本文選題：差異蛋白　切入點(diǎn)：圓頭精子　出處：《中南大學(xué)》2007年碩士論文

【摘要】： 目的：以圓頭精子和正常精子為研究對(duì)象，通過(guò)差異熒光二維電泳技術(shù)(DIGE)和質(zhì)譜技術(shù)尋找和建立圓頭精子和正常精子的差異蛋白圖譜，并對(duì)某些關(guān)鍵性的蛋白進(jìn)行功能分析，以期揭示圓頭精子病理狀態(tài)的本質(zhì)以及細(xì)胞調(diào)控的機(jī)制。 材料和方法：收集已被證實(shí)具有正常生育能力的12位志愿者的精液標(biāo)本，每人捐獻(xiàn)3次；收集經(jīng)巴氏染色分析被證實(shí)為完全圓頭精子癥患者精液20份。所有志愿者都簽署了供精知情同意書。 運(yùn)用Percoll密度梯度離心技術(shù)分離精子，硫脲／尿素法結(jié)合超聲法提取蛋白，，2-D DIGE技術(shù)進(jìn)行差異蛋白質(zhì)組分離。將圓頭精子和正常精子分別用Cy3，Cy5熒光染料標(biāo)記，各取兩樣本的一半混合作為內(nèi)標(biāo)用Cy2標(biāo)記。為了獲得有統(tǒng)計(jì)學(xué)意義的差異蛋白點(diǎn)，我們?cè)賹蓸颖痉聪驑?biāo)記進(jìn)行2D-DIGE。圖像經(jīng)過(guò)DeCyder差異分析軟件進(jìn)行分析。結(jié)合膠內(nèi)酶解和質(zhì)譜鑒定技術(shù)對(duì)分析出的差異蛋白點(diǎn)進(jìn)行身份鑒定。 結(jié)果：本實(shí)驗(yàn)采用了熒光差異二維電泳(DIGE)對(duì)圓頭精子和正常精子蛋白進(jìn)行分離，用DeCyder差異分析軟件分析得到61個(gè)有統(tǒng)計(jì)學(xué)意義的差異點(diǎn)，其中有38個(gè)點(diǎn)在圓頭精子中表達(dá)上調(diào)，23個(gè)蛋白點(diǎn)在圓頭精子中表達(dá)下調(diào)。并對(duì)其中32個(gè)點(diǎn)進(jìn)行身份鑒定，得到了36個(gè)蛋白質(zhì)質(zhì)譜鑒定結(jié)果。 結(jié)論：本次實(shí)驗(yàn)得到了在圓頭精子中存在差異表達(dá)的有統(tǒng)計(jì)學(xué)意義的61個(gè)蛋白點(diǎn)，通過(guò)質(zhì)譜分析，得到了36個(gè)蛋白鑒定結(jié)果，并對(duì)這些蛋白進(jìn)行初步功能分析，推測(cè)其在圓頭精子發(fā)生過(guò)程中可能起作用的機(jī)制。
[Abstract]:Objective: to search and establish differential protein map between round head sperm and normal sperm by differential fluorescence two dimensional electrophoresis (DIGE) and mass spectrometry (MS), and to analyze the function of some key proteins. In order to reveal the nature of the pathological state of sperm and the mechanism of cell regulation. Materials and methods: semen samples of 12 volunteers who had been proved to have normal fertility were collected and donated 3 times each. A total of 20 semen samples were collected from patients with Pap staining and confirmed to be fully polyphoid spermatozoa. All of the volunteers signed the informed consent for spermatozoa. Spermatozoa were separated by Percoll density gradient centrifugation, and differential proteome was separated by thiourea / urea method combined with ultrasonic extraction of protein 2-D DIGE. The sperm of round head and normal sperm were labeled with Cy3 / Cy5 fluorescent dye, respectively. Half of the two samples were mixed as Cy2 markers. In order to obtain statistically significant differential protein spots, Then the two samples were labeled with 2D-DIGE. the images were analyzed by DeCyder differential analysis software, and the differential protein spots were identified by gel endolysis and mass spectrometry. Results: two dimensional fluorescence difference electrophoresis was used to isolate the sperm protein from normal sperm. 61 significant differences were obtained by using DeCyder differential analysis software. Among them, 38 spots were up-regulated in the spermatozoa and 23 protein spots were down-regulated in the spermatozoa. 32 of them were identified and 36 proteins were identified by mass spectrometry. Conclusion: in this experiment, 61 protein spots with different expression in sperm were obtained, and 36 proteins were identified by mass spectrometry, and the function of these proteins was analyzed. The mechanism of its possible role in the spermatogenesis of the head is inferred.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R321

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