本文選題：男性不育 + 血睪屏障　； 參考：《中國(guó)科學(xué)技術(shù)大學(xué)》2014年博士論文

【摘要】：在世界范圍內(nèi)大約有10%-15%的育齡夫婦正在遭受不育癥的折磨。不育癥夫婦中40%的男性是不育的,并且隨著環(huán)境和社會(huì)等因素的影響,男性不育的比例正在逐漸增加。男性不育患者大部分為非梗阻性無(wú)精癥(non-obstructive azoospermia, NOA),其病因不明,對(duì)臨床治療極為不利。隨著對(duì)非梗阻性無(wú)精癥的深入研究,發(fā)現(xiàn)男性不育與血睪屏障(Blood-testis barrier, BTB)的功能異常有關(guān),然而導(dǎo)致血睪屏障功能異常的分子機(jī)制仍有待闡明。 位于生精上皮底部的支持細(xì)胞(Sertoli cell)通過(guò)彼此間的緊密連接構(gòu)建血睪屏障,為精子發(fā)生提供一個(gè)穩(wěn)定的微環(huán)境和獨(dú)特的免疫屏障,并通過(guò)有序的開(kāi)放調(diào)節(jié)精子生成。血睪屏障開(kāi)放機(jī)制的異常使得精子發(fā)生的微環(huán)境和免疫屏障受損,進(jìn)而影響精子生成,導(dǎo)致男性不育。血睪屏障的開(kāi)放機(jī)制受到眾多因子的調(diào)節(jié),而TGF-β3是調(diào)節(jié)血睪屏障開(kāi)放的主要因子之一,因此研究影響TGF-β3的表達(dá)和分泌的因素具有重要意義,這也是本文的第一個(gè)研究課題。 NXF3屬于核輸出因子蛋白家族(nuclear RNA export factor family, NXF),本文研究發(fā)現(xiàn)NXF3在小鼠睪丸的支持細(xì)胞中特異性地表達(dá),并且在附睪頭部、區(qū)的主細(xì)胞中也檢測(cè)到NXF3的表達(dá)。在支持細(xì)胞中首次檢測(cè)到NXF3的表達(dá)是小鼠出生后10天,而此時(shí)正是小鼠血睪屏障形成之時(shí),因此NXF3極有可能與血睪屏障相關(guān),這引起我們的極大興趣。由于TGF-β3是參與血睪屏障調(diào)節(jié)的主要因子之一,我們檢測(cè)了NXF3和TGF-β3在睪丸中的表達(dá),發(fā)現(xiàn)兩者之間具有負(fù)相關(guān)的關(guān)系。進(jìn)一步的實(shí)驗(yàn)也證實(shí)了這一關(guān)系：在用熱或CdC12處理小鼠睪丸后發(fā)現(xiàn),NXF3的表達(dá)下降,而TGF-β3的表達(dá)上升了。隨著研究的深入,發(fā)現(xiàn)NXF3參與調(diào)節(jié)TGF-β3轉(zhuǎn)錄負(fù)反饋的調(diào)控進(jìn)而影響了TGF-β3的表達(dá),即TGF-β信號(hào)通路激活后,NXF3增強(qiáng)了Smad2/3途徑的活性,從而使TGF-P3的轉(zhuǎn)錄受到抑制。通過(guò)更詳細(xì)的研究我們發(fā)現(xiàn)了NXF3的結(jié)合蛋白：STRAP, TGF-β信號(hào)通路的抑制因子。因此NXF3調(diào)節(jié)TGF-p3的機(jī)制得以闡明：TGF-β信號(hào)通路激活后,NXF3與STRAP結(jié)合,抑制了STRAP與Smad7的結(jié)合,影響了TpR1-STRAP-Smad7復(fù)合體的形成,使得Smad2/3途徑激活從而抑制了TGF-β3的轉(zhuǎn)錄,并且Smad2/3途徑的下游靶基因Claudin11、WT1、GATA1和p21也受到調(diào)控. 擬染色小體(chromatoid body)是雄性圓形精子時(shí)期出現(xiàn)的一個(gè)特異性結(jié)構(gòu),在電子顯微鏡下呈纖維狀結(jié)構(gòu),主要由蛋白質(zhì)和RNA組成,不含DNA。由于其含有許多RNA結(jié)合蛋白、mRNA和nicroRNA,擬染色小體被認(rèn)為是精子發(fā)生過(guò)程中的RNA存儲(chǔ)和加工中心,參與精子發(fā)生過(guò)程中的基因表達(dá)調(diào)控,因此非常引人關(guān)注。距1891年擬染色小體首次被報(bào)道,至今已有一百多年,有關(guān)擬染色小體的研究取得了很大的進(jìn)展,許多擬染色小體的蛋白和RNA組分被發(fā)現(xiàn),但是擬染色小體的功能和機(jī)制仍然不清楚,需要進(jìn)一步的探究。 本文發(fā)現(xiàn)CaMKIV (Ca2+/Calmodulin-dependent Protein Kinase IV, CaMKIV)定位在擬染色小體上,是擬染色小體的一個(gè)新的組分。CaMKIV是鈣調(diào)蛋白激酶家族之一,據(jù)報(bào)道CaMKIV表達(dá)在睪丸的精原細(xì)胞和精子細(xì)胞中,并且camkiv基因敲除小鼠由于在精子變形過(guò)程中組蛋白替換異常而不育。研究發(fā)現(xiàn)CaMKIV能夠和擬染色小體中的MVH.MIWI和KIF17b相互作用,并且能夠促進(jìn)MVH、MIWI與KIF17b的結(jié)合。這是首次報(bào)道擬染色小體中蛋白之間的相互作用的調(diào)控模式,對(duì)理解擬染色小體的結(jié)構(gòu)和功能具有重要意義。 綜上所述,本文報(bào)道了NXF3調(diào)控細(xì)胞因子TGF-03在睪丸支持細(xì)胞中的表達(dá)、分泌的機(jī)制,有助于分析非梗阻性無(wú)精癥患者中血睪屏障異常的原因。同時(shí)本文報(bào)道了CaMKIV作為擬染色小體的一個(gè)新的組分,能夠調(diào)節(jié)擬染色小體中的蛋白質(zhì)之間的相互作用,有助于擬染色小體的進(jìn)一步研究。
[Abstract]:Around the world, about 10%-15% of child-bearing age couples are suffering from infertility. 40% of male infertility couples are infertile and the proportion of male infertility is increasing with environmental and social factors. Most male infertility patients are non obstructive azoospermia (non-obstructive azoospermia, NOA). The cause of unknown etiology is extremely adverse to clinical treatment. With the in-depth study of non obstructive azoosinoses, it is found that male infertility is associated with the dysfunction of the Blood-testis barrier (BTB). However, the molecular mechanism that causes the dysfunction of the blood testis barrier remains to be elucidated.
The support cells (Sertoli cell) located at the bottom of the spermatogenic epithelium (Sertoli) construct the blood testis barrier through close connections between each other, providing a stable microenvironment and unique immune barrier for spermatogenesis, and through orderly and open regulation of spermatogenesis. The heterogeneity of the opening mechanism of the blood testis barrier causes the microenvironment and immune barrier of sperm to be damaged. The mechanism of the opening of the blood testis barrier is regulated by many factors, and TGF- beta 3 is one of the main factors regulating the opening of the blood testis barrier. Therefore, it is of great significance to study the factors affecting the expression and secretion of TGF- beta 3, which is the first research topic in this article.
NXF3 belongs to the nuclear RNA export factor family (NXF) family (export factor family, NXF). This study found that NXF3 was expressed specifically in the mouse testis supporting cells, and the expression of NXF3 was detected in the main cells of the epididymis and the main cells of the region. The expression of NXF3 was first detected in the support cells for 10 days after the birth of mice. It is when the mouse blood testis barrier is formed that NXF3 is highly likely to be associated with the blood testis barrier, which is of great interest. Since TGF- beta 3 is one of the major factors involved in the regulation of the blood testis barrier, we detected the expression of NXF3 and TGF- beta 3 in the testis, and found a negative correlation between the two. Further experiments confirmed this One relationship: after the treatment of mouse testicles with heat or CdC12, the expression of NXF3 decreased and the expression of TGF- beta 3 increased. With the further study, it was found that NXF3 was involved in the regulation of the negative feedback of TGF- beta 3 transcription and then influenced the expression of TGF- beta 3, that is, NXF3 enhanced the activity of Smad2/3 pathway, thus enabling TGF-P3. Transcriptional inhibition. Through more detailed studies we found the binding protein of NXF3: STRAP, the inhibitory factor of the TGF- beta signaling pathway. Therefore, the mechanism of NXF3 regulating TGF-p3 is elucidated: the binding of NXF3 to STRAP by the activation of the TGF- beta signaling pathway inhibits the combination of STRAP and Smad7, affecting the formation of the TpR1-STRAP-Smad7 complex and making Sma The activation of d2/3 pathway inhibits transcription of TGF- beta 3, and the downstream target genes Claudin11, WT1, GATA1 and p21 of Smad2/3 pathway are also regulated.
The pseudo coloring body (chromatoid body) is a specific structure in the male round sperm period. It is fibrous structure under the electron microscope, mainly composed of protein and RNA, without DNA. because it contains many RNA binding proteins, mRNA and nicroRNA. The quasi dyed corpuscle is considered to be the RNA storage and processing center in the process of spermatogenesis. It is very concerned about the regulation of gene expression in the process of spermatogenesis. It is first reported in 1891. It has been more than 100 years ago. So far, great progress has been made in the study of quasi dyed corpuscles. Many proteins and RNA components of pseudo dyed bodies have been found, but the function and mechanism of the quasi dyed corpuscles are still unclear. Chu, need further inquiry.
This article found that CaMKIV (Ca2+/Calmodulin-dependent Protein Kinase IV, CaMKIV) is located on the pseudo dyed corpuscle. A new component.CaMKIV of the quasi dyed body is one of the calmodulin kinase family. It is reported that CaMKIV is expressed in spermatogonia and spermatogonial cells of the testis, and the CaMKIV knockout mice are deformed in sperm deformation. It is found that CaMKIV can interact with MVH.MIWI and KIF17b in the pseudo dyed corpuscle and can promote the combination of MVH, MIWI and KIF17b. This is the first report on the interaction between proteins in the pseudo dyed bodies, which is important to understand the structure and function of the quasi dyed corpuscle.
To sum up, this paper reports the expression of NXF3 regulating cytokine TGF-03 in the testis support cells and the mechanism of secretion. It is helpful to analyze the cause of abnormal blood testis barrier in non obstructive azoospermia patients. This article reports that CaMKIV is a new component of the pseudo dyed corpuscle, which can regulate the protein between the pseudo dyed corpuscles. The interaction helps to further study the quasi staining bodies.
【學(xué)位授予單位】：中國(guó)科學(xué)技術(shù)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R698.2

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