本文選題：TGF-β1　切入點(diǎn)：microRNA芯片分析　出處：《湖南大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：男性不育一直是個(gè)世界性醫(yī)學(xué)難題。男性不育與精子發(fā)生異常密切相關(guān)。TGF-β1(transforming growth factor β,TGF-β1)作為TGF-β超家族的主要成員之一,以其復(fù)雜的信號(hào)通路在睪丸發(fā)育不同階段扮演不同角色,參與調(diào)節(jié)精子發(fā)生。因此,尋找并確定生殖細(xì)胞發(fā)育過(guò)程中參與TGF-β1信號(hào)通路的關(guān)鍵分子將有助于更好地詮釋精子發(fā)生機(jī)制。基于此,本文以小鼠精原上皮細(xì)胞GC-1spg為實(shí)驗(yàn)材料,進(jìn)行外源TGF-β1刺激后,開(kāi)展了以下三個(gè)方面的工作： 通過(guò)高通量的microRNA(miRNA)芯片分析和定量PCR驗(yàn)證,篩選出24種受TGF-β1調(diào)節(jié)的miRNA分子,其中重點(diǎn)驗(yàn)證了10個(gè)表達(dá)豐度較高的差異miRNAs,并通過(guò)生物信息學(xué)分析和定量PCR技術(shù)初步驗(yàn)證了這些miRNAs的可能靶基因：miR-5112的可能靶基因?yàn)镠bp1, miR-199a-3p的可能靶基因?yàn)镃elsr2,miR-196a的可能靶基因?yàn)镹r6a1。 通過(guò)高通量的2-DE(two-dimensional gel electrophoresis,2-DE)電泳結(jié)合MALDI-TOF-TOF-MS電泳結(jié)合質(zhì)譜技術(shù),鑒定得到了8個(gè)可能受TGF-β1調(diào)控的差異蛋白：Gelsolin(Gsn),Vinculin(Vcl),Ezrin(Ezr), NDKB(Nme2),PPIA(Ppia),Cofilin-1(Cfl1),TERA(Vcp)和IF5A1(Eif5a)。這8個(gè)差異蛋白在TGF-β1的作用下表達(dá)下調(diào)。 通過(guò)GEO數(shù)據(jù)檢索等生物信息學(xué)方法分析發(fā)現(xiàn),上述預(yù)測(cè)的下游靶基因Nr6a1,Celsr2和Hbp1以及差異蛋白NDKB,COF1和PPIA等在畸形精子癥和精原細(xì)胞瘤等病人組織中存在差異表達(dá),提示它們可能在精子發(fā)生過(guò)程中扮演重要角色。 值得關(guān)注的是,通過(guò)整合miRNA芯片結(jié)果以及蛋白質(zhì)組學(xué)結(jié)果,同時(shí)結(jié)合生物信息學(xué)分析發(fā)現(xiàn),有些差異miRNA與差異蛋白在TGF-β1的作用下,它們的表達(dá)趨勢(shì)符合microRNA負(fù)性調(diào)控機(jī)制下的互反現(xiàn)象：如差異蛋白NDKB可能受miR-199a-3p調(diào)節(jié),而差異蛋白PPIA可能受miR-196a或miR-149調(diào)節(jié),從而響應(yīng)TGF-β信號(hào)通路。結(jié)果暗示我們,在GC-1spg細(xì)胞中,這3個(gè)miRNA可能分別抑制N DKB和PPIA等蛋白的表達(dá),從而參與精子發(fā)生過(guò)程。 綜上所述,我們?cè)贕C-1spg細(xì)胞系中篩選并重點(diǎn)驗(yàn)證了10個(gè)差異表達(dá)的miRNA和8個(gè)差異表達(dá)蛋白,并發(fā)現(xiàn)TGF-β1可能通過(guò)誘導(dǎo)miR-199a-3p,miR-196a和miR-149等的上調(diào)來(lái)抑制其各自的下游靶分子NDKB和PPIA等蛋白的表達(dá),從而參與精子發(fā)生
[Abstract]:Male infertility has always been a worldwide medical problem. Male infertility is closely related to abnormal spermatogenesis. As one of the main members of the TGF- 尾 superfamily, male infertility plays different roles in different stages of testicular development with its complex signaling pathway. Therefore, finding and identifying the key molecules involved in TGF- 尾 1 signaling pathway during germ cell development will contribute to a better interpretation of the mechanism of spermatogenesis. Therefore, GC-1spg of mouse spermatogonial epithelial cells is used as the experimental material. After exogenous TGF- 尾 1 stimulation, the following three aspects were carried out:. Twenty-four miRNA molecules regulated by TGF- 尾 1 were screened by high-throughput microRNA-miRNA-chip analysis and quantitative PCR verification. Among them, 10 differentially expressed miRNAss with high abundance were verified, and the possible target gene of these miRNAs genes: 1 miR-5112 was confirmed by bioinformatics analysis and quantitative PCR technique. The possible target gene of these miRNAs genes was Hbp1, and the possible target gene of miR-199a-3p was Celsr2miR-196a because of Nr6a1. By means of high throughput 2-DEG gel electrophoresistics and MALDI-TOF-TOF-MS electrophoretic mass spectrometry, eight differentially regulated differential proteins (TGF- 尾 1) were identified and down-regulated by TGF- 尾 1. The eight differential proteins were down-regulated by TGF- 尾 _ 1. These proteins were down-regulated by TGF- 尾 _ 1. By GEO data retrieval and other bioinformatics methods, it was found that the predicted downstream target genes Nr6a1, Celsr2 and Hbp1, and the differential protein NDKB#en0# COF1 and PPIA were differentially expressed in the tissues of patients with malformation spermatozoa and seminoma. It suggests that they may play an important role in spermatogenesis. It is worth noting that by integrating the results of miRNA microarray and proteomics, and combining with bioinformatics analysis, some differential miRNA and differential proteins have been found under the action of TGF- 尾 1. Their expression trend is consistent with the reciprocal phenomenon under the negative regulatory mechanism of microRNA: for example, differential protein NDKB may be regulated by miR-199a-3p, while differential protein PPIA may be regulated by miR-196a or miR-149, thus responding to the TGF- 尾 signaling pathway. The results suggest that in GC-1spg cells, These three miRNA may inhibit the expression of N DKB and PPIA proteins, and thus participate in spermatogenesis. In conclusion, we screened 10 differentially expressed miRNA and 8 differentially expressed proteins in GC-1spg cell lines. It was also found that TGF- 尾 1 may inhibit the expression of NDKB and PPIA proteins in their downstream target molecules by inducing the up-regulation of miR-199a-3pnmiR-196a and miR-149, thus participating in spermatogenesis.
【學(xué)位授予單位】：湖南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R698.2

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本文編號(hào)：1574915