藻蓝蛋白改善半乳糖致衰小鼠卵巢功能的转录组分析

发布时间：2018-03-22 21:31

本文选题：卵巢　切入点：半乳糖致衰　出处：《内蒙古大学》2017年硕士论文　论文类型：学位论文

【摘要】：卵巢是女性生殖系统中最重要的器官之一,随着妇女年龄的增加,其功能不断下降,并经历卵泡减少、卵母细胞质量下降、月经不调、卵巢激素缺乏、停止排卵最后导致生育能力下降的过程。而延缓卵巢老化是增加生殖能力的重要手段。藻蓝蛋白(C-Phycocyanin,PC)是来自钝顶螺旋藻的一种具有抗炎症、抗氧化、抗癌症、提高免疫能力和保肝护肾作用的蛋白质,因为它具有多种生物活性而被应用在许多临床疾病研究中。在前期的研究中,我们已经发现PC具有改善D-半乳糖致衰小鼠卵巢功能的作用,但PC发挥这一作用的分子机理还未被充分研究。本研究主要对D-半乳糖致衰小鼠灌胃藻蓝蛋白后,通过转录组测序分析藻蓝蛋白对致衰小鼠卵巢功能改善的分子机理,为临床治疗卵巢功能的研究提供基础数据。实验以B6D2F/1品系小鼠为研究对象,分别采集8周龄小鼠卵巢作为对照组,D-半乳糖致衰小鼠卵巢作为致衰组,藻蓝蛋白灌胃D-半乳糖致衰小鼠卵巢作为致衰改善组。利用转录组测序(RNA-Sequencing,RNA-seq)的方法对这三组不同来源的卵巢进行转录组测序分析。通过基因本体(Gene Ontology,GO)分类及KEGG(Kyoto Encyclopedia of Genes and Genomes)Pathway 进行基因差异表达分析和差异基因的功能分析,得到如下实验结果:(1)按照P0.05筛选差异基因,对照组和致衰组差异分析共筛选出71个差异基因;对照组和致衰改善组共筛选出56个差异基因;致衰组和致衰改善组共筛选出89个差异基因。我们集中分析了致衰组和致衰改善组差异表达基因的功能,发现这些差异基因主要参与调节细胞分化Sfrp2、Efnb2、Runx3、Jun)、细胞凋亡(Tnfrsf9、Btnl2、Ltf、Cdkn1α)、细胞程序性死亡(Tnfrsf9、Ltf、nt、1)、细胞因子产生(Tnfrsf9、Btnl2、Ltf)等与发育相关的过程。KEGG Pathway富集分析发现,这些差异基因显著富集到Wnt信号通路(Wnt signaling pathway)、TNF信号通路(TNF signaling pathway)、p53 信号通路(p53 signaling pathway)及 NF-κB信号通路(NF-kappa B signaling pathway)等与衰老有关的通路。(2)结合上述分析,我们筛选了 Aocl、Cd74、Cox6a2、Cyp4f18、Enpp3、Tnfrsf9、Wnt11、Zfp365等8个与衰老相关的差异基因进行实时荧光定量分析和验证。与RNA-seq的结果相比,两种方法得到的基因表达差异趋势一致,证明了 RNA-seq结果的可靠性,并发现这8个基因与炎症、癌症、免疫相关,在灌胃藻蓝蛋白后,这些基因在致衰改善组中发生显著改变,我们推测这些基因的表达变化可能对衰老卵巢的功能改善起到一定促进作用。综上所述,本研究通过RNA-seq分析发现致衰小鼠的卵巢转录组活动发生较大改变,这些改变在一定程度上可以通过灌胃藻蓝蛋白而恢复,鉴定出的相关差异表达基因参与了免疫、炎症及衰老有关的信号通路,提示我们藻蓝蛋白可能通过这些信号通路发挥对衰老小鼠卵巢功能的改善作用,为未来卵巢功能相关研究提供了有效的靶点。
[Abstract]:The ovary is one of the most important organs in the female reproductive system. As women get older, their functions decline, and they experience follicle decline, oocyte quality decline, irregular menstruation, and ovarian hormone deficiency. Stopping ovulation leads to a decline in fertility, and delaying ovarian aging is an important means of increasing reproductive capacity. Phycocyanin C-Phycocyanin PCC, a form of spirulina platensis, has anti-inflammatory, antioxidant, and anti-cancer properties. A protein that improves immunity and protects the liver and kidney, because it has a variety of biological activities and has been used in many clinical studies. We have found that PC can improve ovarian function of mice induced by D-galactose, but its molecular mechanism has not been fully studied. The molecular mechanism of phycocyanin on ovarian function improvement in aging mice was analyzed by transcriptome sequencing, which provided basic data for clinical treatment of ovarian function. The ovaries of 8-week-old mice were collected as control group. The ovaries of aging mice induced by phycocyanin were treated as the improvement group of aging by intragastric administration of phycocyanin. The transcriptional sequence analysis of these three groups of ovaries from different sources was carried out by the method of transcriptional sequencing. The classification and KEGG(Kyoto Encyclopedia of the three groups of ovaries from different sources were analyzed. Genes and Genomes)Pathway was used to analyze gene differential expression and function of differentially expressed genes. The results are as follows: 1) according to P0.05, 71 differentially genes were screened in control group and senescence group, 56 genes were screened in control group and senescence improvement group, 56 genes in control group and senescence improvement group were screened out. A total of 89 differentially expressed genes were screened from aging group and senescence improvement group. We focused on the function of differentially expressed genes in senescence group and senescence improvement group. It was found that these differentially expressed genes were mainly involved in the processes of regulating cell differentiation, such as regulation of cell differentiation, Sfrp2, Efnb2, Runx3, Tnfrsf9, Btnl2, Tnfrsf9, Tnfrsf9, Tnfrsf9, Tnfrsf9, Tnfrsf9Btnl2, etc., and KEGG Pathway enrichment analysis showed that these genes were mainly involved in the processes related to development, such as the regulation of cell differentiation, Tnfrsf9, Btnl2, Tnfrsf9, Tnfrsf9, Tnfrsf9Btnl2, etc. These differentially expressed genes were significantly enriched in the Wnt signaling pathway, signaling signaling pathway, and NF- 魏 B signal pathway, NF-kappa B signaling pathway, etc., combined with the above analysis. We have screened eight different genes related to senescence, such as Aocl-CD74Cox6a2Cyp4f18, Enpp3Tnfrsf9, Wnt11Zfp365, and carried out real-time fluorescence quantitative analysis and verification. Compared with the results of RNA-seq, the difference trend of gene expression obtained by the two methods is consistent, which proves the reliability of the results of RNA-seq. It was also found that these eight genes were associated with inflammation, cancer, and immunity, and that these genes were significantly altered in the aging improvement group after oral administration of phycocyanin. We speculate that the changes of the expression of these genes may play a role in improving the function of aging ovaries. In conclusion, we found that the transcriptional activity of the ovaries in aging mice changed greatly by RNA-seq analysis. These changes can be recovered in part by oral administration of phycocyanin, and the identified differentially expressed genes are involved in signaling pathways related to immunity, inflammation and aging. These results suggest that phycocyanin may play an important role in improving the ovarian function of aging mice through these signaling pathways and provide an effective target for the future study of ovarian function.
【学位授予单位】：内蒙古大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R711.75

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