發(fā)布時間：2018-02-02 02:27

  本文關(guān)鍵詞： TLR信號 精子運動 線粒體功能 信號通路　出處：《揚州大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：生殖道感染是引起男性不育及女性不孕的最重要原因之一,而感染能啟動固有免疫應(yīng)答。Toll樣受體(TLR)作為固有免疫系統(tǒng)的重要分子,在固有免疫系統(tǒng)抵抗外界干擾方面發(fā)揮著重要作用。近年的研究發(fā)現(xiàn)TLR表達于作為生殖細胞的精子中,而精子運動被認為是成功受孕的關(guān)鍵。為了探索TLR信號對精子生物學(xué)功能的影響,我們首先觀察了不同TLR激動劑對精子運動的影響,發(fā)現(xiàn)TLR激動劑能夠顯著抑制精子運動。本課題擬深入研究TLR信號影響精子運動的具體信號通路,并在此基礎(chǔ)上進一步探究TLR信號通過影響精子的何種生物學(xué)過程最終導(dǎo)致了精子運動減慢。 研究內(nèi)容分為3個部分： 1.不同種類的TLR激動劑對精子生物學(xué)性能的影響 目的：觀察不同病原組分的TLR激動劑對小鼠精子運動的影響,并初步探討TLR激動劑抑制精子運動的可能原因。 方法：采用逆轉(zhuǎn)錄PCR的方法分析不同TLR在精子中的表達。利用計算機輔助精子分析(CASA)的方法觀察不同TLR激動劑與小鼠精子孵育6h后的精子運動情況以及TLR激動劑處理不同時間對正常人精子運動的影響。利用流式細胞術(shù)的方法觀察TLR激動劑處理6h后的小鼠精子凋亡情況。 結(jié)果：除了TLR3表達量極低之外,其它的TLR在精子中均有表達。在TLR激動劑處理小鼠精子6h后,除了TLR3所對應(yīng)的激動劑poly(I：C)對精子運動沒有影響,其他TLR激動劑均能不同程度地抑制精子運動。另外,TLR激動劑抑制精子運動具有時間依賴性,Zymosan與LPS均只能在處理6h后才有效抑制精子運動,而R848在作用精子2h后即有抑制效應(yīng)。與對照組相比,TLR激動劑在6h之內(nèi)不能有效地促進精子凋亡的發(fā)生。 結(jié)論：TLR激動劑能夠有效抑制精子運動并且該效應(yīng)并不由TLR激動劑促進精子凋亡引起的。 2.TLR信號影響精子運動的通路研究 目的：探索TLR信號影響精子運動的確切信號通路 方法：利用MyD88敲除小鼠研究TLR激動劑對該種小鼠精子運動的影響。使用LPS處理小鼠精子2h,4h,6h,提取蛋白,通過Western-blotting觀察小鼠精子IRAK, PI3K, GSK3α, AKT, GSK3β, ERK, p65等分子磷酸化水平的變化,同時使用IRAK, PI3K,MEK/ERK, NF-κB等分子的抑制劑處理小鼠精子半小時后再給予TLR激動劑刺激,CASA觀察信號通路被抑制精子運動的改變。使用PI3K抑制劑處理小鼠精子,Western-Blotting觀察AKT及GSK3a磷酸化水平的變化。 結(jié)果：TLR激動劑對MyD88敲除小鼠的精子運動無影響。TLR激動劑處理小鼠精子后可觀察到IRAK, PI3K, GSK3a等分子磷酸化水平隨處理時間的延長而上升,而AKT,GSK3β, ERK, p65等分子的磷酸化水平無明顯變化。IRAK或PI3K通路被抑制后TLR激動劑不再能夠顯著抑制精子運動,而MEK/ERK, NF-κB通路被抑制后TLR激動劑依然能顯著抑制精子運動。PI3K抑制劑處理小鼠精子后,AKT的磷酸化水平?jīng)]有顯著改變而GSK3α的磷酸化水平受到了顯著地抑制。 結(jié)論：TLR信號以MyD88/IRAK4/PI3K及GSK3a依賴而非AKT依賴的信號通路途徑抑制精子運動。 3.TLR信號影響精子的生物學(xué)過程研究 目的：探討TLR信號通過影響精子的何種生物學(xué)過程最終抑制了精子運動,并將深入分析TLR信號通路在影響該生物學(xué)過程中的作用。 方法：利用熒光素酶反應(yīng)的方法檢測TLR激動劑處理6h后小鼠精子ATP水平的變化,進而使用基因敲除小鼠分析TLR激動劑引起的精子ATP水平的變化是否依賴MyD88及PI3K。采用F1F。ATPase ELISA檢測試劑盒檢測TLR激動劑處理6h后正常人精子F1F。ATPase的活性變化。利用JC-1線粒體膜電位檢測試劑盒通過流式細胞術(shù)檢測TLR激動劑處理不同時間對小鼠精子膜電位的影響,并利用Western-blotting分析相關(guān)信號通路分子在TLR激動劑處理后轉(zhuǎn)移到線粒體的情況。 結(jié)果：與對照組相比,TLR激動劑處理后顯著降低了小鼠精子ATP水平,并且TLR激動劑不能引起MyD88-/-及pik3cd-/-小鼠精子ATP水平的變化。TLR激動劑處理6h后能夠引起正常人精子F1F。ATPase活性的顯著上升,并以時間依賴的方式降低小鼠精子線粒體膜電位。最后,TLR激動劑處理6h后能夠引起PI3K及GSK3a等信號通路分子轉(zhuǎn)移到小鼠精子線粒體中。 結(jié)論：TLR信號通過影響線粒體膜電位的方式間接消耗ATP從而影響精子運動,該影響依賴于MyD88及PI3K通路。
[Abstract]:Toll - like receptor ( TLR ) , as an important molecule of the innate immune system , plays an important role in the innate immune system ' s resistance to external interference . In recent years , TLR expression is considered to be the key to successful pregnancy . The study was divided into 3 parts : 1 . Effects of different types of TLR agonists on sperm biological properties Objective : To observe the effect of TLR agonist on sperm motility in mice and to investigate the possible causes of the inhibition of sperm motility by TLR agonists . Methods : The expression of TLR in sperm was analyzed by reverse transcription polymerase chain reaction ( RT - PCR ) . The effects of TLR agonist and TLR agonist on sperm motility were observed by computer - assisted sperm analysis ( CASA ) . Results : In addition to TLR3 expression , other TLR agonists were expressed in the sperm . After 6 h of TLR agonist treatment , the agonist poly ( I : C ) corresponding to TLR3 had no effect on sperm motility , and other TLR agonists inhibited sperm motility to a varying degree . In addition , TLR agonists inhibited sperm motility after 6 hours of treatment . The TLR agonist did not effectively promote sperm apoptosis within 6 hours compared with the control group . Conclusion : TLR agonists can effectively inhibit sperm motility and the effect is not caused by TLR agonists in promoting sperm apoptosis . 2 . TLR signaling pathway for sperm motility Objective : To explore the exact signal pathway of TLR signaling affecting sperm motility Methods : The effects of TLR agonist on sperm motility in mice were studied by using MyD88 knockout mice . After 2 h , 4 h , 6 h of LPS - treated mice , the changes of phosphorylation level were observed by Western - blotting . Results : TLR agonist has no effect on the sperm motility of MyD88 knockout mice . TLR agonist can be observed after treatment of sperm in mice . Conclusion : TLR signaling can inhibit sperm motility in the pathway of MyD88 / RK4 / PIK and GSK3 - dependent signaling pathway . 3 . Study on the biological process of TLR signaling affecting sperm Objective : To investigate the effect of TLR signaling on sperm motility , and to deeply analyze the role of TLR signaling pathway in the biological process . Methods : The changes of ATP level in human sperm after 6 hours of TLR agonist treatment were detected by luciferase reaction , and the changes of ATP level of sperm induced by TLR agonist were analyzed by using F1F . ATPase ELISA kit . The effect of TLR agonist on sperm membrane potential was detected by flow cytometry and Western - blotting was used to analyze the effect of TLR agonist on sperm membrane potential of mice . Results : Compared with the control group , TLR agonist treatment significantly reduced the level of ATP in the sperm of mouse sperm , and TLR agonist could not induce the change of ATP level of MyD88 - / - and pik3cd - / - mice . Conclusion : TLR signaling indirectly consumes ATP by affecting the mitochondrial membrane potential , which affects sperm motility , which is dependent on the MyD88 and PIK pathways .

【學(xué)位授予單位】：揚州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R321

【共引文獻】

相關(guān)期刊論文 前10條

1 匡威;譚家莉;王橋;李洪濤;伍麗靜;劉琴瑤;;低強度周期性應(yīng)力對骨骼肌細胞增殖分化調(diào)控的機制[J];臨床口腔醫(yī)學(xué)雜志;2014年05期

2 王U喅，

本文編號：1483413