有氧运动干预高脂膳食大鼠生精细胞凋亡miRNA的研究

发布时间：2018-06-23 01:20

本文选题：大鼠 + 睾丸　；参考：《扬州大学》2017年硕士论文

【摘要】：实验目的:探讨mi RNA对高脂膳食诱导的肥胖大鼠生精细胞凋亡的调控,并初步揭示有氧运动对雄性高脂膳食大鼠生精细胞凋亡的mi RNA干预作用。实验方法:将48只雄性健康SD大鼠随机分为4组:普通膳食对照组(A)、高脂膳食组(B)、普通膳食+运动组(C)、高脂膳食+运动组(D),每组各12只;A组和C组,普通饲料喂养;B组和D组,高脂、高热量饲料喂养。C组及D组大鼠进行10周60min无负重游泳运动,每周6天。实验结束后,各组大鼠数量为A组8只,B组11只,C组10只,D组11,测量大鼠体重及睾周脂重。将A组大鼠分为A01、A02、A03三组;B组大鼠分为B01、B02、B03三组;C组大鼠分为C01、C02、C03三组;D组大鼠分为D01、D02、D03三组,对其进行文库构建,共构建12组cDNA文库,利用Solexa高通量测序技术结合生物信息分析鉴定mi RNA并进行差异表达谱的分析,进一步选择部分差异mi RNA,分析其生物学功能。实验结果:1.通过Solexa测序技术,成功构建了大鼠的基因文库,经检测得到每组高质量的纯净序列(Clean Data)均大于20M,均占原始数据87%以上,Q30均在95%以上。对文库中的纯净序列进行了分类注释,获得包含mi RNA的Unannotated reads。并将小RNA的纯净序列定位到大鼠的基因组,以备后续的研究。2.通过生物学分析,从A组、B组、C组、D组四组睾丸组织中共鉴定出464个mi RNA,其中315个为已知mi RNA和149个为新的mi RNA。根据|log2(FC)|≥1进行文库测序的mi RNA差异表达分析显示:B组对A组差异表达显著mi RNA有27个,其中3个上调,24个下调。C组对A组差异表达mi RNA有11个,其中0个上调,11个下调。D组对A组差异表达mi RNA有62个,其中37个上调,25个下调。3.根据|log2(FC)|≥1重点分析A与B组mi RNA的表达差异,B组对A组差异表达mi RNA有27个,其中3个上调,24个下调,共预测到19612个靶基因,其中24个下调mi RNAs为:rno-miR-101a-5p,57 个;rno-miR-101a-3p,4 个;rno-miR-135b-3p,465 个等;3 个上调 miRNAs:rno-miR-130b-5p,164 个;rno-miR-346,2963 个;unconservative_6_3922464,3220 个。4.GO分类可知,对照组A组和高脂膳食组B组存在差异的mi RNA基因,其对应的靶基因在代谢方面、生殖方面、细胞死亡、免疫系统等方面均有所表达;在KEGG通路中分类可知,差异表达 mi RNA 靶基因在 Pathways in cancer、MAPK signaling pathway、Wnt signaling pathway、Axon guidance、Apoptosis 等方面均表达。5.通过与NR、Swiss-Prot、Pfam数据库比对,获得与有性生殖有关的miRNA有:rno-miR-6328;rno-miR-196b-5p;rno-miR-346;unconservative_6_3922464;rno-miR-503-5p。与细胞凋亡有关的 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。进一步发现,以上mi RNA通过Bcl、Bax两个途径进行生精细胞凋亡的。结论:1.本实验通过高通量测序方法成功获得普通膳食大鼠、高脂膳食大鼠、普通运动组和高脂运动组睾丸的mi RNA的cDNA文库。首次对大鼠睾丸组织进行了 micro RNA组学分析,共鉴定出464个mi RNA,其中315个为已知mi RNA和149个为新的mi RNA。2.较多的mi RNA在高脂膳食的大鼠睾丸中呈下调趋势,进行有氧干预后,普通组较多的mi RNA呈上调,总之,差异的mi RNA,都在有氧运动后发生表达量的改变,使其生物学功能发生改变。3.通过GO、KEGG通路中分类可知,对照组A组和高脂膳食组B组差异的mi RNA基因,与有性生殖、细胞死亡等有关。4.通过与NR、Swiss-Prot、Pfam数据库比对,我们认为高脂膳食引起肥胖的大鼠生殖细胞通过 Bcl、Bax 途径凋亡有关的 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;与有性生殖有关的 mi RNA 有:unconservative_6_3922464;rno-miR-196b-5p;mo-miR-6328;rno-miR-346;rno-miR-503-5p;而进行有氧运动干预,可以改变这些差异基因的表达量,起到了有效的改善生精细胞调亡情况。证明有氧运动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.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：G804.2

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