人精液凝固蛋白衍生肽SGI-52抑制精子运动机制的研究

发布时间：2018-05-09 00:14

本文选题：精液凝固蛋白Ⅰ + 膜结构　；参考：《昆明医科大学》2014年硕士论文

【摘要】：目的：前期研究发现人精液凝固蛋白I衍生肽SGI-52有精子运动抑制活性,在此基础上进一步研究其与正常精子、去膜精子是否结合以及部位,膜结构变化,膜电位变化,线粒体膜电位,研究SGI-52肽对人生精细胞内钙离子的变化,探讨SGI-52肽影响精子运动可能的机制。方法：1、通过计算机辅助精液分析系统(CASA)选取2012年9月到2013年12月符合WHO正常人精液标准的标本,运用Percoll不连续密度梯度离心法收集有活力的精子,其中将部分活力的精子用含有0.1%Triton X-100的去膜介质(Demembranation medium, DM)处理模拟精子损伤,最后将全部精子分为正常精子实验组、正常精子对组照、去膜精子实验组、去膜精子对照组四组,对照组加入去离子水,而实验组则加入终浓度为5mg/ml的SGI-52肽。2、精子结合部位检测：用人输卵管培养液(Human tubal fluid, HTF)漂洗重悬并调节精子浓度为1×107/m1,各实验组加入SGI-52-FITC,各对照组则力入BSA-FITC于37℃孵育,并分别在1min、15min、30min取出适量用含4%甲醛及染料Hoechst的PBS固定处理各组精子,用PBS漂洗2次后用70%甘油重悬并制成玻片,采用OLYMPUS FV-1000激光共聚焦显微镜系统镜检并采集荧光图像。3、精子膜结构检测：精子中加入SGI-52肽,37℃孵育15min后加入终浓度为50μg/ml的SYBRGreen I和7-AAD染料充分混合后避光孵育10min, PBS漂洗2次去除未结合的染料,调节精子密度为1×106/ml,用流式细胞仪检测精子质膜完整性。4、精子膜电位检测：将精子悬浮于1ml含有400nM的DiBAC4(3)的人精子缓冲液(Human sperm solution, HSS)中并调整精子密度为3×106/ml,其中加入SGI-52肽,37℃孵育后用流式细胞仪检测精子膜电位。5、精子线粒体膜电位检测：用HTF漂洗各组精子并将精子密度调整为6×105/ml,加入SGI-52肽孵育,采用荧光染料JC-1单色标记法进行流式细胞仪检测精子线粒体膜电位。6、生精细胞钙离子浓度变化：取去势手术切除的睾丸标本,无菌条件下应用机械分离方法和Percoll不连续密度梯度离心法收集人生精细胞,调整细胞浓度为2×103/m1并将100μl细胞悬液置于96孔板37℃细胞培养箱中过夜培养,培养后每孔加入30μ l浓度为2mM的Fura-2AM荧光染料,置于37℃细胞培养箱避光染色1h,染色完毕后移去染液并用不含钙的OR2-Ca2+液洗一遍,然后每孔加入50μl含有2mM钙离子的OR2+Ca2+液,然后分别加入用OR2+Ca2+液溶解的终浓度为1mg/ml、3mg/ml、5mg/ml SGI-52肽,通过钙离子激发荧光显微系统检测生精细胞内钙离子浓度的变化。结果：1、激光共聚焦结果显示SGI-52肽与正常精子及去膜精子均有结合,结合部位在精子膜表面。但是两者结合的程度有区别：lmin时,SGI-52肽与正常精子结合的荧光强度较暗淡且结合的部位主要在精子的尾部,而在去膜精子,可见SGI-52肽明显的结合在头部膜表面及其尾部；在l0min时,正常精子的尾部的荧光逐渐变强,而去膜精子可见SGI-52肽已经渗透到了顶体；15min时,正常精子的头部可见SGI-52肽结合,而去膜精子组可见SGI-52肽已经进入精子内部。 2、SGI-52肽可引起精子膜结构通透性升高。正常精子在经过SGI-52肽处理后的通透性的比率要小于去膜精子。 3、SGI-52肽可引起精子的膜电位降低,即膜电位超极化。去膜精子整体膜电位均高于正常精子组。 4、SGI-52肽可引起精子线粒体膜电位降低。去膜精子整体活力要低于正常精子。 5、SGI-52肽可引起人生精细胞内钙离子浓度的升高。结论：人精液凝固蛋白衍生肽SGI-52通过与精子结合,通过改变精子膜结构,精子膜电位以及线粒体膜电位影响精子的运动,并且对精子获能起到一定的作用。
[Abstract]:Objective: To study the sperm motility inhibitory activity of human seminal coagulin I derived peptide SGI-52, and on this basis, we further study the combination of normal sperm, membrane sperm, membrane structure change, membrane potential change, mitochondrial membrane potential, and study the changes of calcium ion in human sperm cells by SGI-52 peptide and explore the SGI-52 peptide. The mechanism that affects the possible motility of sperm.
Methods: 1, by using the computer assisted semen analysis system (CASA) to select specimens from September 2012 to December 2013 to meet the standard of normal human semen of WHO, Percoll discontinuous density gradient centrifugation was used to collect the active sperm, and some of the sperm with 0.1%Triton X-100 (Demembranation medium, DM) was used. The sperm injury was simulated, and all the sperm were divided into the normal sperm experiment group, the normal sperm test group, the membrane sperm experiment group, the membrane sperm control group four groups, the control group added deionized water, and the experimental group added SGI-52 peptide.2 with the final concentration of 5mg/ml, and the sperm binding site detection: Human tubal fluid (HTF). The sperm concentration was 1 * 107/m1, the experimental group was added to SGI-52-FITC, the experimental groups were added to the SGI-52-FITC, and the control groups were incubated at BSA-FITC at 37, and the sperm were fixed with the PBS containing 4% formaldehyde and the dye Hoechst respectively in 1min, 15min, and 30min, and the glass was overhung with 70% glycerin after 2 times of PBS rinsing, and OLYMPUS FV-1000 stimulated. The light confocal microscope system was examined and the fluorescence image.3 was collected. The sperm membrane structure was detected: the sperm was added to the SGI-52 peptide. After incubating 15min at 37 C, the SYBRGreen I and 7-AAD dye with the final concentration were mixed with the SYBRGreen I and 7-AAD dyes to be fully mixed to avoid the 10min, and PBS rinse 2 times to remove the unbonded dyestuffs. The sperm density was 1 x 106/ml, and the flow cell was used. Sperm plasma membrane integrity.4, sperm membrane potential detection: sperm suspension in 1ml containing 400nM DiBAC4 (3) of human sperm buffer solution (Human sperm solution, HSS) and adjust the sperm density of 3 x 106/ml, including SGI-52 peptide, 37 degrees centigrade incubated with a flow cytometry test sperm membrane potential.5, sperm mitochondrial membrane potential detection: The sperm was rinsed by HTF and the sperm density was adjusted to 6 x 105/ml, and SGI-52 peptide was added to incubate the sperm. The mitochondrial membrane potential.6 of sperm and the calcium concentration of spermatogenic cell were detected by the fluorescent dye JC-1 monochrome labeling method, and the castrated testicular specimens were removed by the castrated operation. The mechanical separation method and Percoll were used under aseptic conditions. Continuous density gradient centrifugation was used to collect human sperm cells. The cell concentration was adjusted to 2 x 103/m1 and 100 L cell suspension was placed in the cell culture box of 96 orifice plate for overnight culture. After culture, the Fura-2AM fluorescent dye with 30 micron l concentration of 2mM was added to each hole, and the cell culture box was placed at 37 centigrade to avoid light and dye 1H. After dyeing, the dye was removed and the calcium containing no calcium was used. The OR2-Ca2+ solution was washed, then each hole was added to the OR2+Ca2+ solution containing 50 mu l containing 2mM calcium ion, and then the final concentration of OR2+Ca2+ solution was added to 1mg/ml, 3mg/ml, 5mg/ml SGI-52 peptide respectively. The calcium ion concentration in spermatogenic cells was detected by the calcium ion excitation fluorescence microscopy.
Results: 1, the results of confocal laser confocal show that SGI-52 peptide combines with normal sperm and spermatozoa, and the binding site is on the surface of the sperm membrane. But the degree of binding is different: when lmin, the fluorescence intensity of the combination of SGI-52 peptide and normal spermatozoon is dim and the binding site is mainly in the tail of the sperm, and the SGI-52 peptide is seen in the spermatozoon. In l0min, the fluorescence of the tail of normal spermatozoa becomes stronger at the time of l0min, while the SGI-52 peptide has penetrated into the acrosome and the head of the normal spermatozoa can be seen in the head of the normal sperm with SGI-52 peptide binding, while the SGI-52 peptide has entered the sperm inside the sperm group.
2, SGI-52 peptide can increase the permeability of sperm membrane structure. The ratio of permeability of normal sperm after SGI-52 peptide treatment is less than that of de membrane sperm.
3, SGI-52 peptide can cause sperm membrane potential to decrease, that is, membrane potential hyperpolarization. The membrane potential of the deactivating sperm is higher than that of the normal sperm group.
4, SGI-52 peptide can cause sperm mitochondrial membrane potential to decrease. The overall viability of the sperm removed is lower than that of normal sperm.
5, SGI-52 peptide can cause the increase of calcium ion concentration in human sperm cells. Conclusion: human seminal coagulin derived peptide SGI-52 can affect sperm movement by changing the structure of sperm membrane, sperm membrane potential and mitochondrial membrane potential by combining with sperm, and it plays a certain role in sperm capacitation.

【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R698.2

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