采用DIGE方法对人类圆头精子和正常精子的差异蛋白质组学研究

发布时间：2018-04-01 00:38

本文选题：差异蛋白　切入点：圆头精子　出处：《中南大学》2007年硕士论文

【摘要】： 目的：以圆头精子和正常精子为研究对象，通过差异荧光二维电泳技术(DIGE)和质谱技术寻找和建立圆头精子和正常精子的差异蛋白图谱，并对某些关键性的蛋白进行功能分析，以期揭示圆头精子病理状态的本质以及细胞调控的机制。材料和方法：收集已被证实具有正常生育能力的12位志愿者的精液标本，每人捐献3次；收集经巴氏染色分析被证实为完全圆头精子症患者精液20份。所有志愿者都签署了供精知情同意书。运用Percoll密度梯度离心技术分离精子，硫脲／尿素法结合超声法提取蛋白，，2-D DIGE技术进行差异蛋白质组分离。将圆头精子和正常精子分别用Cy3，Cy5荧光染料标记，各取两样本的一半混合作为内标用Cy2标记。为了获得有统计学意义的差异蛋白点，我们再将两样本反向标记进行2D-DIGE。图像经过DeCyder差异分析软件进行分析。结合胶内酶解和质谱鉴定技术对分析出的差异蛋白点进行身份鉴定。结果：本实验采用了荧光差异二维电泳(DIGE)对圆头精子和正常精子蛋白进行分离，用DeCyder差异分析软件分析得到61个有统计学意义的差异点，其中有38个点在圆头精子中表达上调，23个蛋白点在圆头精子中表达下调。并对其中32个点进行身份鉴定，得到了36个蛋白质质谱鉴定结果。结论：本次实验得到了在圆头精子中存在差异表达的有统计学意义的61个蛋白点，通过质谱分析，得到了36个蛋白鉴定结果，并对这些蛋白进行初步功能分析，推测其在圆头精子发生过程中可能起作用的机制。
[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.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R321

【参考文献】