本文選題：精液凝固蛋白Ⅰ + 膜結(jié)構(gòu)�。� 參考：《昆明醫(yī)科大學(xué)》2014年碩士論文

【摘要】：目的：前期研究發(fā)現(xiàn)人精液凝固蛋白I衍生肽SGI-52有精子運(yùn)動(dòng)抑制活性,在此基礎(chǔ)上進(jìn)一步研究其與正常精子、去膜精子是否結(jié)合以及部位,膜結(jié)構(gòu)變化,膜電位變化,線粒體膜電位,研究SGI-52肽對(duì)人生精細(xì)胞內(nèi)鈣離子的變化,探討SGI-52肽影響精子運(yùn)動(dòng)可能的機(jī)制。 方法：1、通過計(jì)算機(jī)輔助精液分析系統(tǒng)(CASA)選取2012年9月到2013年12月符合WHO正常人精液標(biāo)準(zhǔn)的標(biāo)本,運(yùn)用Percoll不連續(xù)密度梯度離心法收集有活力的精子,其中將部分活力的精子用含有0.1%Triton X-100的去膜介質(zhì)(Demembranation medium, DM)處理模擬精子損傷,最后將全部精子分為正常精子實(shí)驗(yàn)組、正常精子對(duì)組照、去膜精子實(shí)驗(yàn)組、去膜精子對(duì)照組四組,對(duì)照組加入去離子水,而實(shí)驗(yàn)組則加入終濃度為5mg/ml的SGI-52肽。2、精子結(jié)合部位檢測：用人輸卵管培養(yǎng)液(Human tubal fluid, HTF)漂洗重懸并調(diào)節(jié)精子濃度為1×107/m1,各實(shí)驗(yàn)組加入SGI-52-FITC,各對(duì)照組則力入BSA-FITC于37℃孵育,并分別在1min、15min、30min取出適量用含4%甲醛及染料Hoechst的PBS固定處理各組精子,用PBS漂洗2次后用70%甘油重懸并制成玻片,采用OLYMPUS FV-1000激光共聚焦顯微鏡系統(tǒng)鏡檢并采集熒光圖像。3、精子膜結(jié)構(gòu)檢測：精子中加入SGI-52肽,37℃孵育15min后加入終濃度為50μg/ml的SYBRGreen I和7-AAD染料充分混合后避光孵育10min, PBS漂洗2次去除未結(jié)合的染料,調(diào)節(jié)精子密度為1×106/ml,用流式細(xì)胞儀檢測精子質(zhì)膜完整性。4、精子膜電位檢測：將精子懸浮于1ml含有400nM的DiBAC4(3)的人精子緩沖液(Human sperm solution, HSS)中并調(diào)整精子密度為3×106/ml,其中加入SGI-52肽,37℃孵育后用流式細(xì)胞儀檢測精子膜電位。5、精子線粒體膜電位檢測：用HTF漂洗各組精子并將精子密度調(diào)整為6×105/ml,加入SGI-52肽孵育,采用熒光染料JC-1單色標(biāo)記法進(jìn)行流式細(xì)胞儀檢測精子線粒體膜電位。6、生精細(xì)胞鈣離子濃度變化：取去勢(shì)手術(shù)切除的睪丸標(biāo)本,無菌條件下應(yīng)用機(jī)械分離方法和Percoll不連續(xù)密度梯度離心法收集人生精細(xì)胞,調(diào)整細(xì)胞濃度為2×103/m1并將100μl細(xì)胞懸液置于96孔板37℃細(xì)胞培養(yǎng)箱中過夜培養(yǎng),培養(yǎng)后每孔加入30μ l濃度為2mM的Fura-2AM熒光染料,置于37℃細(xì)胞培養(yǎng)箱避光染色1h,染色完畢后移去染液并用不含鈣的OR2-Ca2+液洗一遍,然后每孔加入50μl含有2mM鈣離子的OR2+Ca2+液,然后分別加入用OR2+Ca2+液溶解的終濃度為1mg/ml、3mg/ml、5mg/ml SGI-52肽,通過鈣離子激發(fā)熒光顯微系統(tǒng)檢測生精細(xì)胞內(nèi)鈣離子濃度的變化。 結(jié)果：1、激光共聚焦結(jié)果顯示SGI-52肽與正常精子及去膜精子均有結(jié)合,結(jié)合部位在精子膜表面。但是兩者結(jié)合的程度有區(qū)別：lmin時(shí),SGI-52肽與正常精子結(jié)合的熒光強(qiáng)度較暗淡且結(jié)合的部位主要在精子的尾部,而在去膜精子,可見SGI-52肽明顯的結(jié)合在頭部膜表面及其尾部；在l0min時(shí),正常精子的尾部的熒光逐漸變強(qiáng),而去膜精子可見SGI-52肽已經(jīng)滲透到了頂體；15min時(shí),正常精子的頭部可見SGI-52肽結(jié)合,而去膜精子組可見SGI-52肽已經(jīng)進(jìn)入精子內(nèi)部。 2、SGI-52肽可引起精子膜結(jié)構(gòu)通透性升高。正常精子在經(jīng)過SGI-52肽處理后的通透性的比率要小于去膜精子。 3、SGI-52肽可引起精子的膜電位降低,即膜電位超極化。去膜精子整體膜電位均高于正常精子組。 4、SGI-52肽可引起精子線粒體膜電位降低。去膜精子整體活力要低于正常精子。 5、SGI-52肽可引起人生精細(xì)胞內(nèi)鈣離子濃度的升高。結(jié)論：人精液凝固蛋白衍生肽SGI-52通過與精子結(jié)合,通過改變精子膜結(jié)構(gòu),精子膜電位以及線粒體膜電位影響精子的運(yùn)動(dòng),并且對(duì)精子獲能起到一定的作用。
[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.

【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R698.2

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