【摘要】：雄性生殖干細(xì)胞(male Germline Stem Cells,mGSCs)是定位于雄性動物睪丸中的曲細(xì)精管基底膜上的成體干細(xì)胞。mGSCs不僅能夠通過自我更新來維持自身群體數(shù)量的穩(wěn)定,還可以通過定向分化產(chǎn)生精子,是唯一一種能夠通過自然生殖過程將雄性動物的遺傳信息傳遞到下一代的成體干細(xì)胞。PLZF是一種高度保守的多功能轉(zhuǎn)錄調(diào)控因子,其表達(dá)具有嚴(yán)格的組織特異性和時(shí)序特異性,參與機(jī)體的發(fā)育、分化等多種重要的生物學(xué)過程。近年來,PLZF作為非常重要的轉(zhuǎn)錄因子,已引起人們的廣泛關(guān)注。PLZF表達(dá)于未分化的mGSCs,對其多能性的維持、自我更新和分化的平衡以及雄性個(gè)體的生育能力維持等具有重要的調(diào)控作用,是被廣泛認(rèn)可的mGSCs的標(biāo)志基因。目前,雖然對PLZF調(diào)控mGSCs自我更新和分化的作用及作用機(jī)制的探索已經(jīng)取得了一些進(jìn)展,但這些研究都是在小鼠等模式動物中取得的。PLZF對山羊等家畜大動物mGSCs的作用及作用機(jī)制卻知之甚少。本研究擬在已有基礎(chǔ)上,探索PLZF對奶山羊mGSCs生物學(xué)特性的的影響,以進(jìn)一步了解其對精子發(fā)生過程的調(diào)節(jié)機(jī)制。本實(shí)驗(yàn)通過構(gòu)建PLZF超表達(dá)和干擾慢病毒載體,轉(zhuǎn)導(dǎo)奶山羊mGSCs,并篩選得到了穩(wěn)定超表達(dá)和干擾PLZF的奶山羊mGSCs細(xì)胞株。并通過免疫熒光染色,qPCR,Western blot,EdU染色,流式細(xì)胞檢測等技術(shù)手段檢測了PLZF對奶山羊mGSCs多能性、自我更新、增殖、分化和凋亡等生物學(xué)特性的影響。1.PLZF超表達(dá)載體及干擾載體的構(gòu)建和穩(wěn)轉(zhuǎn)細(xì)胞株的篩選我們構(gòu)建得到了PLZF慢病毒超表達(dá)載體pPLZF-CDH和PLZF慢病毒干擾載體pshPLZF。然后將慢病毒載體包裝病毒并轉(zhuǎn)導(dǎo)奶山羊mGSC,通過篩選、鑒定得到了超表達(dá)PLZF的穩(wěn)轉(zhuǎn)細(xì)胞株mGSCs-oePLZF及其對照細(xì)胞株mGSCs-pCDH,干擾PLZF的穩(wěn)轉(zhuǎn)細(xì)胞株mGSCs-pshPLZF及其對照細(xì)胞株mGSCs-pSIH。2.PLZF促進(jìn)奶山羊mGSCs維持其自我更新和多能性并抑制其分化通過免疫熒光染色、qPCR和Western blot檢測,研究發(fā)現(xiàn)GFRα1、Vasa、CD49f、CD90、Oct4、Etv5等與mGSCs自我更新和多能性相關(guān)的基因表達(dá)顯著上調(diào),而mGSCs的分化標(biāo)志基因c-Kit表達(dá)水平顯著下調(diào)。另外我們通過qPCR檢測發(fā)現(xiàn)Tet1表達(dá)水平顯著下調(diào),同時(shí)Dnmt3a表達(dá)水平顯著上調(diào),說明PLZF可能促進(jìn)奶山羊mGSCs的DNA甲基化修飾。3.PLZF抑制奶山羊mGSCs增殖,促進(jìn)其凋亡我們通過測定細(xì)胞生長曲線、CCK8檢測、EdU染色、流式細(xì)胞周期檢測、免疫熒光染色、qPCR、Western blot等實(shí)驗(yàn)方法,發(fā)現(xiàn)超表達(dá)PLZF的奶山羊mGSCs細(xì)胞增殖速度變慢、細(xì)胞周期在G0/G1期發(fā)生阻滯,細(xì)胞凋亡相關(guān)基因表達(dá)上調(diào)。通過細(xì)胞移植實(shí)驗(yàn)發(fā)現(xiàn),移植PLZF超表達(dá)穩(wěn)轉(zhuǎn)細(xì)胞的白消安模型小鼠睪丸要明顯比對側(cè)移植對照細(xì)胞的睪丸小,且曲細(xì)精管內(nèi)形成的細(xì)胞克隆也明顯比對照組差;相應(yīng)的,移植PLZF干擾穩(wěn)轉(zhuǎn)細(xì)胞的睪丸明顯比對照組大,曲細(xì)精管內(nèi)形成的細(xì)胞克隆也比對照組好。
[Abstract]:Male reproductive stem cells (male Germline Stem Cells,mGSCs) are adult stem cells located on the basal membrane of the seminiferous tubules located in the testicles of male animals. MGSCs can not only maintain the stability of its population through self-renewal. It is the only adult stem cell that can transmit genetic information from male animals to the next generation through natural reproduction. PLZF is a highly conserved multifunctional transcriptional regulator. Its expression has strict tissue specificity and timing specificity, and participates in many important biological processes, such as development and differentiation. In recent years, PLZF, as a very important transcription factor, has attracted much attention. PLZF is expressed in undifferentiated mGSCs, to maintain its pluripotency. The balance of self-renewal and differentiation and the maintenance of male fertility play an important role in the regulation of mGSCs, which is widely recognized as a marker gene. At present, although some progress has been made in exploring the role and mechanism of PLZF in regulating the self-renewal and differentiation of mGSCs, However, these studies were obtained in mice and other model animals. The effect and mechanism of PLZF on mGSCs in large domestic animals such as goats are not well understood. This study is to explore the effect of PLZF on the biological characteristics of mGSCs in dairy goats on the basis of previous studies in order to further understand its regulatory mechanism on spermatogenesis. In this study, we constructed PLZF overexpression vector and interfered lentivirus vector, then transduced mGSCs, into dairy goat mGSCs cell line which was stable and interfered with PLZF. Immunofluorescence staining, qPCR,Western blot,EdU staining and flow cytometry were used to detect the pluripotency, self-renewal and proliferation of PLZF to dairy goat mGSCs. Effects of biological characteristics such as differentiation and apoptosis. Construction of 1.PLZF superexpression vector and interference vector and screening of stable cell line we constructed PLZF lentivirus superexpression vector pPLZF-CDH and PLZF lentivirus interference vector pshPLZF. Then the lentivirus vector was packaged and transduced into dairy goat mGSC,. The stable cell line mGSCs-oePLZF and the control cell line mGSCs-pCDH, were identified by screening. The stable transformed cell line mGSCs-pshPLZF which interferes with PLZF and its control cell line mGSCs-pSIH.2.PLZF promote the maintenance of self-renewal and pluripotency and inhibit the differentiation of dairy goat mGSCs by immunofluorescence staining, qPCR and Western blot detection. Oct4,Etv5 and other genes related to mGSCs self-renewal and pluripotency were significantly up-regulated, while mGSCs differentiation marker gene c-Kit expression was significantly down-regulated. In addition, we found that the expression of Tet1 was significantly down-regulated and the expression of Dnmt3a was up-regulated by qPCR assay, which suggested that PLZF might promote DNA methylation of mGSCs in dairy goats. 3.PLZF inhibited mGSCs proliferation of dairy goats. By measuring cell growth curve, CCK8 detection, EdU staining, flow cytometry, immunofluorescence staining, qPCR,Western blot and other experimental methods, we found that the proliferation rate of mGSCs cells with overexpression of PLZF became slower. The cell cycle was blocked at G0/G1 stage and the expression of apoptosis-related genes was up-regulated. The results of cell transplantation showed that the testis of the mice transplanted with stable PLZF cells were smaller than those of the contralateral control cells, and the cell clones formed in the seminiferous tubules were significantly worse than those in the control group. Accordingly, the testis of the transplanted PLZF interfering stable cells were significantly larger than that of the control group, and the cell clones formed in the seminiferous tubule were also better than those in the control group.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S827

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本文編號：2409937