【摘要】：二硫化碳(Carbon Disulfide, CS_2)是重要的化工溶劑和原料,它的使用范圍非常廣泛,因此人群和動(dòng)物很容易處于CS2暴露之中。研究報(bào)道,CS2對(duì)哺乳動(dòng)物多種器官、系統(tǒng)具有毒性作用。但在毒性作用機(jī)制的研究方面,關(guān)于CS2對(duì)神經(jīng)系統(tǒng)和心血管系統(tǒng)毒性研究的報(bào)道較多,而關(guān)于CS2對(duì)男(雄)性生殖毒性研究的報(bào)道匱乏。本研究通過體內(nèi)和體外實(shí)驗(yàn)探索CS2誘導(dǎo)雄性睪丸生殖細(xì)胞損傷的機(jī)制,通過隊(duì)列研究探討CS2的男性生殖毒性,并進(jìn)一步通過分子生物學(xué)實(shí)驗(yàn)研究精子細(xì)胞損傷的機(jī)制。為最終闡明CS2暴露誘導(dǎo)男(雄)性生殖細(xì)胞損傷的機(jī)制,及明確CS2職業(yè)暴露與男工性功能和性激素水平的關(guān)系提供理論基礎(chǔ)和科學(xué)依據(jù)。 第一部分線粒體凋亡通路在CS2誘導(dǎo)睪丸生殖細(xì)胞損傷中的作用 目的：探討CS2對(duì)雄性大鼠睪丸超微結(jié)構(gòu)和生殖細(xì)胞的影響,探索線粒體離子通道轉(zhuǎn)換在其中的作用和機(jī)制,并研究線粒體通透性轉(zhuǎn)換孔(MPTP)抑制劑CsA的干預(yù)作用。 方法：48只雄性SD大鼠采用隨機(jī)數(shù)法平均分為6組,前4組根據(jù)預(yù)實(shí)驗(yàn)以CS2遞增濃度(0、50、250、1250mg/m~3)進(jìn)行靜式吸入染毒,共染毒10周。后兩組設(shè)置為CsA(12.5mg/kg)對(duì)照組以及CS2(1250mg/m~3)+CsA(12.5mg/kg)干預(yù)組,動(dòng)物染毒后6周,在吸入CS2的同時(shí)采用灌胃攝入CsA。染毒結(jié)束后,用光鏡和電鏡觀察大鼠睪丸組織結(jié)構(gòu)和超微結(jié)構(gòu)的改變；使用相關(guān)試劑盒檢測(cè)細(xì)胞凋亡率、細(xì)胞內(nèi)鈣離子濃度(Ca2+)、活性氧自由基(ROS)濃度、線粒體跨膜電位(△ψm)、胞內(nèi)ATP含量和MPTP蛋白表達(dá)水平；實(shí)時(shí)定量PCR檢測(cè)相關(guān)基因mRNA表達(dá)水平：Western Blot檢測(cè)蛋白的表達(dá)水平。 結(jié)果：CS2暴露可造成睪丸生殖細(xì)胞超微結(jié)構(gòu)損傷,線粒體腫脹、空泡化；生殖細(xì)胞凋亡顯著增加,細(xì)胞內(nèi)Ca2+蓄積,ROS濃度升高,線粒體呼吸鏈復(fù)合物活性增加。同時(shí),△ψm、胞內(nèi)ATP含量和MPTP開放水平顯著下降。實(shí)時(shí)熒光PCR和Western blot分析結(jié)果顯示,Bcl-2基大mRNA和蛋白1的表達(dá)顯著降低,而Bax,Cyt C基因mRNA和蛋自的表達(dá)隨CS2染毒濃度的增加而增加。而且,MPTP抑制劑CsA對(duì)CS2誘導(dǎo)睪丸組織和細(xì)胞的損傷具有一定的拮抗作用。 結(jié)論：CS2可造成睪丸組織、細(xì)胞和線粒體超微結(jié)構(gòu)的損傷,并通過線粒體凋亡通路誘導(dǎo)睪丸生殖細(xì)胞凋亡。MPTP在其中發(fā)揮重要作用。另外,本研究結(jié)果提示CsA對(duì)CS2誘導(dǎo)睪丸細(xì)胞損傷可能具有潛在的治療作用。 第二部分內(nèi)質(zhì)網(wǎng)凋亡通路在CS2誘導(dǎo)睪丸支持細(xì)胞損傷中的作用 目的：建立睪丸支持細(xì)胞原代培養(yǎng)體系,并探索內(nèi)質(zhì)網(wǎng)凋亡通路是否在CS2誘導(dǎo)睪丸支持細(xì)胞凋亡中發(fā)揮作用。 方法：32只雄性SD大鼠隨機(jī)分為4組,以不同濃度CS2(0,50,250,1250mg/m3)靜式吸入染毒,共4周。染毒結(jié)束后,取大鼠部分睪丸組織制片觀察,其余部分睪丸組織進(jìn)行支持細(xì)胞原代培養(yǎng)。采用富爾根染色法對(duì)體外培養(yǎng)的支持細(xì)胞進(jìn)行鑒定,并在倒置顯微鏡下動(dòng)態(tài)觀察支持細(xì)胞在體外的生長(zhǎng)情況。對(duì)原代培養(yǎng)支持細(xì)胞進(jìn)行Caspase3活性、胞內(nèi)Ca2+濃度、內(nèi)質(zhì)網(wǎng)凋亡通路相關(guān)基因(Calpain2, Cleaved-Caspase12, GRP78和CHOP) mRNA和蛋白表達(dá)的檢測(cè)。 結(jié)果：暴露4周后,光學(xué)顯微鏡下觀察發(fā)現(xiàn)大鼠睪丸曲細(xì)精管結(jié)構(gòu)松散,細(xì)胞排列紊亂。電子顯微鏡觀察到睪丸支持細(xì)胞超微結(jié)構(gòu)損傷,染色質(zhì)變性,并發(fā)現(xiàn)內(nèi)質(zhì)網(wǎng)腫脹形成空泡。對(duì)支持細(xì)胞進(jìn)行原代培養(yǎng)發(fā)現(xiàn),CS2暴露組細(xì)胞凋亡率與對(duì)照組相比顯著增加,且凋亡率隨著CS2暴露濃度增加而增加。Caspase3活性和細(xì)胞內(nèi)Ca2+的濃度均顯著增加，內(nèi)質(zhì)網(wǎng)凋亡相關(guān)分子(Calpain2, Cleaved-Caspase12, GRP78和CHOP)的基因mRNA和蛋白表達(dá)顯著增加。 結(jié)論：本研究發(fā)現(xiàn)內(nèi)質(zhì)網(wǎng)凋亡通路在CS2誘導(dǎo)睪丸支持細(xì)胞凋亡中發(fā)揮重要作用。 第三部分職業(yè)性CS2暴露對(duì)男性工人性功能、性激素水平和精子質(zhì)量的影響 一、職業(yè)性CS2暴露對(duì)男工性功能和性激素水平的影響 目的：研究職業(yè)性CS2暴露對(duì)男工性功能和性激素水平的影響。 方法：對(duì)76名CS2暴露男工和94名非CS2暴露男工的基本情況和性功能進(jìn)行問卷調(diào)查：對(duì)其血清中性激素結(jié)合球蛋白(SHBG)、卵泡刺激素(FSH)、黃體生成素(LH)和睪酮(T)等激素水平進(jìn)行實(shí)驗(yàn)室檢測(cè)。并且對(duì)男工性功能和性激素水平與影響因素進(jìn)行單因素分析。 結(jié)果：男性工人性功能調(diào)查結(jié)果顯示,CS2暴露組男工存在性厭惡感和性功能障礙的人數(shù)比例顯著高于對(duì)照組男工,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。男性工人性激素水平檢測(cè)結(jié)果顯示,與對(duì)照組相比,CS2暴露組男工FSH和LH水平顯著增高,血清SHBG和T水平顯著降低(P0.05)。 結(jié)論：職業(yè)性CS2暴露可導(dǎo)致男性工人發(fā)生性功能障礙和性激素水平異常比例增高。 二、職業(yè)性CS2暴露對(duì)男工精子質(zhì)量的影響 目的：探討職業(yè)性CS2暴露對(duì)男工精子質(zhì)量的影響及導(dǎo)致?lián)p傷的分子機(jī)制。 方法：選擇人口學(xué)特征基本相似的CS2暴露男工76名和非CS2暴露男工94名,采集精液進(jìn)行精子常規(guī)分析、精液抗氧化能力、精子線粒體膜電位、MPTP蛋白表達(dá)和精子線粒體呼吸酶復(fù)合物等指標(biāo)的實(shí)驗(yàn)室研究。并且對(duì)男工精子質(zhì)量與影響因素進(jìn)行單因素分析。 結(jié)果：與對(duì)照組相比,職業(yè)性CS2暴露男工精液液化時(shí)間延長(zhǎng),精子畸形率升高,精子活率降低,精子活動(dòng)力減弱,精子細(xì)胞凋亡率和染色質(zhì)斷裂精子比例均升高。而且精液總抗氧化能力、精子蛋白表達(dá)和精子線粒體呼吸酶復(fù)合物Ⅱ和Ⅳ活性均降低。 結(jié)論：職業(yè)性CS2暴露可導(dǎo)致男工精子質(zhì)量顯著降低,而線粒體功能損傷是導(dǎo)致男性工人精子質(zhì)量降低的重要因素。
[Abstract]:Carbon disulfide (CS2) is an important chemical solvent and raw material. It has a wide range of applications, so people and animals are easy to be exposed to CS2. Studies have reported that CS2 has toxic effects on many mammalian organs. However, in the study of the mechanism of toxicity, CS2 on the nervous system and cardiovascular system. There are many reports on systemic toxicity, but there are few reports on the toxicity of CS2 to male (male) reproduction. This study explored the mechanism of CS2-induced male testicular germ cell damage in vivo and in vitro, explored the male reproductive toxicity of CS2 through cohort study, and further studied sperm cell damage through molecular biological experiments. The results provide theoretical and scientific basis for clarifying the mechanism of CS2 exposure induced male (male) sexual germ cell injury and the relationship between CS2 occupational exposure and sexual function and sex hormone level of male workers.
Part one the role of mitochondrial apoptotic pathway in CS2 induced testicular germ cell injury
AIM: To investigate the effects of CS2 on the ultrastructure and germ cells of testis in male rats, explore the role and mechanism of mitochondrial ion channel transition, and study the intervention of CsA, an inhibitor of mitochondrial permeability transition pore (MPTP).
Methods: 48 male SD rats were divided into 6 groups by random number method. The first 4 groups were treated with CS2 incremental concentration (0,50,250,1250 mg/m 3) by static inhalation for 10 weeks according to the preliminary experiment. After administration of CsA, the histological and ultrastructural changes of rat testis were observed by light and electron microscopy; the apoptosis rate, intracellular calcium ion concentration (Ca2+), reactive oxygen species (ROS), mitochondrial transmembrane potential (_m), intracellular ATP content and MPTP protein expression were detected by relevant kits; and the expression level of ATP and MPTP protein was measured by real-time quantitative PCR. The expression level of mRNA was detected by Western Blot.
Results: CS2 exposure could cause ultrastructural damage of testicular germ cells, mitochondria swelling and vacuolation; germ cell apoptosis increased significantly, intracellular Ca2+ accumulation, ROS concentration increased, and mitochondrial respiratory chain complex activity increased. The results showed that the expression of Bcl-2-based large mRNA and protein-1 decreased significantly, while the expression of Bax, Cyt-C gene mRNA and egg self-expression increased with the increase of CS2 concentration.
Conclusion: CS2 can damage the ultrastructure of testicular tissue, cells and mitochondria, and induce testicular germ cell apoptosis through mitochondrial apoptosis pathway. MPTP plays an important role in CS2-induced testicular germ cell apoptosis.
The second part is the role of endoplasmic reticulum apoptosis pathway in CS2 induced Sertoli cell injury.
Objective: To establish a primary culture system of testicular Sertoli cells and explore whether the endoplasmic reticulum apoptosis pathway plays a role in CS2-induced apoptosis of testicular Sertoli cells.
Methods: Thirty-two male SD rats were randomly divided into four groups and exposed to CS2 (0,50,250,1250 mg/m3) for 4 weeks. After exposure, some testicular tissues were taken for observation and the rest of testicular tissues were cultured for primary culture. Caspase 3 activity, intracellular Ca2+ concentration, mRNA and protein expression of endoplasmic reticulum apoptosis pathway related genes (Calpain 2, Cleaved-Caspase 12, GRP78 and CHOP) were detected in primary cultured Sertoli cells.
Results: After 4 weeks of exposure, the structure of testicular seminiferous tubules was loosened and the cells were disordered. The ultrastructural damage of Sertoli cells, chromatin degeneration and endoplasmic reticulum swelling were observed under electron microscope. The apoptosis rate of Sertoli cells in CS2 exposed group was compared with that in control group. Caspase 3 activity and intracellular Ca 2+ concentration increased significantly, and the mRNA and protein expression of endoplasmic reticulum apoptosis-related molecules (Calpain 2, Cleaved-Caspase 12, GRP78 and CHOP) increased significantly.
CONCLUSION: The endoplasmic reticulum apoptosis pathway plays an important role in CS2-induced apoptosis of Sertoli cells.
The third part is the effect of occupational CS2 exposure on sexual function, sex hormone levels and sperm quality in male workers.
Effect of occupational exposure to CS2 on sexual function and sex hormone levels in male workers
Objective: To study the effects of occupational CS2 exposure on sexual function and sex hormone levels in male workers.
Methods: Seventy-six CS2 exposed male workers and 94 non-CS2 exposed male workers were investigated by questionnaire. The levels of sex hormone binding globulin (SHBG), follicle stimulating hormone (FSH), luteinizing hormone (LH) and testosterone (T) in their serum were measured in laboratory, and the sex function and sex hormone levels in male workers were determined. Univariate analysis was performed.
Results: The sexual function survey showed that the proportion of male workers with sexual aversion and sexual dysfunction in CS2 exposed group was significantly higher than that in control group (P And T level decreased significantly (P0.05).
CONCLUSION: Occupational CS2 exposure can lead to sexual dysfunction and abnormal sex hormone levels in male workers.
Two, the effect of occupational CS2 exposure on sperm quality of male workers.
Objective: To explore the effect of occupational CS2 exposure on sperm quality and molecular mechanism of male workers.
Methods: Seventy-six CS2 exposed male workers and 94 non-CS2 exposed male workers with similar demographic characteristics were selected to collect semen for routine sperm analysis, antioxidant capacity, mitochondrial membrane potential, MPTP protein expression and mitochondrial respiratory enzyme complex. Univariate analysis was performed.
Results: Compared with the control group, CS2 exposed male workers had longer liquefaction time, higher sperm deformity rate, lower sperm motility, lower sperm apoptosis rate and higher percentage of chromatin breakage sperm. Sex decreased.
CONCLUSION: Occupational exposure to CS2 can result in significant reduction of sperm quality in male workers, and mitochondrial dysfunction is an important factor leading to the decrease of sperm quality in male workers.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R114

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