本文選題：硫化氫 + 精子�。� 參考：《南京醫(yī)科大學(xué)》2014年博士論文

【摘要】：男性不育是造成人類生殖障礙的重要原因。不育男性中超過85%均能夠產(chǎn)生精子,但是大多質(zhì)量低下。精子發(fā)生過程異常,會影響精子的功能,造成男性不育。除了基因突變和染色體異常等遺傳缺陷,環(huán)境因素和感染等疾病因素是導(dǎo)致精子發(fā)生和功能障礙的主要原因。這其中氧化應(yīng)激與炎癥反應(yīng)是最關(guān)鍵的損傷機(jī)制。目前為止,男性不育在臨床上缺乏有效的或者針對性的治療手段。硫化氫(Hydrogen Sulfide, H2S)是繼一氧化氮(Nitic Oxide, NO)和一氧化碳(Carbon Monoxide, CO)后的第三種氣體信號分子,在哺乳動物體內(nèi),H2S主要由胱硫醚-γ-裂解酶(CSE)、胱硫醚-pˉ合成酶(CBS)催化L-半胱氨酸生成。研究證實(shí),生理濃度的H2S對于清除氧自由基,控制機(jī)體的氧化應(yīng)激水平,對抗炎癥反應(yīng)具有十分重要的生理意義。在一些病理狀況下,外源性補(bǔ)充H2S,可以明顯減輕氧化應(yīng)激和炎癥造成的組織和器官的功能障礙。在關(guān)于女性生殖的研究中,H2S對于卵細(xì)胞的發(fā)育和成熟具有重要意義,并對胎兒的出生和孕婦心血管功能的保護(hù)具有重要價(jià)值。在男性的生殖系統(tǒng)中,同樣也存在著內(nèi)源性的H2S合成酶,但對于其生理意義的研究報(bào)道多局限于改善男性的勃起功能障礙。內(nèi)源性的H2S合成酶在男性的睪丸和精子中均有表達(dá),可能對于調(diào)控男性精子發(fā)生和精子功能具有重要的潛在價(jià)值。目前為止,H2S對男性生育障礙的治療和預(yù)防,以及對人類精子質(zhì)量的影響尚無報(bào)道。本研究的主要目標(biāo)是探討H2S對精子發(fā)生過程及精子功能是否具有保護(hù)作用,并進(jìn)一步研究其發(fā)揮作用的可能機(jī)制。我們首先使用自由基檢測儀評價(jià)了H2S在正常人精漿和育力低下病人精漿中含量的差異,用Western-blot的方法比較了內(nèi)源性H2S合成酶在不同來源精子中表達(dá)的差異。結(jié)果表明低水平H2S的精漿常常伴隨著較差活力的精子,弱精子中內(nèi)源性H2S合成酶CBS的表達(dá)顯著降低,提示H2S可能參與了男性不育的病理過程。我們用動物實(shí)驗(yàn)進(jìn)一步驗(yàn)證H2S對男性生殖的意義：我們利用LPS誘導(dǎo)的炎癥和STZ誘導(dǎo)的糖尿病,建立常見的精子發(fā)生障礙模型,建模成功后,腹腔注射外源性給予GYY4137或NaHS兩種不同的H2S供體,給藥結(jié)束后,用電腦輔助精液分析儀(CASA)評價(jià)小鼠附睪尾部的精子質(zhì)量,并用HE染色觀察小鼠睪丸生精結(jié)構(gòu)的變化,結(jié)果表明H2S可以顯著抑制炎癥和糖尿病引起的精子質(zhì)量的下降并改善睪丸結(jié)構(gòu)的損傷。我們用Real-Time PCR和Western-blot的方法檢測了內(nèi)源性H2S合成酶CSE和CBS的mRNA水平和蛋白表達(dá)水平,并用化學(xué)法檢測了睪丸中CBS和CSE的催化活性,結(jié)果顯示外源性給予H2S可以改善炎癥損傷引起的內(nèi)源性H2S合成的障礙,其中CBS可能是影響睪丸中內(nèi)源性H2S水平主要的限速酶。小鼠睪丸行冰凍切片,DHE熒光染色測定超氧陰離子(02-)水平,結(jié)果顯示,H2S可抑制睪丸02的生成。Tunel染色提示H2S可減輕炎癥引起的生精細(xì)胞的凋亡。免疫熒光測定血睪屏障緊密連接蛋白,結(jié)果顯示,H2S可維護(hù)血睪屏障的完整。ELISA方法檢測小鼠血清睪酮水平,H2S能夠抑制炎癥造成的血清睪酮水平的降低。進(jìn)一步研究H2S保護(hù)精子發(fā)生的的機(jī)制：Real-Time PCR的結(jié)果表明,H2S降低了炎癥引起的睪丸中促炎因子IL-1p、IL-6、TNF-amRNA水平的升高,并提高抗炎因子ILˉ10的mRNA水平,同時(shí)改善炎癥引起的睪酮合成相關(guān)蛋白StAR、P450scc、3β-HSD和P450c17mRNA水平的降低。試劑盒檢測睪丸中的MDA水平,GSH/GSSG,以及SOD和caspase-3的活性,結(jié)果表明H2S可抑制炎癥引起的MDA水平的增高,并維持GSH/GSSG的水平和SOD的活性,同時(shí)抑制caspase-3的激活。Western blot檢測結(jié)果發(fā)現(xiàn),H2S可以抑制IκB的降解和NF-κB的核轉(zhuǎn)位(凝膠遷移率實(shí)驗(yàn)進(jìn)一步證實(shí)H2S抑制了炎癥狀態(tài)下NF-κB活性的增高),抑制iNOS的表達(dá),促進(jìn)Nrf2的核轉(zhuǎn)位并提高Nrf2在胞漿中的水平,H2S可以提高Bcl2/Bax的水平,同時(shí)H2S能夠顯著抑制MAPK信號通路蛋白JNK、ERK、P38的磷酸化水平。為了進(jìn)一步證實(shí)內(nèi)源性H2S對睪丸精子發(fā)生的意義,我們利用腺病毒轉(zhuǎn)染的方式構(gòu)建右側(cè)睪丸組織過表達(dá)CBS的小鼠模型,在LPS刺激的條件下,CSAS的檢測結(jié)果顯示CBS過表達(dá)的右側(cè)附睪中,精子質(zhì)量明顯高于左側(cè)附睪。同時(shí)HE染色和免疫熒光染色的結(jié)果提示,在LPS造成炎癥損傷的條件下,CBS過表達(dá)的右側(cè)睪丸,其生精結(jié)構(gòu)和血睪屏障的完整性與左側(cè)睪丸相比均有明顯改善。我們利用人精子細(xì)胞實(shí)驗(yàn)觀測H2S對人類精子活力的影響：CASA檢測結(jié)果顯示內(nèi)源性H2S合成酶CBS抑制劑AOAA顯著抑制正常精子的活力水平和超激活運(yùn)動能力,而外源性補(bǔ)充H2S能夠改善CBS被抑制引起的精子活力和超激活運(yùn)動能力的下降；合適濃度的H2S對于改善精漿中H2S水平較低的弱精癥病人的精子活力具有一定效果。以上結(jié)果表明：①H2S對男性精子功能具有重要意義,外源性補(bǔ)充H2S有助于改善因H2S缺乏造成的精子功能障礙。②動物實(shí)驗(yàn)研究結(jié)果表明,H2S能夠改善炎癥和糖尿病引起的精子質(zhì)量的下降,保護(hù)睪丸的生精結(jié)構(gòu),維持血睪屏障的完整,改善睪酮合成的障礙,維護(hù)正常精子發(fā)生。③H2S對精子發(fā)生和功能障礙的改善,依賴其可以通過抑制NF-κB的活性,控制睪丸組織中的炎癥水平；通過促進(jìn)Nrf2的核轉(zhuǎn)位,提高抗氧化因子的表達(dá)抑制睪丸組織中氧化應(yīng)激水平的增高；通過抑制線粒體誘導(dǎo)的凋亡信號通路的激活,減輕生精細(xì)胞的凋亡；通過抑制MAPK信號通路的激活,調(diào)控睪酮的合成和血睪屏障的完整。本研究為男性生育障礙的防治提供了新的思路。
[Abstract]:Male infertility is an important cause of human reproductive disorders. More than 85% of male sterile men can produce sperm, but most of them have low quality. The abnormal process of spermatogenesis affects sperm function and causes male infertility. In addition to genetic defects such as gene mutation and chromosome abnormality, environmental factors and infection factors are the causes of sperm. The main cause of occurrence and dysfunction. This is the most critical mechanism of oxidative stress and inflammation. So far, male infertility is clinically lacking in effective or targeted treatment. Hydrogen Sulfide (H2S) is third after nitric oxide (Nitic Oxide, NO) and carbon monoxide (Carbon Monoxide, CO). In mammals, in mammals, H2S mainly catalyzes the formation of L- cysteine by cystathion - gamma lyase (CSE) and cystathion -p synthetase (CBS). It is proved that the physiological concentration of H2S is of great physiological significance to scavenging oxygen free radicals, controlling the oxidative stress level of the body, and against the inflammatory reaction. In the study of female reproduction, H2S is of great significance to the development and maturation of egg cells in the study of female reproduction, and is of great value for the protection of fetal birth and pregnant women's cardiovascular function. In the male reproductive system, the H2S is the same. There are also endogenous H2S synthetases, but reports on their physiological significance are mostly limited to improving male erectile dysfunction. Endogenous H2S synthetases are expressed in male testicles and spermatozoa, and may have important potential value in regulating male spermatogenesis and sperm function. So far, H2S has been used for male birth. The main objective of this study is to explore the protective effect of H2S on the process of spermatogenesis and sperm function, and to further study the possible mechanism of its role. First, we used a free radical detector to evaluate H2S in normal human seminal plasma and fertility. The differences in the content of the seminal plasma in the lower patients were compared. The differences in the expression of endogenous H2S synthase in different sources were compared by Western-blot. The results showed that the seminal plasma of the low level H2S was often accompanied by poor vitality, and the expression of endogenous H2S synthase CBS in the weak spermatozoa decreased significantly, suggesting that H2S might be involved in male infertility. Pathological process. We use animal experiments to further verify the significance of H2S for male reproduction: we use LPS induced inflammation and STZ induced diabetes to establish a common spermatogenesis disorder model. After the modeling success, two different H2S donors are given intraperitoneally to GYY4137 or NaHS. After the drug is finished, computer assisted semen analysis is used. The quality of spermatozoa in the tail of epididymis of mice was evaluated by CASA, and the changes of spermatogenic structure in mice testis were observed by HE staining. The results showed that H2S could significantly inhibit the decrease of sperm quality caused by inflammation and diabetes and improve the damage of testicular structure. The endogenous H2S synthase CSE and CB were detected by Real-Time PCR and Western-blot method. The level of mRNA and protein expression of S and the catalytic activity of CBS and CSE in testis were detected by chemical method. The results showed that exogenous H2S could improve the obstacle of endogenous H2S synthesis caused by inflammatory injury. CBS may be the limiting enzyme that affects endogenous H2S level in the testis. Frozen section of mice testis and DHE fluorescence staining The level of superoxide anion (02-) was measured. The results showed that H2S could inhibit the formation of.Tunel in testis 02, suggesting that H2S could reduce the apoptosis of spermatogenic cells caused by inflammation. The immunofluorescence assay of blood testis barrier closely linked protein, the results showed that H2S could maintain the serum testosterone level of mice by the complete.ELISA method of maintaining the blood testis barrier, and H2S could inhibit the inflammation. The decrease in serum testosterone levels caused by the disease. Further study the mechanism of H2S protection of spermatogenesis: the results of Real-Time PCR suggest that H2S reduces inflammation induced IL-1p, IL-6, TNF-amRNA level, increases the mRNA level of IL - 10, and improves the testosterone synthesis associated protein StA of inflammation. R, P450scc, 3 beta -HSD and P450c17mRNA levels decreased. The kit detected the MDA levels, GSH/GSSG, and the activity of SOD and Caspase-3 in the testis. The results showed that H2S could inhibit the increase of MDA levels caused by inflammation, and maintain GSH/GSSG level and SOD activity. The degradation of I kappa B and the nuclear transposition of NF- kappa B (gel migration rate experiments further confirmed that H2S inhibited the increase of NF- kappa B activity in the inflammatory state), inhibited the expression of iNOS, promoted the nuclear transposition of Nrf2 and enhanced the level of Nrf2 in the cytoplasm, so that H2S could increase the level of Bcl2/Bax. In order to further confirm the significance of endogenous H2S to testicular spermatogenesis, we constructed the mouse model of the right testicular tissue over expression of CBS by adenovirus transfection. Under the condition of LPS stimulation, the results of CSAS detection showed that the quality of the right epididymis in the right epididymis was significantly higher than that of the left epididymis by CBS. Meanwhile, the sperm quality was significantly higher than that of the left epididymis. At the same time, HE staining was found. The results of color and immunofluorescence staining suggest that under the condition of LPS causing inflammatory damage, CBS overexpressed right testicles, the integrity of the spermatogenic structure and the blood testis barrier have improved significantly compared with the left testis. We used human sperm cells to observe the effect of H2S on human sperm motility: CASA results showed endogenous H2S The enzyme CBS inhibitor AOAA significantly inhibits the activity of normal spermatozoa and the activity of hyperactivation. Exogenous H2S can improve the decrease of sperm motility and hyperactivation induced by the inhibition of CBS, and the appropriate concentration of H2S can improve the sperm vitality of the asthenosin patients with low H2S level in the seminal plasma. The results showed that: (1) H2S is of great significance for male sperm function. Exogenous supplementation of H2S helps to improve sperm dysfunction caused by H2S deficiency. 2. Experimental results showed that H2S could improve the decrease of sperm quality caused by inflammation and diabetes, protect the spermatogenesis structure of the testis, maintain the integrity of the blood testis barrier and improve the integrity of the blood testis barrier. The obstacle of testosterone synthesis to maintain normal spermatogenesis. (3) the improvement of spermatogenesis and dysfunction by H2S depends on the inhibition of the activity of NF- kappa B to control the level of inflammation in the testicular tissue; by promoting the nuclear transposition of the Nrf2, the expression of antioxidant factors is increased to inhibit the increase of oxidative stress in the testis tissue; The activation of apoptosis signaling pathway induced by mitochondria reduces the apoptosis of spermatogenic cells, regulates the synthesis of testosterone and the integrity of the blood testis barrier by inhibiting the activation of MAPK signaling pathway. This study provides a new way of thinking for the prevention and control of male fertility disorders.

【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R698.2

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6 汪文;做個(gè)準(zhǔn)爸爸，，你準(zhǔn)備好了嗎？[N];江蘇科技報(bào);2000年

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10 黃力;準(zhǔn)爸爸要早做準(zhǔn)備[N];保健時(shí)報(bào);2003年

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