發(fā)布時(shí)間：2018-04-03 01:13

  本文選題：水牛　切入點(diǎn)：睪丸曲精細(xì)管　出處：《廣西大學(xué)》2016年博士論文

【摘要】：睪丸內(nèi)曲精細(xì)管是精子發(fā)生的重要場所,曲精細(xì)管組織主要由支持細(xì)胞和各級(jí)生精細(xì)胞組成,支持細(xì)胞為生精細(xì)胞提供營養(yǎng)物質(zhì)并對(duì)精母細(xì)胞最終發(fā)育成完整形態(tài)的精子具有至關(guān)重要的作用。精子發(fā)生是一個(gè)冗長、復(fù)雜的過程,其參與了遺傳信息的傳遞,哺乳動(dòng)物精子發(fā)生主要由三部分組成：精母細(xì)胞發(fā)生、減數(shù)分裂及精子形成,這一增殖分化過程由各種各樣的分泌因子所調(diào)控并受制于生精細(xì)胞和支持細(xì)胞間大量基因和蛋白的相互調(diào)節(jié)作用。曲精細(xì)管中的多種蛋白質(zhì)在精子發(fā)生過程中發(fā)揮了重要作用,但究竟有哪些蛋白參與及其具體分子調(diào)節(jié)機(jī)制目前尚未清楚。水牛是廣西重要的經(jīng)濟(jì)物種,具有耐粗飼、飼料轉(zhuǎn)化率高、乳品營養(yǎng)價(jià)值高等特點(diǎn),但是其繁殖率低且整體研究水平不高,開展深入的基礎(chǔ)研究將有助于實(shí)現(xiàn)其更大的經(jīng)濟(jì)價(jià)值。本研究將首次利用差異蛋白質(zhì)組學(xué)的方法來比較不同時(shí)期(性成熟前期、性成熟期、性成熟后期)水牛睪丸曲精細(xì)管蛋白表達(dá)的差異,探索生精細(xì)胞和支持細(xì)胞在這三個(gè)不同發(fā)育時(shí)期蛋白質(zhì)組成、含量等方面的變化,找出與水牛精子發(fā)生過程及其雄性生殖生理相關(guān)的標(biāo)志性蛋白。主要采用雙向電泳技術(shù)和液相色譜分離兩大主流技術(shù)對(duì)不同時(shí)期水牛睪丸曲精細(xì)管差異蛋白進(jìn)行研究,根據(jù)研究方法的不同,主要分為以下兩章：第一章,采用雙向電泳技術(shù)分析不同時(shí)期水牛睪丸曲精細(xì)管差異蛋白質(zhì)組。主要包括兩個(gè)部分,首先是水牛睪丸曲精細(xì)管雙向電泳體系的建立和優(yōu)化。通過對(duì)不同IPG膠條(線性與非線性)及膠條pH值、蛋白質(zhì)上樣量這三個(gè)參數(shù)進(jìn)行比較和優(yōu)化,建立適合于水牛睪丸曲精細(xì)管蛋白質(zhì)分離的雙向電泳體系。結(jié)果顯示,采用上樣量350μg的曲精細(xì)管總蛋白、24 cm且pH 4-7的線性IPG膠條能夠更好的分離水牛睪丸曲精細(xì)管蛋白質(zhì),獲得質(zhì)量較好且分辨率較高的雙向電泳圖譜,通過對(duì)圖像的分析檢測,可得到大約486個(gè)蛋白點(diǎn)。其次,采用已優(yōu)化并建立的雙向電泳體系對(duì)不同時(shí)期(性成熟前期、性成熟期、性成熟后期)水牛睪丸曲精細(xì)管蛋白質(zhì)進(jìn)行分離,獲取差異蛋白質(zhì)并進(jìn)行質(zhì)譜鑒定。結(jié)果顯示,以性成熟期水牛睪丸曲精細(xì)管蛋白表達(dá)圖譜作為對(duì)照,篩選出性成熟前期與性成熟期水牛睪丸曲精細(xì)管差異蛋白150個(gè),性成熟期與性成熟后期水牛睪丸曲精細(xì)管差異蛋白92個(gè)。質(zhì)譜成功鑒定出性成熟前期與性成熟期水牛睪丸曲精細(xì)管差異蛋白點(diǎn)4個(gè),對(duì)應(yīng)4種蛋白質(zhì),在性成熟前期水牛睪丸曲精細(xì)管中均表現(xiàn)為下調(diào)；鑒定出性成熟期與性成熟后期水牛睪丸曲精細(xì)管差異蛋白點(diǎn)15個(gè)并對(duì)應(yīng)13種蛋白質(zhì),在性成熟后期水牛睪丸曲精細(xì)管中表達(dá)上調(diào)的有8個(gè),下調(diào)4個(gè),只在性成熟后期水牛睪丸曲精細(xì)管中表達(dá)的有1個(gè)。本章試驗(yàn)建立了良好的水牛睪丸曲精細(xì)管雙向電泳體系,分析并鑒定了一批水牛睪丸曲精細(xì)管差異蛋白質(zhì),并找出可能與水牛精子發(fā)生相關(guān)的差異蛋白質(zhì),如超氧化物歧化酶、熱休克蛋白HSPA2、波形蛋白、抗增殖蛋白、細(xì)胞角蛋白KRT8及半乳凝集素1等。第二章,利用TMT定量標(biāo)記結(jié)合二維液相色譜／質(zhì)譜(2DLC-MS/MS)技術(shù)分析不同時(shí)期水牛睪丸曲精細(xì)管差異蛋白質(zhì)組。主要包括三個(gè)部分,首先對(duì)不同時(shí)期(性成熟前期、性成熟期、性成熟后期)水牛睪丸曲精細(xì)管蛋白質(zhì)酶解肽段進(jìn)行TMT標(biāo)記、液相分離和質(zhì)譜鑒定。結(jié)果顯示,四次重復(fù)標(biāo)記試驗(yàn)共獲得1290個(gè)重復(fù)蛋白質(zhì),差異倍數(shù)≥2.0的重復(fù)差異蛋白304個(gè),以性成熟期水牛睪丸曲精細(xì)管作為對(duì)照,其中只在性成熟前期水牛睪丸曲精細(xì)管中表達(dá)上調(diào)的蛋白有252個(gè),下調(diào)16個(gè)；只在性成熟后期水牛睪丸曲精細(xì)管中表達(dá)上調(diào)的蛋白有1個(gè),下調(diào)26個(gè)；在性成熟前期和性成熟后期均表達(dá)下調(diào)的蛋白有9個(gè)。其次,對(duì)三個(gè)時(shí)期水牛睪丸曲精細(xì)管差異蛋白質(zhì)進(jìn)行生物信息學(xué)分析。主要進(jìn)行GO分析及信號(hào)通路分析,同時(shí)結(jié)合UniProU SpermatogenesisOnline 1.0等數(shù)據(jù)庫進(jìn)行綜合檢索,發(fā)現(xiàn)27個(gè)與精子發(fā)生相關(guān)及26個(gè)與細(xì)胞生長發(fā)育、老化增殖相關(guān)的差異蛋白質(zhì)。最后,從這些重要差異蛋白質(zhì)中選取7個(gè)進(jìn)行一系列驗(yàn)證。對(duì)這7個(gè)蛋白(CABS1、HSPA2、EEF1G、AKAP3、ROPN1、 ODF2、SPERT)進(jìn)行Western blot和實(shí)時(shí)熒光定量PCR驗(yàn)證以及進(jìn)一步的細(xì)胞定位分析。發(fā)現(xiàn)Western blot結(jié)果與TMT定量結(jié)果一致；HSPA2、EEF1G、AKAP3和SPERT在不同發(fā)育時(shí)期水牛睪丸曲精細(xì)管中的蛋白表達(dá)模式與mRNA表達(dá)模式一致；免疫組化分析結(jié)果發(fā)現(xiàn),EEF1G主要定位于圓形精子細(xì)胞核上,同時(shí)在支持細(xì)胞和間質(zhì)細(xì)胞核中有少量表達(dá),HSPA2在細(xì)胞質(zhì)、精原細(xì)胞及曲精細(xì)管基膜上有表達(dá),CABS1主要在精子細(xì)胞胞質(zhì)中表達(dá)；對(duì)其余的四個(gè)蛋白進(jìn)行精子的免疫熒光分析發(fā)現(xiàn),SPERT位于精子頂體、頸部和中段起始部分,AKAP3和ODF2主要位于精子頂體部分,ROPN1主要位于精子尾部主段。本章試驗(yàn)采用TMT結(jié)合2D LC-MS/MS技術(shù)能夠?qū)Σ煌瑫r(shí)期水牛睪丸曲精細(xì)管蛋白質(zhì)進(jìn)行有效地分離和鑒定,更加全面地展示水牛睪丸曲精細(xì)管在不同發(fā)育時(shí)期蛋白質(zhì)的表達(dá)情況和變化規(guī)律。綜上所述,本研究分別采用雙向電泳和液相色譜技術(shù)對(duì)不同時(shí)期(性成熟前期、性成熟期、性成熟后期)水牛睪丸曲精細(xì)管蛋白質(zhì)進(jìn)行分離和質(zhì)譜鑒定,首次建立了三個(gè)不同發(fā)育時(shí)期水牛睪丸曲精細(xì)管差異蛋白質(zhì)表達(dá)譜。該研究結(jié)果將有助于闡明精子發(fā)生的分子機(jī)制,并找到可能與水牛精子發(fā)生相關(guān)的標(biāo)志性蛋白,為后續(xù)水牛生殖調(diào)控的研究打下堅(jiān)實(shí)基礎(chǔ)。
[Abstract]:In this study , the difference of protein expression in testis of buffalo was studied by means of two - dimensional electrophoresis and two - dimensional electrophoresis .
In the second chapter , we established the two - dimensional electrophoresis system of spermatic tubules of bovine testis in the late stage of sexual maturation . The results showed that there were a number of different proteins , such as superoxide dismutase , heat shock protein HSPA2 , wave form protein , anti - proliferative protein , cytokeratin KRT8 and galectin 1 . The results showed that there were only two hundred and twenty - two different protein subunits , including superoxide dismutase , heat shock protein HSPA2 , wave form protein , anti - proliferative protein , cytokeratins KRT8 , and galectin 1 .
There were 1 and 26 downregulated proteins in the spermatic tubules of buffalo testis only in the late stage of sexual maturation .
In the early and late stage of sexual maturation , there were 9 proteins down - regulated in the late stage and late stage of sexual maturation .
The protein expression pattern of HSPA2 , EEF1G , AKAP3 and SPERT was consistent with that of mRNA expression pattern during different developmental stages .
The results of immunohistochemistry showed that EEF1G was mainly located on the nucleus of circular sperm , and the expression of HSPA2 was expressed in the cytoplasm , spermatogonia and convoluted tubules , and CABS1 was mainly expressed in the cytoplasm of sperm cells .
In this chapter , two - dimensional electrophoresis and liquid chromatography ( LC - MS / MS ) technique was used to separate and identify the protein in the testis of buffalo testis . The results of this study could help clarify the molecular mechanism of the spermatids and find the marker proteins which may be related to the spermatids of buffalo .

【學(xué)位授予單位】：廣西大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S823.83
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本文編號(hào)：1702952