發(fā)布時(shí)間：2018-01-30 13:17

  本文關(guān)鍵詞： 精子DNA損傷 SCSA 獲能 頂體反應(yīng) 氧化應(yīng)激 不明原因復(fù)發(fā)性流產(chǎn)　出處：《大連醫(yī)科大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：探究不明原因復(fù)發(fā)性流產(chǎn)女性配偶的精子在體外獲能和頂體反應(yīng)過(guò)程中與精子DNA損傷之間的相關(guān)性，以及精子DNA損傷的可能機(jī)制。 方法:分別對(duì)不明原因復(fù)發(fā)性流產(chǎn)組（n=25）及正常生育組（n=25）的精子，經(jīng)培養(yǎng)液和鈣離子載體A23187（Calciumionophore A23187，Sigma，USA）取0.5h,1h,2h,4h在體外誘導(dǎo)獲能和頂體反應(yīng)處理后，用考馬斯亮藍(lán)染液檢測(cè)誘導(dǎo)不同時(shí)間點(diǎn)的頂體反應(yīng)率（AR）以及用流式細(xì)胞儀檢測(cè)誘導(dǎo)不同時(shí)間點(diǎn)的DFI和ROS。整個(gè)實(shí)驗(yàn)過(guò)程中，活性氧（reactive oxygen species,ROS）經(jīng)雙氫羅丹明123（DHR）孵育不同時(shí)間點(diǎn)0.5h,1h,2h,4h誘導(dǎo)獲能和頂體反應(yīng)后的精子，經(jīng)流式細(xì)胞儀檢測(cè)后，用相對(duì)光單位（relative light unit,RLU）表示。臨床使用的“金標(biāo)準(zhǔn)”精子染色質(zhì)結(jié)構(gòu)分析（sperm chromatin structure assay,SCSA）檢測(cè)精子DNA損傷，以及DNA斷裂指數(shù)（DNA fragmentation index,DFI）表示。 結(jié)果：首先，在未經(jīng)誘導(dǎo)獲能和頂體反應(yīng)前，RSA組的DFI高于正常生育組的DFI，分別是（19.3±4.56)%v.s.(12.2±2.87)%, P0.05，差異有統(tǒng)計(jì)學(xué)意義。然后，我們檢測(cè)在37℃分別孵育0.5h,1h,2h,4h后兩組的DFI值，差異沒有統(tǒng)計(jì)學(xué)意義。接下來(lái),我們檢測(cè)誘導(dǎo)獲能和頂體反應(yīng)發(fā)生的時(shí)間分別是0.5h,1h,2h,4h后RSA組和正常生育組的DFI,發(fā)現(xiàn)兩組的DFI都有增加，但在0.5h,1h,2h時(shí),兩組分別增加的DFI差異無(wú)統(tǒng)計(jì)學(xué)意義，到4h時(shí)，我們發(fā)現(xiàn)RSA組增加的DFI率高于正常生育組增加的DFI率，分別是（60.02±6.7）%v.s.（49.08±8.6）%，差異有統(tǒng)計(jì)學(xué)意義。最后，我們檢測(cè)RSA組和正常生育組經(jīng)雙氫羅丹明123（DHR）在37℃孵育不同的時(shí)間點(diǎn)0.5h,1h,2h,4h后上流式細(xì)胞儀檢測(cè)其對(duì)應(yīng)時(shí)間點(diǎn)的ROS，發(fā)現(xiàn)兩組的差異無(wú)統(tǒng)計(jì)學(xué)意義。同時(shí)，我們檢測(cè)兩組經(jīng)雙氫羅丹明123（DHR）誘導(dǎo)獲能和頂體反應(yīng)發(fā)生不同的時(shí)間點(diǎn)0.5h,1h,2h,4h后上流式細(xì)胞儀檢測(cè)其對(duì)應(yīng)時(shí)間點(diǎn)的ROS，，呈現(xiàn)和兩組檢測(cè)DFI時(shí)同樣的變化趨勢(shì)，隨著誘導(dǎo)獲能和頂體反應(yīng)時(shí)間的增加，兩組的ROS都有增加，但在0.5h,1h,2h時(shí)，兩組增加的ROS差異無(wú)統(tǒng)計(jì)學(xué)意義，到4h時(shí)，RSA組的ROS水平高于正常生育組的ROS水平，分別是36.4±3.1v.s.27.3±2.8，差異有統(tǒng)計(jì)學(xué)意義。 結(jié)論：1.男性精子DNA損傷與不明原因復(fù)發(fā)性流產(chǎn)有一定的相關(guān)性，提示精子DNA損傷或許是不明原因性復(fù)發(fā)性流產(chǎn)的男性因素之一。2.不明原因復(fù)發(fā)性流產(chǎn)患者配偶的精子在獲能和頂體反應(yīng)的過(guò)程中產(chǎn)生更多的精子DNA損傷，并且這種損傷會(huì)隨著體外獲能和頂體反應(yīng)的時(shí)間延長(zhǎng)而逐漸增加，誘導(dǎo)時(shí)間到4h時(shí)可觀察到顯著的增加。3.其發(fā)生機(jī)制可能是在獲能和頂體反應(yīng)的過(guò)程中，體內(nèi)高活性分子ROS產(chǎn)生過(guò)多，氧化物的清除速率沒有產(chǎn)生氧化物的速度快，使得氧化系統(tǒng)和抗氧化系統(tǒng)的平衡被打破，導(dǎo)致精子完整性受到損傷，精子的染色體DNA斷裂。
[Abstract]:Aim: to investigate the relationship between sperm DNA damage and sperm capacitation and acrosome reaction in female spouses with recurrent abortion and the possible mechanism of sperm DNA damage. Methods: the spermatozoa of the patients with recurrent abortion of unknown cause (n = 25) and normal fertility group (n = 25) were studied. The culture medium and calcium carrier A231877 Calciumophore A23187 Sigmaa USA were used to extract 0.5 h for 1 h and 2 h for 2 h. 4 h after induction of capacitation and acrosome reaction in vitro. The acrosome reaction rate at different time points was detected by Coomassie brilliant blue dye and the DFI and ROSs at different time points were detected by flow cytometry. Reactive oxygen (Ros) was incubated with Rhodamine 123DHRs for 1 h and 2 h at different time points. Spermatozoa induced by capacitation and acrosome reaction for 4 h were detected by flow cytometry, then relative light unit was used as relative unit of light. "Gold Standard" sperm chromatin structure analysis of Sperm chromatin structure assay for clinical use, RLU said. The DNA damage of sperm and the DNA breakage index (DNA fragmentation index DFI) were detected by SSA. Results: first, the DFI of DFI group was higher than that of normal fertility group before induced capacitation and acrosome reaction. The difference was statistically significant (P 0.05). Then we measured the incubation time at 37 鈩

本文編號(hào)：1476327