本文選題：弱畸形精子癥 + microRNA�。� 參考：《南方醫(yī)科大學(xué)》2015年博士論文

【摘要】：全球范圍內(nèi)大概有15%的夫妻存在不孕不育,其中男性因素導(dǎo)致的不育占50%。在精液分析中,弱精癥及弱畸形精子癥是一個(gè)常見(jiàn)的精液異常指標(biāo)；首先,我們采用人類(lèi)全基因組基因芯片獲取弱精癥與正常男性精子的基因表達(dá)譜,通過(guò)生物信息學(xué)篩選差異表達(dá)基因,發(fā)現(xiàn)SEMG1基因可能是弱精癥的候選基因之一；同時(shí)證實(shí)發(fā)現(xiàn)CRISP2在弱精子癥中低表達(dá),而且其他研究發(fā)現(xiàn)該蛋白為結(jié)構(gòu)蛋白,在精卵結(jié)合的頂體反應(yīng)及配子融合中其重要作用；其次,我們進(jìn)一步評(píng)估CRISP2在弱畸形精子癥精子中的表達(dá)及臨床意義,發(fā)現(xiàn)CRISP2蛋白在弱畸形精子癥中低表達(dá),而Q-PCR結(jié)果無(wú)差異,提示在弱畸形精子癥患者中,CRISP2蛋白的差異性表達(dá)是一種轉(zhuǎn)錄后的調(diào)控。因此,本向研究擬進(jìn)一步評(píng)估該蛋白在弱畸形精子癥低表達(dá)的分子機(jī)制。我們知道基因的異常表達(dá)常常因?yàn)檫z傳機(jī)制和表觀遺傳機(jī)制導(dǎo)致：表觀遺傳是指DNA序列不發(fā)生變化,但基因表達(dá)卻發(fā)生了可遺傳的改變。這種改變是細(xì)胞內(nèi)除了遺傳信息以外的其他可遺傳物質(zhì)發(fā)生的改變,且這種改變?cè)诎l(fā)育和細(xì)胞增殖過(guò)程中能穩(wěn)定傳遞。表觀遺傳分子機(jī)制包括DNA甲基化,組蛋白修飾,染色體改型及RNA干擾(MICRNA, LNCRNA)。我們對(duì)CRISP2基因的甲基化進(jìn)行研究,采用甲基化特異性的PCR及亞硫酸鹽序列測(cè)定法兩種方法檢測(cè)CRISP2的甲基化,在10例正常對(duì)照組和弱畸形精子癥組中均未發(fā)現(xiàn)CRISP2基因啟動(dòng)子區(qū)CpG島存在甲基化情況；CRISP2基因在20例正常精子及弱畸形精子癥中精子中Q-PCR結(jié)果無(wú)差異,CRISP2蛋白表達(dá)顯著下調(diào)。結(jié)合臨床資料分析,發(fā)現(xiàn)CRISP2蛋白表達(dá)下調(diào)與精子形態(tài)、精子活力及男性不育呈正相關(guān)。CRISP2蛋白在弱畸形精子癥患者中低表達(dá),結(jié)合我們之前的CRISP2基因的甲基化的研究,未發(fā)現(xiàn)CRISP2基因啟動(dòng)子區(qū)CpG島存在甲基化情況,表明CRISP2蛋白的表達(dá)受轉(zhuǎn)錄后的調(diào)控。進(jìn)而我們將工作轉(zhuǎn)向CRISP2基因/蛋白的靶向microRNA的研究。通過(guò)生物信息學(xué)軟件miRDB、miRWalk、miRTargetscan預(yù)測(cè)作用于CRISP2的microRNA,選取評(píng)分最高、共同交集的microRNA來(lái)進(jìn)一步研究：發(fā)現(xiàn)miR-27a、miR-27b、miR-340、miR-502-3p、miR-510、miR-640及miR-767-5p可以特異性結(jié)合CRISP2 3'-UTR區(qū)并調(diào)控其表達(dá),有文獻(xiàn)證實(shí)miR-27a在弱精癥中高表達(dá),因此,我們?cè)?0例正常精子及弱畸形精子癥中精子中Q-PCR檢測(cè)miR-27a表達(dá),發(fā)現(xiàn)miR-27a在弱畸形精子癥中精子中Q-PCR表達(dá)顯著增加。結(jié)合臨床資料分析,發(fā)現(xiàn)miR-27a表達(dá)下調(diào)與精子形態(tài)、精子活力及男性不育呈互相關(guān)。為此,我們進(jìn)一步探討是否miR-27a通過(guò)抑制CRISP2表達(dá)而導(dǎo)致弱畸形精子癥的發(fā)生。在293細(xì)胞中進(jìn)行熒光素酶報(bào)告基因Luciferase以驗(yàn)證篩選出的miR-27a和CRISP2基因的靶向性。通過(guò)將293細(xì)胞、含有CRISP2 3'-UTR(克隆的)pEZX-MT05 plasmid及miR-27a進(jìn)行轉(zhuǎn)染,取上清液進(jìn)行蛋白熒光測(cè)定,測(cè)定結(jié)果提示miR-27a通過(guò)結(jié)合CRISP2 3'-UTR導(dǎo)致弱畸形精子癥的發(fā)生可能。本研究的主要研究方法、內(nèi)容和結(jié)論包括以下幾個(gè)方面：第1部分基因芯片及生物信息學(xué)篩選精子活力不足相關(guān)差異表達(dá)基因目的：應(yīng)用基因芯片技術(shù)獲取成年男性精子活力不足和精子活力正常的精子基因表達(dá)譜,采用生物信息學(xué)篩選精子活力不足相關(guān)的差異表達(dá)基因,確定下一步研究目標(biāo)。方法：1.收集成年男性弱精子癥患者精液12例,活力正常精液12例作為對(duì)照組,Percoll密度梯度離心分離純化后,提取精子總RNA,通過(guò)體外轉(zhuǎn)錄合成cRNA,逆轉(zhuǎn)錄標(biāo)記cRNA,純化后與Agilent 4110B芯片雜交,雜交信號(hào)經(jīng)掃描后,Feature Extraction軟件對(duì)掃描結(jié)果進(jìn)行處理。2.將精子活力不足基因表達(dá)譜采用BRB-ArrayTools及GenSpring10.0分析工具進(jìn)行分析,差異基因篩選采用Algorithm:GeneSpring10---t-Test Against Zero檢驗(yàn),檢驗(yàn)結(jié)果由芯片公司提供。選取兩種分析工具具有共同的交集的基因。3.我們采用如NextBio及MEDLINE和FACTA生物信息學(xué)軟件工具對(duì)差異表達(dá)基因進(jìn)一步篩選并通過(guò)熒光定量PCR對(duì)芯片檢測(cè)結(jié)果進(jìn)行驗(yàn)證。結(jié)果：1.將精子活力不足基因表達(dá)譜采用BRB-ArrayTools及GenSpring10.0分析工具進(jìn)行分析,其中BRB-ArrayTools篩選得到的261個(gè)差異表達(dá)基因,而GenSpring10.0篩選得到的1265個(gè)差異表達(dá)基因,兩種分析結(jié)果重疊的基因(表達(dá)上調(diào)或下調(diào)),即精子活力不足的分子標(biāo)簽,共有71個(gè),其中10個(gè)基因共同表達(dá)上調(diào),61個(gè)基因共同表達(dá)下調(diào)；2.我們?cè)贜extBio下載了畸精癥和正常射精精子的基因芯片表達(dá)譜數(shù)據(jù)文件及其報(bào)道的差異基因,與我們分析得到的精子活力不足相關(guān)基因進(jìn)行了進(jìn)一步分析比較,發(fā)現(xiàn)21個(gè)在弱精癥中特異表達(dá)的差異基因；3.進(jìn)一步通過(guò)MEDLINE和FACTA軟件工具分析我們發(fā)現(xiàn)PGAP1和SEMG1與男性不育有關(guān),其中PGAP1與精子活力無(wú)關(guān)；4. Real-time PCR試驗(yàn)證實(shí)SEMG1基因的表達(dá)水平與芯片的結(jié)果基本一致。因此,我們提出SEMG1可能是精子活力不足的候選基因。結(jié)論：1.我們獲得了精子活力不足的基因表達(dá)譜,芯片結(jié)果告訴我們精子活力與精子中的基因的表達(dá)水平可能有著密切的聯(lián)系；2.篩選出的具有特征性的和新發(fā)現(xiàn)的基因可能會(huì)成為潛在的診斷的標(biāo)記物；如生物信息學(xué)分析發(fā)現(xiàn)SEMG1可能是精子活力不足的候選基因；CRISP2基因在精子活力不足患者精子中表達(dá)顯著下調(diào),說(shuō)明CRISP2與精子活力密切相關(guān)；這些基因的進(jìn)一步功能研究可能會(huì)幫助我們解釋精子活力不足的發(fā)病機(jī)制。第2部分CRISP2在弱畸形精子癥中的表達(dá)及臨床意義目的：探討弱畸形精子癥患者精子中富含半胱氨酸的分泌蛋白2(CRISP2)mRNA及蛋白的表達(dá)水平,明確其與弱畸形精子癥關(guān)系,探討其表達(dá)水平在臨床中的意義。方法：1.收集成年男性弱畸形精子癥患者精液20例,正常對(duì)照組精液20例作為對(duì)照組,50% Percoll離心分離純化后,提取精子總RNA及總蛋白,采用SYBR Green實(shí)時(shí)定量PCR和免疫印跡(Western Blot)技術(shù)檢測(cè)CRISP2 mRNA及蛋白相對(duì)表達(dá)量。2.通過(guò)回顧性調(diào)查弱畸形精子癥及正常對(duì)照組1年,觀察各組夫妻最終是否生育。采用Spearman相關(guān)系數(shù)(r)分析CRISP2 mRNA及蛋白相對(duì)表達(dá)量與弱畸形精子癥及生育的相關(guān)性。所有數(shù)據(jù)采用GraphPad Prism 5軟件進(jìn)行統(tǒng)計(jì)分析。結(jié)果：1. CRISP2基因在弱畸形精子癥患者精子中的表達(dá)量與其在正常對(duì)照組中的表達(dá)量間差異無(wú)統(tǒng)計(jì)學(xué)意義(P=-0.8578)。2. CRISP2蛋白在弱畸形精子癥患者精子中的表達(dá)量與其在正常對(duì)照組中的表達(dá)量差異具有統(tǒng)計(jì)學(xué)意義(P=0.0156)。3. CRISP2蛋白表達(dá)量和精子前向運(yùn)動(dòng)率(r=0.6240,P=0.0011)及形態(tài)正常率(r=0.5845,P=0.0027)呈顯著正相關(guān);而CRISP2蛋白表達(dá)水平與年齡、精液量、pH值、精子濃度等無(wú)顯著相關(guān)性。4.弱畸形精子癥組中的不育率(73%)高于正常對(duì)照組(24%),且差異具有統(tǒng)計(jì)學(xué)意義(P=0.0188); CRISP2蛋白相對(duì)低表達(dá)組的不育率(67%)高于CRISP2蛋白相對(duì)高表達(dá)組的不育率(17%),差異具有統(tǒng)計(jì)學(xué)意義(P=0.0361)。結(jié)論：1. CRISP2蛋白表達(dá)水平與弱畸形精子癥患者精子活力、形態(tài)呈顯著正相關(guān),而CRISP2蛋白低表達(dá)與弱畸形精子癥患者生育預(yù)后呈正相關(guān)。2. CRISP2可能是弱畸形精子癥發(fā)病機(jī)制研究的一個(gè)重要的分子靶標(biāo),值得進(jìn)一步深入研究其作用及調(diào)控機(jī)制。第3部分Mir-27a在弱畸形精子癥中的表達(dá)及臨床意義目的：探討弱畸形精子癥患者精子中Mir-27a的表達(dá)水平,明確其與弱畸形精子癥關(guān)系,探討其表達(dá)水平在臨床中的意義。方法：1.收集成年男性弱畸形精子癥患者精液20例,正常對(duì)照組精液20例作為對(duì)照組,50%Percoll離心分離純化后,提取精子總RNA,采用SYBR Green實(shí)時(shí)定量PCR檢測(cè)Mir-27a RNA及蛋白相對(duì)表達(dá)量。3.通過(guò)回顧性調(diào)查弱畸形精子癥及正常對(duì)照組1年,觀察各組夫妻最終是否生育。采用相關(guān)性分析Mir-27a RNA表達(dá)量與弱畸形精子癥及生育的相關(guān)性。所有數(shù)據(jù)采用GraphPad Prism 5軟件進(jìn)行統(tǒng)計(jì)分析。結(jié)果：1. Mir-27a在弱畸形精子癥患者精子中的表達(dá)量與其在正常對(duì)照組中的表達(dá)量間具有統(tǒng)計(jì)學(xué)差異,表現(xiàn)為顯著性高表達(dá)(P=0.0179)。2. Mir-27a基因和精子前向運(yùn)動(dòng)率(r=-0.3236,P=0.0417)及形態(tài)正常率(r=-0.4560,P=0.0031)呈顯著負(fù)相關(guān);而miR-27a表達(dá)水平與年齡、精液量、pH值、精子濃度等無(wú)顯著相關(guān)性。3. Mir-27a基因相對(duì)低表達(dá)組的不育率(25%)顯著低于miR-27a相對(duì)高表達(dá)組的不育率(69%,P=0.0320)。這些數(shù)據(jù)提示高水平的miR-27a與不育密切相關(guān)。結(jié)論：1. miR-27a基因表達(dá)水平與弱畸形精子癥患者精子活力、形態(tài)呈顯著負(fù)相關(guān),而miR-27a高表達(dá)與弱畸形精子癥患者生育預(yù)后呈負(fù)相關(guān)。2.提示miR-27a可能是弱畸形精子癥發(fā)病機(jī)制研究的一個(gè)重要的分子靶標(biāo),值得進(jìn)一步深入研究其作用及調(diào)控機(jī)制。第4部分Mir-27a介導(dǎo)CRISP2表達(dá)導(dǎo)致弱畸形精子癥的分子機(jī)制目的：探討弱畸形精子癥患者精子中CRISP2表達(dá)下調(diào)的分子機(jī)制,明確Mir-27a是否與CRISP2有關(guān),及其是否調(diào)控CRISP2的表達(dá)。方法：1.收集成年男性弱畸形精子癥患者精液10例,正常對(duì)照組精液10例作為對(duì)照組,50%Percoll離心分離純化后,提取精子DNA, PC擴(kuò)增并利用BSP法直接擴(kuò)增出硫化處理后的目的片段并測(cè)序,檢測(cè)CRISP2基因的CpG島甲基化情況。2.采用Spearman相關(guān)系數(shù)(r)分析弱畸形精子癥中CRISP2基因／蛋白相對(duì)表達(dá)量與Mir-27a表達(dá)量的相關(guān)性。3.采用熒光素酶報(bào)告基因Luciferase驗(yàn)證miR-27a調(diào)控CRISP2,通過(guò)將293T細(xì)胞、含有CRISP2 3'-UTR(克隆的)pEZX-MT05 plasmid及miR-27a進(jìn)行轉(zhuǎn)染,取上清液進(jìn)行蛋白熒光測(cè)定。所有數(shù)據(jù)采用GraphPad Prism 5軟件進(jìn)行統(tǒng)計(jì)分析。結(jié)果：1.10例正常組樣本和10例弱畸形精癥組樣本CRISP2基因的CpG島均未發(fā)生甲基化；2.弱畸形精子癥組精液精子中miR-27a基因含量與CRISP2蛋白表達(dá)量呈顯著負(fù)相關(guān)(r=-0.4330, P=0.0345); miR-27a基因含量與CRISP2基因表達(dá)量無(wú)相關(guān)(P0.05)；3.發(fā)現(xiàn)加入了miR-27a的含CRISP2基因3'UTR克隆組質(zhì)粒的熒光活性顯著低于其他組的活性,提示：miR-27a可以結(jié)合CRISP2基因3'UTR并抑制其表達(dá)。結(jié)論：1. CRISP2基因在弱畸形精子癥及正常對(duì)照組中無(wú)表達(dá)差異,CRISP2基因在弱畸形精子癥及正常對(duì)照組中無(wú)甲基化異常,而CRISP2蛋白在弱畸形精子癥及正常對(duì)照組中表達(dá)存在顯著差異,表達(dá)顯著下調(diào)；2.通過(guò)生物信息學(xué)及Q-PCR驗(yàn)證發(fā)現(xiàn)miR-27a在弱畸形精子癥及正常對(duì)照組中表達(dá)存在顯著差異,表達(dá)顯著上調(diào),CRISP2蛋白與miR-27a兩者之間存在顯著負(fù)相關(guān)；3.進(jìn)一步實(shí)驗(yàn)發(fā)現(xiàn)miR-27a對(duì)CRISP2蛋白的表達(dá)具有顯著的抑制作用,這為下一步研究CRISP2及miR-27a的功能打下基礎(chǔ),為深入研究CRISP2信號(hào)通路提供了新的依據(jù)。
[Abstract]:About 15% of spouses worldwide have infertility, among which male infertility accounts for 50%. in semen analysis. Asthenospermia and asthenospermia is a common semen anomaly. First, we use human genome microarray to obtain gene expression profiles of asthenospermia and normal male sperm, through biology. Screening differentially expressed genes by informatics shows that SEMG1 gene may be one of the candidate genes for asthenospermia, and that CRISP2 is found to be low in asthenospermia, and other studies have found that the protein is structural protein, which is important in the acrosome reaction and gamete fusion of sperm oocyte binding. Secondly, we further evaluate CRISP2 in the presence of spermatozoa. The expression and clinical significance of spermatozoa in asthenospermia and the low expression of CRISP2 protein in asthenospermia, and no difference in Q-PCR results, suggesting that the differential expression of CRISP2 protein is a post transcriptional regulation in the patients with asthenospermia. Therefore, this study intends to further evaluate the low expression of the protein in the asthenospermia. The molecular mechanism. We know that the abnormal expression of genes is often caused by genetic and epigenetic mechanisms: epigenetics is that the DNA sequence does not change, but the gene expression has a genetic change. This change is a change in the genetic material other than the genetic information in the cell, and this change is The epigenetic molecular mechanisms include DNA methylation, histone modification, chromosome modification and RNA interference (MICRNA, LNCRNA) in the process of development and cell proliferation. We studied the methylation of the CRISP2 gene and detected the methylation of CRISP2 by two methods of methylation specific PCR and sulfite sequencing, at 10 The methylation of CpG island in the promoter region of CRISP2 gene was not found in the normal control group and the asthenospermia group. The Q-PCR results of the CRISP2 gene in the sperm of 20 normal spermatozoa and the asthenospermia were not different, and the expression of the CRISP2 protein was down significantly. The expression of CRISP2 protein and the morphology of the sperm were found by the analysis of the clinical data. The positive correlation between sperm motility and male infertility is a low expression of.CRISP2 protein in patients with asthenospermia. Combined with our previous methylation of the CRISP2 gene, the methylation of the CpG island of the CRISP2 gene promoter was not found, indicating that the expression of the CRISP2 protein was regulated after the transcriptional regulation. And then we turned the work to the CRISP2 gene. The study of protein targeting microRNA. Through the bioinformatics software miRDB, miRWalk, and miRTargetscan, it predicts the microRNA of CRISP2, and selects the highest grade and common intersection of microRNA to further study: miR-27a, miR-27b, miR-340, miR-502-3p, miR-510, etc. The expression of miR-27a was proved to be highly expressed in asthenospermia. Therefore, we detected the expression of miR-27a in the sperm of 20 normal spermatozoa and asthenospermia, and found that the expression of Q-PCR in the spermatozoa was significantly increased in the asthenospermia of the asthenospermia. The expression of miR-27a was significantly increased in the spermatozoa of the weak malformed spermatozoa. The expression of miR-27a and the morphology of sperm, the vitality of the sperm and the activity of sperm were found. Male infertility is interrelated. To this end, we further explore whether miR-27a leads to the occurrence of asthenospermia by inhibiting CRISP2 expression. The luciferase reporter gene Luciferase is used in 293 cells to verify the targeting of the selected miR-27a and CRISP2 genes. By 293 fine cells, CRISP2 3'-UTR (cloned) pEZX-MT05 pl Asmid and miR-27a were transfected and extracted from the supernatant for protein fluorescence. The results suggested that miR-27a could lead to the occurrence of asthenospermia by combining with CRISP2 3'-UTR. The main research methods, contents and conclusions of this study include the following aspects: first parts of gene core and bioinformatics screening sperm motility insufficiency The purpose of differentially expressed genes is to use gene chip technology to obtain the sperm gene expression profiles of adult male sperm motility insufficiency and normal sperm motility. The differential expression genes related to sperm motility are screened by bioinformatics to determine the next step of research. Methods: 1., 12 cases of semen of adult male asthenospermia were collected, and the vitality of the spermatozoa was collected. 12 normal semen were used as control group. After separation and purification of Percoll density gradient centrifugation, the total RNA of sperm was extracted, cRNA was transcribed in vitro and cRNA was marked by reverse transcription. After purification, hybridization with Agilent 4110B chip. After the hybridization signal was scanned, Feature Extraction software treated the scanned fruit by Feature Extraction software to extract the gene expression profile of sperm motility. BRB-ArrayTools and GenSpring10.0 analysis tools were used to analyze the differential gene screening using Algorithm:GeneSpring10---t-Test Against Zero test. The test results were provided by the chip company. We selected two analysis tools with a common intersection of gene.3.. We used NextBio and MEDLINE and FACTA bioinformatics software tools to do bad. The differentially expressed genes were further screened and tested by fluorescence quantitative PCR. Results: 1. the expression profiles of sperm motility insufficiency genes were analyzed by BRB-ArrayTools and GenSpring10.0 analysis tools, of which 261 differentially expressed genes were screened by BRB-ArrayTools, and 1265 differences were obtained by GenSpring10.0 screening. Different expression genes, two kinds of overlapping genes (up-regulated or down-regulated), that is, the molecular label of sperm motility insufficiency, there are 71, of which 10 genes are up regulated together, 61 genes are down regulated together; 2. we downloaded the gene chip expression profiles of abnormal spermatozoa and normal ejaculatory sperm in NextBio and reports The difference gene, which was further analyzed and compared with the gene of sperm motility insufficiency we analyzed, found 21 differentially expressed genes in asthenospermia. 3. further through MEDLINE and FACTA software tools, we found that PGAP1 and SEMG1 were associated with male infertility, and PGAP1 was not related to sperm motility; 4. Real-time PCR test verified that the expression level of the real SEMG1 gene is basically consistent with the results of the chip. Therefore, we suggest that SEMG1 may be a candidate gene for the deficiency of sperm motility. Conclusion: 1. we have obtained the gene expression profiles of insufficient sperm motility, and the results of the chip tell us that the sperm vitality may be closely linked to the level of the gene expression in the sperm. The characteristic and newly discovered genes screened by 2. may be a potential marker for diagnosis; for example, bioinformatics analysis found that SEMG1 may be a candidate gene for insufficient sperm motility, and the CRISP2 gene has a significant downregulation in sperm motility in patients with sperm motility, indicating that CRISP2 is closely related to sperm vitality; Further functional study of genes may help us explain the pathogenesis of insufficient sperm motility. Second the expression and clinical significance of CRISP2 in asthenospermia: To explore the expression level of cysteine rich protein 2 (CRISP2) mRNA and protein in spermatozoa of asthenospermia Methods: 1. to collect 20 cases of semen of adult male asthenospermia, 20 cases of normal control group and 20 cases of normal control group as control group. After 50% Percoll centrifugation, the total RNA and total protein of sperm were extracted. The real-time quantitative PCR and Western blot (Western Blot) technique of SYBR Green was used to detect the sperm. The relative expression of CRISP2 mRNA and protein.2. was examined by a retrospective survey of asthenospermia and normal control group for 1 years. The correlation between CRISP2 mRNA and relative expression of CRISP2 mRNA and protein relative expression was analyzed by Spearman correlation coefficient (R). All data were carried out by GraphPad Prism 5 software. Results: there was no significant difference between the expression of 1. CRISP2 gene in the sperm of the asthenospermia patients and the normal control group (P=-0.8578) the expression of.2. CRISP2 protein in the sperm of the asthenospermia patients was significantly different from that in the normal control group (P=0.0156) There was a significant positive correlation between the expression of.3. CRISP2 protein and the motility of sperm (r=0.6240, P=0.0011) and the normal rate of morphology (r=0.5845, P=0.0027), but there was no significant correlation between the expression level of CRISP2 protein and age, semen volume, pH, and sperm concentration in the.4. asthenospermia group (73%), which was higher than that of the normal control group (24%). Study significance (P=0.0188); the sterility rate of the relatively low expression group of CRISP2 protein (67%) was higher than that of the relatively high expression group of CRISP2 protein (17%), and the difference was statistically significant (P=0.0361). Conclusion: the expression level of 1. CRISP2 protein was positively correlated with the motility of spermatozoa in the patients with asthenospermia, and the low expression of CRISP2 protein and weak malformation. Positive correlation.2. CRISP2 may be an important molecular target for the study of the pathogenesis of asthenospermia. It is worth further studying its role and regulation mechanism. Third the expression and clinical significance of Mir-27a in asthenospermia, and its clinical significance: To explore the Mir-27 in spermatozoa of the weak malformed spermatozoa A expression level, clarify its relationship with asthenospermia and explore the significance of its expression level in clinical. Methods: 1. the semen of adult male asthenospermia was collected in 20 cases, and 20 cases of normal control group were used as control group. After 50%Percoll centrifugation was separated and purified, the total RNA of spermatozoa was extracted, and Mir-27a was used to detect Mir-27a by SYBR Green real-time quantitative PCR The relative expression of RNA and protein.3. was examined by a retrospective study of the asthenospermia and the normal control group for 1 years. The correlation between Mir-27a RNA expression and asthenospermia and fertility was analyzed by correlation analysis. All data were analyzed by GraphPad Prism 5 software. Results: 1. Mir-27a was weak. There was a statistical difference between the expression of sperm in the patients with malformed spermatozoa and the normal control group, which showed significant negative correlation between the significant high expression (P=0.0179).2. Mir-27a gene and the sperm forward movement rate (r=-0.3236, P=0.0417) and the normal rate of morphology (r=-0.4560, P= 0.0031); and the level of miR-27a expression and age, semen. There was no significant correlation between the amount, pH value, sperm concentration and so on. The sterility rate of the relatively low expression group of.3. Mir-27a (25%) was significantly lower than that of the relatively high expression group of miR-27a (69%, P=0.0320). These data suggest that the high level of miR-27a is closely related to infertility. Conclusion: the expression level of the 1. miR-27a gene and the sperm motility of the asthenospermia patients The high expression of miR-27a is negatively correlated with the prognosis of asthenospermia patients with negative correlation.2. suggesting that miR-27a may be an important molecular target for the study of the pathogenesis of asthenospermia. It is worth further studying its role and regulatory mechanism. The fourth part of Mir-27a mediates CRISP2 expression leading to asthenospermia. Molecular mechanism Objective: To investigate the molecular mechanism of down regulation of CRISP2 expression in sperm of asthenospermia patients, to determine whether Mir-27a is related to CRISP2 and to regulate the expression of CRISP2. Methods: 1. the semen of adult male asthenospermia was collected in 10 cases, and 10 cases of normal control group were used as control group, and 50%Percoll centrifugation was used for isolation and purification. Then, the sperm DNA, PC were amplified and the BSP method was used to directly amplify the target fragments after the vulcanization. The CpG island methylation of the CRISP2 gene was detected by Spearman correlation coefficient (R), and the correlation between the relative expression of the CRISP2 gene / protein and the expression of Mir-27a was analyzed by the Spearman correlation coefficient (R), and the luciferase reporter base was adopted. CRISP2 was regulated by miR-27a by Luciferase, and 293T cells were transfected with CRISP2 3'-UTR (cloned) pEZX-MT05 plasmid and miR-27a. The supernatant was used for the determination of protein fluorescence. All data were analyzed by GraphPad Prism 5 software. Results: 1.10 normal group samples and 10 cases of weak malformed spermatosis group were sampled. There was no methylation in the CpG islands, and the expression of miR-27a gene and CRISP2 protein in semen spermatozoa of 2. asthenozoospermia group were significantly different.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R698.2

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