本文選題：精原干細胞 + 性原細胞��； 參考：《西北農(nóng)林科技大學》2017年博士論文

【摘要】：具有干細胞特性的性原細胞及精原細胞統(tǒng)稱為雄性生殖干細胞,這類干細胞可以自我更新維持干細胞庫,也可以分化產(chǎn)生精子。精原干細胞(spermatogonial stem cells,SSCs)是唯一可以將親本遺傳信息傳遞給后代的成體干細胞。SSCs可以分化為精原細胞并起始精子發(fā)生,這個過程需要嚴密的基因表達調控�；虻谋磉_調控受到組蛋白修飾的影響。組蛋白H3和H4的賴氨酸殘基上可以發(fā)生三種甲基化修飾,包括一、二和三甲基化。通常H3K4和H3K36的甲基化與基因的轉錄激活相關,而H3K9,H3K27和H4K20的甲基化與轉錄抑制相關。這些組蛋白的甲基化修飾受到甲基轉移酶或去甲基化酶的催化。組蛋白甲基轉移酶SETDB1(SET domain,bifurcated 1),也稱為ESET,可通過H3K9me2/3調控異染色質的形成,抑制基因表達。SETDB1對于胚胎干細胞的維持、神經(jīng)細胞的存活具有重要作用。本研究利用蛋白質免疫共沉淀、染色質免疫共沉淀、組織免疫熒光等技術研究了SETDB1調控小鼠精原干細胞存活的分子機制,以及SETDB1對豬性原細胞存活的調控作用。主要結果如下:(1)Setdb1敲低可引起C18-4細胞線粒體膜電位的下降,細胞膜磷脂酰絲氨酸的外翻及凋亡晚期DNA的斷裂,且Setdb1-KD誘導了促凋亡基因Bax、Apaf1、p53、Caspase9表達的上調,抑凋亡基因XIAP表達下調。說明Setdb1干擾可以通過調節(jié)凋亡相關基因的表達調控細胞凋亡。(2)Setdb1-KD可激活PTEN,進而抑制AKT及FOXO1的磷酸化。Setdb1及Pten的雙敲低可挽救細胞凋亡的發(fā)生,且反轉了由于Setdb1-KD誘導的凋亡及通路相關蛋白的表達變化。說明了PTEN/AKT/FOXO1通路參與了Setdb1-KD誘導的小鼠精原干細胞凋亡。(3)SETDB1可與AKT相互作用,且SETDB1可以增強AKT對下游靶蛋白FOXO1的調控,抑制FOXO1的入核,從而抑制其自身啟動子的轉錄活性。Setdb1-KD可誘導FOXO1進核,并激活促凋亡基因Bim和Puma的表達。說明SETDB1可以協(xié)同AKT調控FOXO1活性并影響下游靶基因的表達。(4)H3K9me3-ChIP結果顯示,Setdb1-KD導致了基因Pten、Foxo3和Bim啟動子區(qū)域H3K9me3水平的降低,而Foxo1、Puma、Bax和Bcl2啟動子區(qū)域的H3K9me3無顯著變化。另外,SETDB1-ChIP顯示,SETDB1可以直接結合到Pten、Bim、Puma和Bax基因啟動子區(qū)域。結果說明,SETDB1只負責部分基因啟動子上H3K9me3修飾,且SETDB1可以通過直接作用于Pten及Bim啟動子區(qū)域催化H3K9me3修飾,從而調控小鼠精原干細胞的存活。(5)在豬睪丸發(fā)育過程中,SETDB1的表達量隨著豬的發(fā)育逐漸升高,而H3K9me3的表達水平在出生7天豬睪丸中表達量最高。在7天和兩月齡豬的睪丸組織中,SETDB1在性原細胞和精原干細胞中呈胞質分布,而H3K9me3呈核周分布。在成年豬睪丸組織中,SETDB1定位于精原干細胞和分化的精原細胞的細胞核,而H3K9me3在精原干細胞中呈核周分布,在分化的精原細胞中呈斑點狀分布。(6)為了研究SETDB1在豬性原細胞中的功能,通過差異貼壁富集性原細胞,富集后的性原細胞純度可達到76.5%±3.9%。通過Western及RT-PCR結果發(fā)現(xiàn),SETDB1主要表達于性原細胞。說明SETDB1在調控豬性原細胞的存活上可能發(fā)揮著重要功能。(7)SETDB1干擾引起豬性原細胞凋亡,且凋亡的發(fā)生不依賴于H3K9me3;另外,SETDB1可與催化H3K27甲基化的甲基轉移酶EZH2結合,且SETDB1敲低引起H3K27me3水平的降低,說明SETDB1干擾調控豬性原細胞的凋亡是通過H3K27me3水平調控的。綜上所述,通過PTEN/AKT/FOXO1通路及H3K9me3水平,Setdb1-KD誘導小鼠精原干細胞的凋亡;SETDB1協(xié)同AKT參與對下游靶蛋白FOXO1的活性調控。同時,SETDB1-KD也誘導豬性原細胞凋亡,但是H3K9me3水平并未改變,而與SETDB1結合的組蛋白甲基轉移酶EZH2催化的H3K27me3水平的降低有關。本研究揭示了表觀遺傳機制和凋亡信號通路之間的關系,并填補了表觀遺傳調控雄性生殖干細胞存活研究領域的空缺,為今后雄性不育的研究提供了新的思路。
[Abstract]:The stem cells and spermatogonial cells, which have the characteristics of stem cells, are called male reproductive stem cells. These types of stem cells can self renew and maintain a stem cell bank and can produce sperm. Spermatogonial stem cells (SSCs) is the only adult stem cell that can transmit the genetic information to the offspring,.SSCs can be differentiated into Spermatogonial cells and initiating spermatogenesis, this process requires strict regulation of gene expression. The regulation of gene expression is affected by histone modification. Three kinds of methylation modification can occur on the lysine residues of histone H3 and H4, including one, two and trimethylation. The methylation of H3K4 and H3K36 is usually associated with gene transcription activation, and H3K 9, the methylation of H3K27 and H4K20 is related to transcriptional inhibition. Methylation modification of these histones is catalyzed by methyltransferase or demethylation. Histone methyltransferase SETDB1 (SET domain, bifurcated 1), also known as ESET, can regulate the formation of heterochromatin through H3K9me2/3 and inhibit the gene expression of.SETDB1 for embryonic stem cells. In this study, the molecular mechanism of SETDB1 regulating the survival of mouse spermatogonial stem cells and the regulation of SETDB1 on the survival of porcine spermatogonial cells were studied by protein immunoprecipitation, chromatin immunofluorescence, and tissue immunofluorescence. The main results are as follows: (1) C18- in Setdb1 can cause C18- The decrease of mitochondrial membrane potential in 4 cells, ectropion of phosphatidylserine in cell membrane and rupture of late apoptosis DNA, and Setdb1-KD induced up regulation of apoptosis gene Bax, Apaf1, p53, Caspase9 expression and down regulation of apoptosis gene XIAP expression. It shows that Setdb1 interference can regulate apoptosis by regulating the expression of apoptosis related genes. (2) Setdb1-KD Activation of PTEN, and then inhibition of AKT and FOXO1 phosphorylation.Setdb1 and Pten double knock low can save the occurrence of apoptosis, and reverse the Setdb1-KD induced apoptosis and the expression of pathway related proteins. It shows that PTEN/AKT/FOXO1 pathway participates in Setdb1-KD induced mouse spermatogonial stem cell apoptosis. (3) SETDB1 can interact with AKT And SETDB1 can enhance the regulation of AKT to the downstream target protein FOXO1, inhibit the nucleation of FOXO1, inhibit the transcriptional activity.Setdb1-KD of its own promoter, induce FOXO1 entry and activate the expression of Bim and Puma of the apoptotic gene. It shows that SETDB1 can regulate FOXO1 activity with AKT and influence the expression of the downstream target gene. (4) H3K9me3-ChIP results Setdb1-KD resulted in a decrease in the H3K9me3 level in the promoter region of the gene Pten, Foxo3 and Bim, while H3K9me3 in Foxo1, Puma, Bax and Bcl2 promoter region did not change significantly. Me3 modification, and SETDB1 can regulate the survival of mouse spermatogonial stem cells by catalyzing H3K9me3 modification directly in Pten and Bim promoter regions. (5) during the development of pig testicles, the expression of SETDB1 increased gradually with the development of pigs, while the expression level of H3K9me3 was highest in the 7 days of pig testicles at birth. In 7 days and 2 month old, the expression level of H3K9me3 was the highest. In the testis of pigs, SETDB1 is distributed in the cytoplasm of the proogenic and spermatogonial stem cells, and the H3K9me3 is perinuclear. In adult pig testis, SETDB1 is located in the nucleus of the spermatogonial stem cells and the differentiated spermatogonial cells, while H3K9me3 is distributed in the spermatogonial stem cells and is distributed in the differentiated spermatogonial cells. (6) In order to study the function of SETDB1 in porcine proto cells, the purity of the enriched primary cells can reach 76.5% + 3.9%. through Western and RT-PCR through differentially adhered preconcentration cells. SETDB1 is mainly expressed in the primary cells. It shows that SETDB1 may play an important role in regulating the survival of porcine primitive cells. (7) SETDB1 interference. It causes the apoptosis of porcine procells, and the occurrence of apoptosis is not dependent on H3K9me3; in addition, SETDB1 can be combined with the methyltransferase EZH2 that catalyzes H3K27 methylation, and the SETDB1 knockdown causes the decrease of H3K27me3 level. It indicates that the apoptosis of the porcine primary cells by SETDB1 interference is regulated by the level of H3K27me3. In summary, the PTEN/AKT/FOXO1 pathway is described. And H3K9me3 level, Setdb1-KD induced the apoptosis of mouse spermatogonial stem cells; SETDB1 coordinated AKT to regulate the activity of FOXO1 in the downstream target protein. At the same time, SETDB1-KD also induced the apoptosis of porcine proogenic cells, but the H3K9me3 level did not change, but the decrease of H3K27me3 level catalyzed by the histone methyltransferase, which was combined with SETDB1, was related to the decrease of the level of H3K27me3. It reveals the relationship between epigenetic and apoptotic signaling pathways, and fills the vacancy in the study of epigenetic regulation of male reproductive stem cell survival, and provides a new way of thinking for the future research of male infertility.
【學位授予單位】：西北農(nóng)林科技大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：Q23

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