、A02、A03三組;B組大鼠分為B01、B02、B03三組;C組大鼠分為C01、C02、C03三組;D組大鼠分為D01、D02、D03三組,對(duì)其進(jìn)行文庫(kù)構(gòu)建,共構(gòu)建12組cDNA文庫(kù),利用Solexa高通量測(cè)序技術(shù)結(jié)合生物信息分析鑒定mi RNA并進(jìn)行差異表達(dá)譜的分析,進(jìn)一步選擇部分差異mi RNA,分析其生物學(xué)功能。實(shí)驗(yàn)結(jié)果:1.通過Solexa測(cè)序技術(shù),成功構(gòu)建了大鼠的基因文庫(kù),經(jīng)檢測(cè)得到每組高質(zhì)量的純凈序列(Clean Data)均大于20M,均占原始數(shù)據(jù)87%以上,Q30均在95%以上。對(duì)文庫(kù)中的純凈序列進(jìn)行了分類注釋,獲得包含mi RNA的Unannotated reads。并將小RNA的純凈序列定位到大鼠的基因組,以備后續(xù)的研究。2.通過生物學(xué)分析,從A組、B組、C組、D組四組睪丸組織中共鑒定出464個(gè)mi RNA,其中315個(gè)為已知mi RNA和149個(gè)為新的mi RNA。根據(jù)|log2(FC)|≥1進(jìn)行文庫(kù)測(cè)序的mi RNA差異表達(dá)分析顯示:B組對(duì)A組差異表達(dá)顯著mi RNA有27個(gè),其中3個(gè)上調(diào),24個(gè)下調(diào)。C組對(duì)A組差異表達(dá)mi RNA有11個(gè),其中0個(gè)上調(diào),11個(gè)下調(diào)。D組對(duì)A組差異表達(dá)mi RNA有62個(gè),其中37個(gè)上調(diào),25個(gè)下調(diào)。3.根據(jù)|log2(FC)|≥1重點(diǎn)分析A與B組mi RNA的表達(dá)差異,B組對(duì)A組差異表達(dá)mi RNA有27個(gè),其中3個(gè)上調(diào),24個(gè)下調(diào),共預(yù)測(cè)到19612個(gè)靶基因,其中24個(gè)下調(diào)mi RNAs為:rno-miR-101a-5p,57 個(gè);rno-miR-101a-3p,4 個(gè);rno-miR-135b-3p,465 個(gè)等;3 個(gè)上調(diào) miRNAs:rno-miR-130b-5p,164 個(gè);rno-miR-346,2963 個(gè);unconservative_6_3922464,3220 個(gè)。4.GO分類可知,對(duì)照組A組和高脂膳食組B組存在差異的mi RNA基因,其對(duì)應(yīng)的靶基因在代謝方面、生殖方面、細(xì)胞死亡、免疫系統(tǒng)等方面均有所表達(dá);在KEGG通路中分類可知,差異表達(dá) mi RNA 靶基因在 Pathways in cancer、MAPK signaling pathway、Wnt signaling pathway、Axon guidance、Apoptosis 等方面均表達(dá)。5.通過與NR、Swiss-Prot、Pfam數(shù)據(jù)庫(kù)比對(duì),獲得與有性生殖有關(guān)的miRNA有:rno-miR-6328;rno-miR-196b-5p;rno-miR-346;unconservative_6_3922464;rno-miR-503-5p。與細(xì)胞凋亡有關(guān)的 mi RNA 有:rno-miR-6328;rno-miR-3075;rno-miR-346;rno-miR-130b-5p;rno-miR-205;rno-miR-31a-5p;rno-miR-503-5p;unconservative_1_540112;unconservative_6__3922464。進(jìn)一步發(fā)現(xiàn),以上mi RNA通過Bcl、Bax兩個(gè)途徑進(jìn)行生精細(xì)胞凋亡的。結(jié)論:1.本實(shí)驗(yàn)通過高通量測(cè)序方法成功獲得普通膳食大鼠、高脂膳食大鼠、普通運(yùn)動(dòng)組和高脂運(yùn)動(dòng)組睪丸的mi RNA的cDNA文庫(kù)。首次對(duì)大鼠睪丸組織進(jìn)行了 micro RNA組學(xué)分析,共鑒定出464個(gè)mi RNA,其中315個(gè)為已知mi RNA和149個(gè)為新的mi RNA。2.較多的mi RNA在高脂膳食的大鼠睪丸中呈下調(diào)趨勢(shì),進(jìn)行有氧干預(yù)后,普通組較多的mi RNA呈上調(diào),總之,差異的mi RNA,都在有氧運(yùn)動(dòng)后發(fā)生表達(dá)量的改變,使其生物學(xué)功能發(fā)生改變。3.通過GO、KEGG通路中分類可知,對(duì)照組A組和高脂膳食組B組差異的mi RNA基因,與有性生殖、細(xì)胞死亡等有關(guān)。4.通過與NR、Swiss-Prot、Pfam數(shù)據(jù)庫(kù)比對(duì),我們認(rèn)為高脂膳食引起肥胖的大鼠生殖細(xì)胞通過 Bcl、Bax 途徑凋亡有關(guān)的 mi RNA 有:mo-miR-6328;rno-miR-3075;rno-miR-130b-5p;rno-miR-346;rno-miR-503-5p;mo-miR-205;rno-miR-31a-5p;unconservative_6_3922464;unconservative_1_540112;與有性生殖有關(guān)的 mi RNA 有:unconservative_6_3922464;rno-miR-196b-5p;mo-miR-6328;rno-miR-346;rno-miR-503-5p;而進(jìn)行有氧運(yùn)動(dòng)干預(yù),可以改變這些差異基因的表達(dá)量,起到了有效的改善生精細(xì)胞調(diào)亡情況。證明有氧運(yùn)動(dòng)是改善男性不育的有效方法之一。
[Abstract]:Objective: To investigate the regulation of MI RNA on the apoptosis of spermatogenic cells in obese rats induced by high fat diet, and to reveal the effect of MI RNA on the apoptosis of spermatogenic cells in male high fat diet rats. Experimental methods: 48 male healthy SD rats were randomly divided into 4 groups: normal diet control group (A), high fat diet group (B), ordinary diet + Exercise group (C), high fat diet + exercise group (D), 12 rats in each group, group A and C, common feed, B and D, high fat and high calorie diet feeding.C group and D group for 10 weeks without weight negative swimming exercise, 6 days a week. The number of rats in each group was 8 in A group, 11 in B group, 10 in C group and 11 in group 11, measured weight and weight of rats in rats. Group A rats were divided into three groups: A01, A02, A03; B group rats were divided into B01, B02, B03 three groups; C group rats were divided into C01, C02, C03 three groups. The rats were divided into three groups, and 12 groups of library were constructed. Step selected partial difference mi RNA to analyze its biological function. Experimental results: 1. through Solexa sequencing technology, the gene library of rats was constructed successfully, and the high quality pure sequence of each group (Clean Data) was more than 20M, all of which accounted for more than 87% of the original data and more than 95% of Q30. The purified sequences in the library were classified and annotated. We have to include the Unannotated reads. of MI RNA and locate the pure sequence of small RNA into the genome of the rat, so as to prepare a follow-up study of.2. through biological analysis, 464 mi RNA were identified from four groups of A group, B group, C group and D group, of which 315 were known and 149 were sequenced. The differential expression analysis of I RNA showed that there were 27 significant mi RNA expressions in group A, of which 3 were up-regulated, and 24.C groups expressed 11 mi RNA in A group, of which 0 were up-regulated and 62 in 11 down-regulation groups, including 37 up and 25 down 1 In group B, there were 27 differentially expressed mi RNA in group A, of which 3 were up and 24 down-regulated, and 19612 target genes were predicted, of which 24 mi RNAs were rno-miR-101a-5p, 57, rno-miR-101a-3p, 4, rno-miR-135b-3p, 465, 3 up miRNAs:rno-miR-130b-5p, 164, rno-miR-3462963; unconservative_6_39224643220. 4.GO classification shows that the MI RNA gene in the control group A group and the high fat diet group B group is different, and the corresponding target gene is expressed in the aspects of metabolism, reproduction, cell death, and immune system. In the KEGG pathway, the differentially expressed mi RNA target gene is in Pathways in cancer. MAPK Athway, Axon guidance, Apoptosis and other aspects all express.5. by comparing with NR, Swiss-Prot, Pfam database to obtain miRNA related to sexual reproduction: rno-miR-6328; rno-miR-196b-5p; rno-miR-346. 0b-5p; rno-miR-205; rno-miR-31a-5p; rno-miR-503-5p; unconservative_1_540112; unconservative_6__3922464. further found that the above mi RNA carried out spermatogenic cell apoptosis through Bcl, Bax two ways. Conclusion: 1. the experiment was successfully obtained by high throughput sequencing method to obtain common diet rats, high fat diet rats, ordinary exercise groups and high fat. The cDNA Library of MI RNA in the testicle of the exercise group. 464 mi RNA were identified for the first time in the rat testis tissue, and 464 mi RNA were identified. 315 of them were known as mi RNA and 149 new mi RNA.2. MI were down downward in the testicles of high fat diet. The difference of MI RNA, all of the changes in the expression of expression after aerobic exercise, make its biological function change.3. through GO, the classification of KEGG pathway in the A group and the high fat diet group B group, MI RNA gene, and sexual reproduction, cell death, and other.4. through the NR, Swiss-Prot, database comparison, we think high fat diet. Bcl, Bax pathway apoptosis related to MI RNA of rat germ cells that cause obesity in rats: mo-miR-6328; rno-miR-3075; rno-miR-130b-5p; rno-miR-346; rno-miR-503-5p; mo-miR-205; rno-miR-31a-5p; unconservative_6_3922464; B-5p; mo-miR-6328; rno-miR-346; rno-miR-503-5p; and aerobic exercise intervention can change the expression of these differentially genes and effectively improve the apoptosis of spermatogenic cells. It is proved that aerobic exercise is one of the effective ways to improve male infertility.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：G804.2

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