本文選題：差異蛋白　切入點：圓頭精子　出處：《中南大學》2007年碩士論文

【摘要】： 目的：以圓頭精子和正常精子為研究對象，通過差異熒光二維電泳技術(DIGE)和質譜技術尋找和建立圓頭精子和正常精子的差異蛋白圖譜，并對某些關鍵性的蛋白進行功能分析，以期揭示圓頭精子病理狀態(tài)的本質以及細胞調控的機制。 材料和方法：收集已被證實具有正常生育能力的12位志愿者的精液標本，每人捐獻3次；收集經(jīng)巴氏染色分析被證實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。 運用Percoll密度梯度離心技術分離精子，硫脲／尿素法結合超聲法提取蛋白，，2-D DIGE技術進行差異蛋白質組分離。將圓頭精子和正常精子分別用Cy3，Cy5熒光染料標記，各取兩樣本的一半混合作為內標用Cy2標記。為了獲得有統(tǒng)計學意義的差異蛋白點，我們再將兩樣本反向標記進行2D-DIGE。圖像經(jīng)過DeCyder差異分析軟件進行分析。結合膠內酶解和質譜鑒定技術對分析出的差異蛋白點進行身份鑒定。 結果：本實驗采用了熒光差異二維電泳(DIGE)對圓頭精子和正常精子蛋白進行分離，用DeCyder差異分析軟件分析得到61個有統(tǒng)計學意義的差異點，其中有38個點在圓頭精子中表達上調，23個蛋白點在圓頭精子中表達下調。并對其中32個點進行身份鑒定，得到了36個蛋白質質譜鑒定結果。 結論：本次實驗得到了在圓頭精子中存在差異表達的有統(tǒng)計學意義的61個蛋白點，通過質譜分析，得到了36個蛋白鑒定結果，并對這些蛋白進行初步功能分析，推測其在圓頭精子發(fā)生過程中可能起作用的機制。
[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.
【學位授予單位】：中南大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R321

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