本文選題：雄性不育 + FK506�。� 參考：《南昌大學(xué)》2017年碩士論文

【摘要】：研究背景與目的根據(jù)WHO的標(biāo)準(zhǔn)定義,不育是指夫妻同居一年以上,在未采取主動(dòng)避孕而致女方不能懷孕的情況。近年來(lái),隨著工業(yè)化進(jìn)程的加速,人們的生活方式也在發(fā)生改變,如過(guò)重的心里壓力、不健康的飲食、惡化的環(huán)境等因素均可導(dǎo)致夫妻不孕不育,這一現(xiàn)象已引起社會(huì)的高度關(guān)注。目前,男性不育約占整個(gè)人類不孕不育的40%,但其發(fā)病原因及其機(jī)制尚不清楚。研究表明,FK506通過(guò)抑制鈣調(diào)磷酸酶(calcineurin)活性,可引起未成熟精子的中端僵化,從而導(dǎo)致雄性小鼠不育。FKBP12.6是FK506結(jié)合蛋白家族中的一個(gè)重要成員,研究表明,該蛋白可與FK506結(jié)合,即可通過(guò)直接抑制calcineurin活性發(fā)揮免疫抑制作用,同時(shí)又可通過(guò)調(diào)節(jié)鈣離子釋放通道(ryanodine receptor 2,RyR2)而調(diào)控calcineurin活性。因此,本課題借助FKBP12.6敲除小鼠,旨在研究FKBP12.6在FK506所致雄鼠不育過(guò)程中的作用及其機(jī)制,從而為治療男性不育提供新的靶點(diǎn)。方法與結(jié)果1.敲除FKBP12.6基因可明顯緩解FK506引起的成年雄性小鼠不育:為了確定FKBP12.6在FK506所致雄鼠不育中的作用,我們首先將成年野生型(WT)和FKBP12.6基因敲除(FKBP12.6KO)雄性小鼠皮下注射FK506,結(jié)果顯示,給予WT小鼠FK506后,精子發(fā)生中端僵化,受精比率急劇下降,而FKBP12.6KO鼠給藥后與WT相比精子中端僵化明顯減少,受精情況顯著改善。結(jié)果表明FKBP12.6缺失在FK506所致雄鼠不育過(guò)程中起重要保護(hù)作用。2.FKBP12.6在睪丸組織內(nèi)質(zhì)網(wǎng)高表達(dá):睪丸是精子發(fā)生的場(chǎng)所,為了明確FKBP12.6或FKBP12在雄性成年鼠睪丸中對(duì)精子發(fā)生發(fā)育的影響,我們分別提取雄性小鼠的睪丸總蛋白和微粒體(microsome,主要為內(nèi)質(zhì)網(wǎng)蛋白),并檢測(cè)其表達(dá)情況。Western blot檢測(cè),結(jié)果顯示:雖然FKBP12在睪丸中的表達(dá)較FKBP12.6更為豐富,但FKBP12.6在內(nèi)質(zhì)網(wǎng)上的相對(duì)表達(dá)量顯著高于FKBP12。表明FKBP12.6在睪丸鈣離子調(diào)控中可能起重要作用。3.FKBP12.6抑制睪丸特異性calcieneurin(PPP3CC/PPP3R2)活性:有研究表明,calcineurin的PPP3CC/PPP3R2亞基在睪丸組織特異性表達(dá)。為了進(jìn)一步明確FKBP12.6和FKBP12對(duì)睪丸calcineurin活性的影響,我們首先采用體外原核細(xì)胞系統(tǒng)表達(dá)和純化小鼠GST-FKBP12.6和GST-FKBP12融合蛋白,并通過(guò)親和層析實(shí)驗(yàn)(pull down)觀察FKBP12.6和FKBP12蛋白與睪丸組織裂解液或體外純化的PPP3CC/PPP3R2蛋白的特異性結(jié)合。結(jié)果顯示FKBP12.6與睪丸PPP3CC/PPP3R2蛋白的結(jié)合能力顯著高于FKBP12,提示FKBP12.6在FK506抑制睪丸calcineurin活性過(guò)程中可能起主要作用。結(jié)論:我們的研究表明,FKBP12.6缺失可明顯減輕給予FK506引起的雄性成年鼠不育,其機(jī)制可能與其在睪丸組織上表達(dá)缺失,解除了FK506對(duì)成年雄性鼠睪丸calcineurin活性的抑制,進(jìn)而促進(jìn)精子的正常發(fā)育有關(guān)。
[Abstract]:Background & objective according to the standard definition of WHO, infertility refers to couples cohabiting for more than one year and failing to conceive because they have not taken active contraception. In recent years, with the acceleration of industrialization, people's way of life is also changing, such as excessive psychological stress, unhealthy diet, deterioration of the environment and other factors can lead to infertility couples, This phenomenon has aroused the high attention of the society. At present, male sterility accounts for about 40% of infertility, but its pathogenesis and mechanism are not clear. It has been shown that FK506 can induce ossification of immature spermatozoa by inhibiting calcineurin activity, which leads to male sterility. FKBP12.6 is an important member of FK506 binding protein family. It has been shown that this protein can bind to FK506. The activity of calcineurin can be regulated by direct inhibition of calcineurin activity and regulation of ryanodine receptor 2 RyR2). Therefore, the purpose of this study was to study the role of FKBP12.6 in the process of male infertility induced by FK506 and its mechanism with the help of FKBP12.6 knockout mice, so as to provide a new target for the treatment of male sterility. Methods and results 1. Knockout of FKBP12.6 gene significantly alleviated male sterility induced by FK506 in adult male mice. In order to determine the role of FKBP12.6 in FK506 induced male sterility, we first injected FK506 subcutaneously into adult wild-type male mice and FKBP12.6 gene knockout FKBP12.6KOO male mice. After administration of FK506 in WT mice, the middle end of spermatogenesis became ossified, and the fertilization rate decreased sharply, while the ossification of sperm in FKBP12.6KO mice decreased significantly compared with WT, and the fertilization situation was improved significantly. The results show that FKBP12.6 deletion plays an important protective role in the male infertility induced by FK506. 2. FKBP12.6 is highly expressed in the endoplasmic reticulum of testis. Testis are the place where spermatogenesis occurs. In order to clarify the effect of FKBP12.6 or FKBP12 on spermatogenesis and development in the testis of adult male rats, FKBP12.6 was found in the testis of male mice. We extracted total testicular protein and microsomal microsome from male mice, mainly endoplasmic reticulum protein, and detected their expression. Western blot analysis showed that, although the expression of FKBP12 in testis was more abundant than that of FKBP12.6, However, the relative expression of FKBP12.6 in endoplasmic reticulum was significantly higher than that in FKBP12. It is suggested that FKBP12.6 may play an important role in the regulation of calcium ion in testis. 3. FKBP12.6 inhibits the activity of testicular specific calcieneurin PPP3CC / PPP3R2. Some studies have shown that the PPP3CC/PPP3R2 subunit of calcineurin is specifically expressed in testis. In order to further clarify the effect of FKBP12.6 and FKBP12 on testicular calcineurin activity, we first expressed and purified mouse GST-FKBP12.6 and GST-FKBP12 fusion protein by prokaryotic cell system in vitro. The specific binding of FKBP12.6 and FKBP12 proteins to testicular tissue lysate or purified PPP3CC/PPP3R2 protein in vitro was observed by affinity chromatography. The results showed that the binding ability of FKBP12.6 to testicular PPP3CC/PPP3R2 protein was significantly higher than that of FKBP12, suggesting that FKBP12.6 might play a major role in the inhibition of testicular calcineurin activity by FK506. Conclusion: our study shows that FKBP12.6 deletion can significantly alleviate the male infertility induced by FK506, and its mechanism may be similar to the loss of expression in testis, thus relieving the inhibition of calcineurin activity in testis of adult male rats by FK506. And then promote the normal development of sperm related.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R698.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 陸金春;黃宇烽;呂年青;;《世界衛(wèi)生組織人類精液分析實(shí)驗(yàn)室技術(shù)手冊(cè)》與我國(guó)男科實(shí)驗(yàn)室現(xiàn)狀[J];中華男科學(xué)雜志;2010年10期

，

本文編號(hào)：1938007