PAHs致男（雄）性生殖损害中端粒和线粒体损伤效应和机制研究

发布时间：2018-01-16 10:43

本文关键词：PAHs致男（雄）性生殖损害中端粒和线粒体损伤效应和机制研究　出处：《第三军医大学》2017年博士论文　论文类型：学位论文

【摘要】：研究背景多环芳烃(Polycyclic aromatic hydrocarbons,PAHs)是一类重要的环境污染物,能通过呼吸、饮食、饮水和皮肤等多种途径进入人体。PAHs分布广泛,多种PAHs化学物并具有致癌性和致突变性,被国际癌症研究中心(IARC)划分为很可能的人类致癌物。此外,研究发现PAHs及其活性中间产物也能影响雄性生殖功能,最终导致不育。动物实验和人群流行病学研究均报道PAHs暴露水平升高与精液质量下降和精子DNA损伤相关,增加男性不育的风险。然而,PAHs对男性生殖能力的潜在影响仍然存在不确定性,尤其是在普通人群低剂量接触暴露中,PAHs暴露与雄性生殖损伤之间的弱效应,可能仅用常规的精液质量难以衡量,而需要寻找环境PAHs低暴露条件下致雄性生殖损伤的更敏感的生物标志物。此外,PAHs诱发雄性生殖毒性的分子事件及其潜在的机制仍不清楚。因此,对遗传损伤的分子机制进行溯源,发现PAHs造成生殖细胞损害的分子靶点,将为探索反映PAHs雄性生殖损害的生物标志物提供重要的依据。端粒和线粒体DNA在维持基因组完整性和细胞正常生理功能中起重要作用。既往的研究表明,PAHs能诱导细胞端粒DNA和线粒体损伤,提示端粒和线粒体结构和功能的异常可能是PAHs造成雄性生殖损害的重要靶点。本研究中,以本课题组开展的大学生生殖健康队列研究(MARHCS)为基础,分析PAHs环境暴露与精子端粒DNA和线粒体DNA损伤的关联。进一步选择PAHs的典型代表物B[a]P及其代谢产物BPDE开展体外和体内实验,探讨其对雄性生殖细胞端粒和线粒体结构和功能的损伤效应及其在雄性生殖损害中的作用及机制。研究内容1.重庆市大学生人群PAHs暴露与精子遗传损伤和精液质量的关联研究以本课题组在2013-2015年开展的MARHCS队列研究为基础,采用2014年第一次随访的人群(n=666)生物样本(尿液和精液)进行PAHs内暴露水平和精子多项损伤指标的评价。采用GC-MS检测尿液中PAHs代谢产物水平,荧光定量PCR检测精子端粒长度和精子mtDNAcn,JC-1检测精子线粒体膜电位,Long-PCR检测mt DNA完整性,Annexin V-FITC检测精子凋亡以及常规精液质量指标。在此基础上,分析了PAHs尿代谢产物与上述精子损伤相关指标的关联。通过人群研究探讨PAHs环境暴露致男性生殖损伤的生物标志物。2.BPDE和B[a]P诱导生精细胞衰老和凋亡过程中端粒损伤机制研究建立了BPDE染毒的小鼠精母细胞株GC-2模型。采用台盼蓝染色法检测细胞存活率,Annexin V/PI结合流式细胞仪分析检测细胞凋亡,SA-β-Gal染色的方法检测细胞衰老,用于评价BPDE对GC-2细胞的毒性效应;进一步采用荧光定量PCR和末端限制片段长度分析(TRF)的方法检测细胞端粒长度,FISH结合γ-H2AX免疫荧光方法检测端粒DNA断裂,TRAP-PCR-ELISA检测端粒酶活性以及Western blot检测端粒酶逆转录酶(TERT)的蛋白表达水平,用于评价BPDE对GC-2细胞的端粒损伤效应。另外,采用Western blot检测DNA损伤反应通路相关蛋白的表达,观察BPDE对GC-2细胞内DNA损伤反应的激活情况。在此基础上,采用sh RNA-TERT和p LV-EGFP-TERT载体构建了TERT干扰和再表达的GC-2转染细胞模型,研究TERT基因在BPDE诱导的细胞衰老和凋亡中的作用。在细胞实验结果基础上,我们建立了B[a]P(0、5、10和20 mg/kg)灌胃染毒SD大鼠7天动物模型,进一步验证体外GC-2细胞实验的结果。3.BPDE诱导生精细胞凋亡过程中线粒体损伤机制研究建立了BPDE染毒的GC-2细胞模型。采用NAO染色方法检测线粒体质量、RT-PCR检测mtDNAcn的改变、JC-1检测线粒体膜电位的变化、Western blot检测线粒体呼吸链关键蛋白COX IV、转录共激活因子PGC-1α以及线粒体凋亡通路相关蛋白,如Cyt C、caspase-9、caspase-3的表达水平,以评价BPDE对生精细胞的线粒体功能、线粒体生物合成和线粒体途径凋亡的影响。在此基础上,采用PGC-1α激活剂ZLN005预处理GC-2细胞后,检测BPDE对上述线粒体损伤相关指标的影响,以明确PGC-1α在BPDE诱导的生精细胞线粒体损伤和细胞凋亡中的作用。此外,利用上述构建的TERT干扰和再表达转染细胞,进一步研究BPDE诱导的生精细胞线粒体损伤效应,探讨端粒调控与线粒体损伤之间的联系,以明确TERT在BPDE诱导的生精细胞线粒体损伤中的作用。研究结果1.人群PAHs暴露与精子端粒长度和精子线粒体拷贝数的相关性本研究共检测了8种PAHs尿代谢产物,包括1-OHNap、2-OHNap、1-OHPhe、2-OHPhe、3-OHPhe、4-OHPhe、2-OHFlu和1-OHPyr。研究结果显示,尿中1-OHPyr和1-OHNap与精子端粒长度呈负相关,1-OHPyr(β=-0.385;95%可信区间[CI]:-0.749~-0.021;1-OHNap(-0.079;95%CI:-0.146~-0.011)。尿中2-OHPhe、3-OHPhe、∑Phe metabolites和2-OHFlu水平与精子mtDNAcn呈负相关,2-OHPhe(-9.427;95%CI:-17.586~-0.459);3-OHPhe(-11.488;95%CI:-19.462~-2.725);∑Phe metabolites(-9.635;95%CI:-17.965~-0.688);2-OHFlu(-11.692;95%CI:-19.647~-2.949)。尿中PAHs代谢物与精子线粒体膜电位和mt DNA完整性之间没有统计学上显著关联。此外,未观察到这8种PAHs尿代谢产物与精子凋亡和常规精液质量指标(包括精液量、精液密度、精子总数和精子前向运动百分率)之间的相关性。上述结果提示,低水平PAHs暴露可能造成精子端粒和线粒体损伤,并且精子端粒长度和精子mtDNAcn改变可能作为低剂量PAHs环境暴露致男性生殖损伤的敏感指标。2.BPDE和B[a]P诱导生精细胞衰老和凋亡过程中端粒损伤机制研究BPDE对GC-2细胞具有显著的毒性效应,能引起生精细胞增殖抑制、衰老和凋亡。进一步研究发现,BPDE处理能诱导GC-2细胞端粒损伤,包括端粒长度缩短,端粒DNA断裂,以及DNA损伤反应通路(ATM/Chk1/p53/p21)的激活。端粒酶是调节雄性生殖细胞端粒长度的重要因素,研究结果显示BPDE处理可引起细胞端粒酶活性以及端粒酶活性限速因子TERT的蛋白表达水平的降低,提示端粒结构和功能异常是BPDE诱导雄性生殖细胞损伤的靶点。此外,采用建立的TERT转染细胞模型发现,干扰TERT的表达,可加重BPDE诱导的端粒损伤、细胞衰老、细胞凋亡以及DNA损伤反应通路的激活,恢复TERT的表达则部分对抗了BPDE的损伤效应。这些结果提示TERT参与介导了BPDE诱导的端粒DNA损伤相关的生精细胞衰老和凋亡。此外,体内动物实验结果显示B[a]P染毒可引起大鼠生精细胞端粒缩短,TERT蛋白表达水平降低以及DNA损伤反应的激活,并诱发睾丸毒性,包括生精细胞凋亡和衰老,睾丸组织结构改变等,这些结果进一步验证了细胞实验结果。3.BPDE诱导生精细胞凋亡过程中线粒体损伤机制研究BPDE处理引起GC-2细胞线粒体质量降低,mtDNAcn下降,线粒体膜电位降低,线粒体生物合成和氧化磷酸化相关蛋白COX IV和PGC-1α的表达水平降低,线粒体凋亡途径相关蛋白Cyt C、caspase-9和caspase-3的表达升高,表明BPDE可导致线粒体功能损伤和生物合成障碍。GC-2细胞预处理PGC-1α激活剂ZLN005后降低了BPDE诱导的线粒体损伤和细胞凋亡,提示PGC-1α参与调控BPDE诱导的线粒体损伤相关的生精细胞凋亡。此外,进一步分析BPDE诱导的端粒与线粒体损伤的联系,采用TERT转染细胞模型研究显示:TERT抑制可加重BPDE诱导的线粒体损伤,包括线粒体质量、mtDNAcn、线粒体膜电位以及线粒体凋亡途径相关蛋白的表达.;相反,恢复TERT表达则部分对抗了BPDE对线粒体损伤效应,提示TERT通过调控PGC-1α的表达,参与调控BPDE诱导的线粒体损伤。研究结论流行病学研究显示,重庆市大学生人群中PAHs内暴露水平与精子端粒长度缩短和精子mtDNAcn降低存在显著的关联,提示精子端粒DNA和线粒体DNA可能是PAHs化学物造成男性生殖损伤的敏感靶点。小鼠精母细胞GC-2和大鼠染毒实验进一步证实,PAHs的典型代表物B[a]P及其活性代谢产物BPDE可诱导生精细胞的端粒和线粒体结构及功能异常,最终导致细胞的衰老和凋亡等生殖毒性。这一过程中,BPDE可抑制TERT表达,通过端粒损伤及DNA损伤反应途径造成生殖毒性;另一方面BPDE可抑制PGC-1α表达,通过线粒体损伤和凋亡途径造成生殖毒性。其中TERT基因在这两条途径中起关键调控作用。
[Abstract]:The research background of polycyclic aromatic hydrocarbons (Polycyclic aromatic, hydrocarbons, PAHs) is a kind of important environmental pollutants, through breathing, eating, drinking and skin and other ways to enter the body of.PAHs PAHs are widely distributed, a variety of chemical substances and has carcinogenicity and mutagenicity, by International Cancer Research Center (IARC) classified as probable human carcinogen. In addition, the study found that PAHs and its active intermediates can also affect male reproductive function, resulting in infertility. Epidemiological survey and animal experiments have reported that elevated PAHs exposure level associated with the decline in semen quality and sperm DNA damage, increase the risk of male infertility. However, the potential effect of PAHs on male reproductive ability still exists the uncertainty, especially in the general population exposure to low dose exposure, PAHs exposure and weak effect between male reproductive damage, may only use the conventional semen quality It is difficult to measure, and the need to find PAHs low environmental exposure induced more sensitive biomarkers of injury to male reproductive conditions. In addition, the molecular events of male reproductive toxicity induced by PAHs and its potential mechanism is still not clear. Therefore, the molecular mechanism of genetic damage to the source, the molecular target of germ cell damage caused by PAHs that will provide an important basis for exploring the biomarkers reflecting PAHs male reproductive damage. Telomere and mitochondrial DNA plays an important role in maintaining genomic integrity and normal cell physiological function. Previous studies indicated that PAHs cells and mitochondrial damage induced by telomere DNA, suggesting that telomere and mitochondrial structure and function abnormalities may be important target PAHs caused male reproductive damage. In this study, the research on College Students' reproductive health research group carried out the queue (MARHCS) based on analysis of environmental exposure and PAHs Related damage to sperm telomere DNA and mitochondrial DNA in vitro and in vivo. To carry out the typical B[a]P further choice of PAHs and its metabolite BPDE, investigate the male germ cells of telomere and mitochondrial structure and function in male reproductive damage effects and damage of the function and mechanism. The MARHCS cohort study associated with the sperm genetic damage and semen quality by our research group carried out in 2013-2015 exposure of PAHs 1. content of college students in Chongqing city population based, by 2014 the first follow-up group (n=666) in biological samples (urine and semen) to evaluate the PAHs exposure level and sperm number index. The metabolites of PAHs to detect the level of GC-MS in urine fluorescence quantitative PCR detection of sperm, sperm telomere length and mtDNAcn, JC-1 detection of sperm mitochondrial membrane potential, Long-PCR detection of MT DNA integrity, Annexin V-FIT C detection of sperm apoptosis and normal semen quality indicators. On this basis, analyzed the related indexes related to PAHs urinary metabolites and the sperm damage. Through the crowd of PAHs exposure induced male reproductive damage biomarkers of.2.BPDE and B[a]P induced spermatogenic cell aging and apoptosis in the process of telomere damage mechanism of mice spermatocyte cell line GC-2 induced by BPDE model. Using trypan blue staining assay cell viability, Annexin combined with V/PI cell apoptosis was detected by flow cytometry analysis, -Gal staining method of SA- beta cell senescence, for evaluation of BPDE on GC-2 cell toxicity; further analysis using fluorescence quantitative PCR and terminal restriction fragment the length (TRF) method for detection of telomere length of FISH cells, combined with gamma -H2AX immunofluorescence method for detecting telomere DNA fracture, TRAP-PCR-ELISA detection of telomerase activity and Wester N blot detection of telomerase reverse transcriptase (TERT) protein expression levels, for evaluation of the effect of BPDE on telomere damage of GC-2 cells. In addition, the expression of Western blot detected the DNA damage response pathway, activation of BPDE was observed in GC-2 cells in response to injury of DNA. On this basis, the model of TERT GC-2 transfected cells then the interference and expression by SH RNA-TERT and P LV-EGFP-TERT vector. The effect of TERT gene on BPDE induced cell senescence and apoptosis of the cells. Based on the experimental results, we established B[a]P (0,5,10 mg/ and 20 kg) by gavage for 7 days in SD rats animal model, further validation of GC-2 cells in vitro experiment results.3.BPDE induced apoptosis of spermatogenic cells in the process of mitochondrial damage mechanism of establishment of GC-2 cell model by BPDE. NAO was used to detect the quality of mitochondrial staining method for detection of mtDNAcn modified RT-PCR Change, change of mitochondrial membrane potential detection of JC-1, Western and blot to detect the mitochondrial respiratory chain key protein COX IV transcriptional coactivator PGC-1 alpha and mitochondrial apoptosis pathway related proteins such as Cyt, C, caspase-9, the expression level of Caspase-3, BPDE to evaluate the mitochondrial function of spermatogenic cells, affect mitochondrial biogenesis and mitochondrial pathway of apoptosis. On this basis, using PGC-1 alpha activators ZLN005 pretreated GC-2 cells, influence index correlation detection BPDE on the mitochondrial damage, in order to clear the PGC-1 alpha in BPDE induced spermatogenic cell mitochondrial damage and apoptosis. In addition, the constructed by TERT interference and expression in transfected cells, further research the effect of mitochondrial damage of spermatogenic cells induced by BPDE, between telomere regulation and mitochondrial damage linked to clear TERT in BPDE induced spermatogenic cell mitochondria The role of injury. Results: 1. people exposed to PAHs and sperm telomere length and sperm mitochondrial copy number between this study detected 8 PAHs urinary metabolites, including 1-OHNap, 2-OHNap, 1-OHPhe, 2-OHPhe, 3-OHPhe, 4-OHPhe, 2-OHFlu and 1-OHPyr. the results of study showed that urinary 1-OHPyr and 1-OHNap and sperm telomere length negative correlation, 1-OHPyr (beta =-0.385; 95% CI: [CI] -0.749~-0.021; 1-OHNap (-0.079; 95%CI:-0.146~-0.011). Urinary 2-OHPhe, 3-OHPhe, Phe, metabolites and 2-OHFlu and sigma level of sperm mtDNAcn was negatively correlated with 2-OHPhe (-9.427; 95%CI:-17.586~-0.459); 3-OHPhe (-11.488; 95%CI:-19.462~-2.725); Sigma Phe metabolites (-9.635; 95%CI:-17.965~-0.688); 2-OHFlu (-11.692; 95%CI:-19.647~-2.949). No statistically significant correlation between urinary PAHs metabolites and sperm mitochondrial membrane potential and MT DNA integrity. In addition, not Observed the 8 PAHs urinary metabolites and sperm apoptosis and sperm quality index (including semen volume, sperm density, sperm count and sperm motility). The correlation between these results suggest that low levels of PAHs exposure may cause sperm damage and mitochondrial telomere, telomere length and sperm and sperm mtDNAcn as may change low dose PAHs exposure induced male reproductive damage sensitive index.2.BPDE and B[a]P induce senescence and apoptosis of spermatogenic cells in the process of telomere damage mechanism of BPDE toxicity effect on GC-2 cells, can cause spermatogenic cell proliferation, senescence and apoptosis. Further studies showed that BPDE treatment can telomere GC-2 induced cell injury, including the length of the telomere, telomere DNA damage and DNA damage response pathway (ATM/Chk1/p53/p21). The activation of telomerase is regulated in male reproductive cell An important factor in the length, the results showed that BPDE treatment could induce lower expression of telomerase activity and telomerase activity rate limiting factor TERT protein, suggesting that telomere structure and function abnormalities are targets of male germ cells injury induced by BPDE. In addition, the TERT transfected cell model showed that expression of TERT interference, can aggravate the telomere BPDE damage induced cell senescence, apoptosis and activation of the DNA damage response pathway, the expression of TERT is part of the recovery against the damage effect of BPDE. These results suggest that TERT is involved in telomere DNA mediated BPDE induced injury of spermatogenic cell senescence and apoptosis related. In addition, in vivo animal experiments showed that B[a]P exposure induced rat spermatogenic cell telomere shortening, and reduce the expression level of activation of the DNA damage response protein TERT, and induced testicular toxicity, including apoptosis of spermatogenic cells And aging, testicular tissue structure changes, these results further verify the experimental results of cell apoptosis of spermatogenic cells in the process of mitochondrial damage mechanism of BPDE induced mitochondrial GC-2 cells induced by.3.BPDE quality decreased, mtDNAcn decreased the mitochondrial membrane potential was decreased and the expression level of mitochondrial biogenesis and oxidative phosphorylation related protein COX, IV and PGC-1 decreased alpha the mitochondrial apoptosis pathway related protein, Cyt C, increased expression of caspase-9 and Caspase-3, found that BPDE can cause.GC-2 damage and mitochondrial function disorder biosynthesis in cells pretreated with PGC-1 alpha activators ZLN005 reduced mitochondrial damage and apoptosis induced by BPDE, suggesting that PGC-1 is involved in the regulation of alpha BPDE induced mitochondrial injury related to apoptosis of spermatogenic cells. In addition, the further analysis of BPDE induced mitochondrial damage and telomere links using TERT transfected cell model Show: TERT can increase the inhibition of BPDE induced mitochondrial injury, including mitochondrial mass, mtDNAcn, mitochondrial membrane potential and mitochondrial apoptosis pathway related protein expression.; instead, restoration of TERT expression is part of against the damage effect of BPDE on mitochondria, suggesting that TERT regulate the expression of PGC-1 alpha, involved in the regulation of BPDE induced mitochondrial injury epidemiology. The research results show that, Chongqing City College Students PAHs exposure levels and sperm telomere shortening and sperm mtDNAcn decreased significant association, suggesting that sperm telomere DNA and mitochondrial DNA may be a sensitive target for male reproductive damage caused by PAHs. GC-2 chemical exposure experiments of spermatocytes in mice and rats confirmed that PAHs is the typical representative of B[a]P and its active metabolite BPDE can induce spermatogenic cell telomere and mitochondrial structure and function abnormalities, leading to fine The reproductive toxicity of cell senescence and apoptosis. In this process, BPDE can inhibit the expression of TERT by telomere damage and DNA damage response pathway caused by reproductive toxicity; on the other hand, BPDE can inhibit PGC-1 expression, mitochondrial damage and apoptosis caused by way of reproductive toxicity. The TERT gene plays a key role in the two in the way.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R114

【相似文献】