【摘要】：精原干細(xì)胞(SSCs)的分化與自我更新決定了精子發(fā)生的起始,SSCs的增殖是維持大量精子產(chǎn)生的基礎(chǔ)。SSCs的分化與自我更新是一個(gè)受到嚴(yán)密調(diào)控的過(guò)程,研究參與SSCs自我更新與增殖過(guò)程中的因子對(duì)研究精子發(fā)生具有重大的意義。近期研究表明早幼粒白血病鋅指蛋白(PLZF),趨化因子受體4(CXCR4)以及miR-146a與SSCs的自我更新與增殖相關(guān)。本實(shí)驗(yàn)以PLZF-miR-146a-CXCR4為研究主線,以分離純化的奶山羊精原干細(xì)胞(gSSCs)為實(shí)驗(yàn)材料,通過(guò)Western Blot,免疫熒光染色,qRT-PCR,單熒光素酶載體報(bào)告系統(tǒng),雙熒光素酶載體報(bào)告系統(tǒng)以及流式細(xì)胞術(shù)研究三者之間的互相作用關(guān)系以及對(duì)gSSCs增殖的調(diào)控作用。研究發(fā)現(xiàn)PLZF通過(guò)抑制miR-146a的轉(zhuǎn)錄來(lái)促進(jìn)CXCR4的表達(dá)致使ERK1/2的磷酸化增強(qiáng),最終促進(jìn)gSSCs的自我更新和增殖。1.PLZF,miR-146a和CXCR4在雄性奶山羊睪丸中表達(dá)規(guī)律的研究通過(guò)qRT-PCR技術(shù)對(duì)3月齡,6月齡,9月齡,12月齡以及18月齡奶山羊睪丸進(jìn)行了轉(zhuǎn)錄水平的檢測(cè),發(fā)現(xiàn)PLZF與CXCR4的轉(zhuǎn)錄趨勢(shì)基本一致,但與miR-146a的轉(zhuǎn)錄趨勢(shì)相反。通過(guò)免疫熒光染色技術(shù)對(duì)PLZF和CXCR4的定位以及CXCR4在3月齡,6月齡,9月齡和18月齡的睪丸組織的表達(dá)進(jìn)行了研究,結(jié)果表明PLZF可與CXCR4共同定位于精原干細(xì)胞,CXCR4的蛋白檢測(cè)與mRNA檢測(cè)結(jié)果相近。2.PLZF,miR-146a和CXCR4之間相互作用關(guān)系的研究在gSSCs中超表達(dá)和干擾PLZF,通過(guò)qRT-PCR和Western Blot檢測(cè)其對(duì)mi R-146a和CXCR4轉(zhuǎn)錄水平和蛋白水平的調(diào)控。發(fā)現(xiàn)PLZF的超表達(dá)可以促進(jìn)CXCR4的表達(dá),同時(shí)抑制miR-146a的轉(zhuǎn)錄,抑制PLZF的表達(dá)則會(huì)出現(xiàn)相反的結(jié)果。隨后,通過(guò)單熒光素酶載體報(bào)告系統(tǒng)在293T細(xì)胞中檢測(cè)PLZF與miR-146a啟動(dòng)子區(qū)域的互作關(guān)系,證明PLZF靶向miR-146a的啟動(dòng)子區(qū)抑制其轉(zhuǎn)錄。在gSSCs中轉(zhuǎn)染mi R-146a模擬物、抑制物及無(wú)義物,通過(guò)qRT-PCR和Western Blot檢測(cè)其CXCR4的表達(dá)變化,發(fā)現(xiàn)miR-146a會(huì)抑制CXCR4的表達(dá),之后通過(guò)雙熒光素酶載體報(bào)告系統(tǒng)檢測(cè)miR-146a是否靶向調(diào)控CXCR4,結(jié)果證實(shí)miR-146a間接調(diào)控CXCR4的表達(dá)。3.PLZF,miR-146a和CXCR4對(duì)gSSCs自我更新和增殖的影響通過(guò)Western Blot檢測(cè)PLZF,miR-146a和CXCR4對(duì)ERK1/2磷酸化水平的影響,結(jié)果表明PLZF和CXCR4表達(dá)量上升會(huì)促進(jìn)ERK1/2的磷酸化;而降低PLZF的表達(dá),則會(huì)降低ERK1/2的磷酸化。通過(guò)流式細(xì)胞周期檢測(cè)技術(shù)研究對(duì)gSSCs增殖的影響,發(fā)現(xiàn)PLZF,CXCR4的表達(dá)量上升會(huì)促進(jìn)gSSCs增殖。
[Abstract]:The differentiation and self-renewal of spermatogonial stem cells (SSCs) determine the initiation of spermatogenesis and the proliferation of SSCs is the basis of maintaining a large number of spermatozoa. The differentiation and self-renewal of SSCs is a tightly regulated process. The study of the factors involved in the process of self-renewal and proliferation of SSCs is of great significance to the study of spermatogenesis. Recent studies have shown that zinc finger protein (PLZF), chemokine receptor 4 (CXCR4) and miR-146a are associated with self-renewal and proliferation of SSCs in promyelocytic leukemia. In this experiment, PLZF-miR-146a-CXCR4 was used as the main line of study, and the purified dairy goat spermatogonial stem cells (gSSCs) were used as experimental materials. Western blot, immunofluorescence staining, qRT-PCRand single luciferase carrier report system were used. Double luciferase vector reporting system and flow cytometry were used to study the interaction and regulation of GSSCs proliferation. It was found that PLZF promoted the expression of CXCR4 by inhibiting the transcription of miR-146a, resulting in increased phosphorylation of ERK1 / 2. Finally promote the self-renewal and proliferation of gSSCs. 1. The expression of PLZFN miR-146a and CXCR4 in the testis of male dairy goats was studied. The transcription level of testis was detected by qRT-PCR at the age of 3 months, 6 months, 9 months, 12 months, and 18 months, respectively. It was found that the transcription trend of PLZF and CXCR4 was basically the same, but contrary to that of miR-146a. The localization of PLZF and CXCR4 and the expression of CXCR4 in testis at the age of 3 months, 6 months, 9 months and 18 months were studied by immunofluorescence staining. The results showed that PLZF could co-locate with CXCR4 in spermatogonial stem cell line CXCR4. 2. The interaction between PLZF and CXCR4 was studied in gSSCs by overexpression and interference with PLZF4. The results of qRT-PCR and Western blot were used to detect the expression of MIR-146a and CXCR4 in gSSCs. The regulation of transcription and protein levels. It was found that the overexpression of PLZF could promote the expression of CXCR4, inhibit the transcription of miR-146a, and inhibit the expression of PLZF. Then, the interaction between PLZF and miR-146a promoter region was detected in 293T cells by a single luciferase vector reporting system, which proved that PLZF-targeted promoter region of miR-146a inhibited its transcription. The expression of CXCR4 was detected by qRT-PCR and Western Blot. It was found that miR-146a could inhibit the expression of CXCR4. The effect of miR-146a on the expression of CXCR4. 3. The effects of miR-146a and CXCR4 on the self-renewal and proliferation of gSSCs were detected by Western Blot. The results showed that the increased expression of PLZF and CXCR4 increased the phosphorylation of ERK1 / 2, but decreased the expression of PLZF, decreased the phosphorylation of ERK1 / 2. The effect of flow cytometry on the proliferation of gSSCs was studied. It was found that the increased expression of PLZFG CXCR4 could promote the proliferation of gSSCs.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S814

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 李冬梅;秦曉東;;精原干細(xì)胞自我更新與分化的調(diào)控[J];中華男科學(xué)雜志;2013年11期

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本文編號(hào)：2134185