DNA损伤修复基因Xrcc1和代表性纳米材料对雄性生殖系统的影响及机制研究

发布时间：2018-04-28 13:27

本文选题：Xrcc1 + 精子发生　；参考：《南京医科大学》2017年博士论文

【摘要】：不孕不育一直是生殖健康研究领域一个亟待解决的重大科学问题。据世界卫生组织预测,在21世纪,不孕不育将成为仅次于肿瘤和心脑血管病的第三大疾病。研究表明,半个多世纪以来,人类精液质量显著下降,精子生成障碍已成为男性不育最常见的病因,也是我国成年男性生殖健康面临的最主要威胁。精子生成障碍可能与遗传因子改变,环境化学污染物暴露等密切相关。精子发生是一个连续的细胞分裂分化过程,包括精原细胞分裂增殖、精母细胞减数分裂和精子细胞变态三个主要阶段,最终形成成熟的精子。在这个复杂的过程中,生物体与生俱来的DNA损伤修复机制能够及时修复来源于外界环境或生物体自身产生的损伤。本实验室前期通过原发性无精子症病例对照研究发现,DNA损伤修复基因X射线修复交叉互补蛋白1(X-Ray Repair Cross Complementing 1,XRCC1)单核苷酸多态性是原发性无精子症的遗传易感因素,但无法解释精子生成障碍的分子机制。本研究首先构建了 Xrcc1基因敲除小鼠模型研究其在精子发生中的作用;由于人类遗传物质相对稳定,短时间内的人类的遗传变异是有限的,因此精子生成障碍也有可能受到环境因素的影响,环境因素种类众多,进而我们选择在生殖系统中缺乏安全性评价的纳米材料作为研究对象,来评价纳米材料对雄性生殖的影响。我们的研究假设是:1.DNA损伤修复基因Xrcc1在小鼠精子发生中有着至关重要的作用;2.代表性纳米材料可能对生殖细胞DNA造成损伤,进而影响精子发生过程。为验证上述假设:1.基于Xrcc1全敲除小鼠胚胎致死的报道,我们采用Cre-Loxp条件性基因敲除策略构建原始生殖细胞特异的Xrcc1基因敲除小鼠模型,综合评价该模型中小鼠精子发生过程及其状态;2.通过小鼠精原干细胞体外培养,探讨Xrcc1在其正常生理功能的作用;3.通过纳米材料的体外生殖细胞系的评价,探讨环境因素在精子生成障碍中的作用。第一部分DNA损伤修复基因Xrcc1在雄性小鼠生殖功能中的作用及机制研究目的XRCC1能够作为DNA损伤修复过程中的招募蛋白,与DNA损伤修复过程中的修复蛋白相互作用,发挥DNA损伤修复能力。以往研究表明DNA损伤修复过程在生殖细胞中的意义重大。然而,Xrcc1在维持精子发生以及生殖系统中的确切功能尚不清楚。方法我们通过Cre-Loxp原理构建原始生殖细胞条件性基因敲除Xrcc1的小鼠模型,结合生殖功能评价实验、病理学检测、以及分子机制研究,综合阐明Xrcc1在小鼠精子发生过程中的作用。结果通过交配生育实验发现Xrcc1完全敲除的小鼠出现不育的表型。与对照组相比,Xrcc1敲除组小鼠的精子数目和精子活力均显著降低。睾丸组织病理结果提示Xrcc1敲除组曲细精管有明显的病理改变。Xrcc1敲除组小鼠精原细胞发现凋亡的发生、睾丸组织氧化应激的增加和线粒体功能紊乱。此外,Xrcc1敲除组小鼠的精原细胞干性因子受到损伤。结论缺失Xrcc1后,小鼠的精原细胞干性发生紊乱,小鼠睾丸的线粒体功能受损以及出现凋亡现象。Xrcc1参与小鼠精子发生的正常生理过程。第二部分DNA损伤修复基因Xrcc1在小鼠精原干细胞中的作用及机制研究目的为进一步明确小鼠原始生殖细胞中Xrcc1的缺失,线粒体功能异常、及细胞内氧化应激状态的关系,采用体外精原干细胞(Spermatogonia Stem Cells,SSCs)模型来验证这一假设。方法通过建立SSCs模型,同时构建Xrcc1干扰RNA包装的腺病毒,通过腺病毒敲减SSCs中的Xrcc1,同时处理抗氧剂N-乙酰半胱氨酸(N-acety1-L-cysteine,NAC)来评价SSCs中的氧化应激状态及线粒体功能指标。结果Xrcc1缺乏可以引起SSCs的自我更新和自我分化因子减少,氧化应激相关指标增加,造成线粒体基因组上编码呼吸链复合物的转录本减少、细胞ATP水平降低,并且降低精子发生相关基因的表达。NAC的补充可以缓解因Xrcc1的减少而引起的氧化应激和线粒体基因组上编码呼吸链复合物的转录本减少和细胞ATP水平的降低,但对自我更新和自我分化因子和精子发生相关基因表达没有作用。结论在SSCs中,Xrcc1的减少可以通过氧化应激来影响细胞线粒体功能,以及通过其他途径来影响SSCs的干性维持和精子发生。第三部分代表性纳米材料暴露对小鼠精母细胞DNA的影响第一节多壁碳纳米管对小鼠精母细胞的影响目的多壁碳纳米管在许多领域广泛使用,已有研究报道其高剂量可以造成雄性小鼠生殖系统可逆性损伤。然而,低剂量多壁碳纳米管的生殖效应数据有限。方法运用鼠源性精母细胞系模型(GC-2spd)评价多壁碳纳米管的生殖毒性效应。采用细胞活力实验选择不影响细胞活力的剂量,透射电子显微镜观察多壁碳纳米管在细胞中的蓄积情况,PCR方法检测多壁碳纳米管对生殖细胞DNA的影响。结果发现0.5μg/mL的多壁碳纳米管在精母细胞系中不引起细胞活力降低。在这个剂量下,细胞周期和细胞活性氧(Reactive Oxygen Species,ROS)水平均没有影响。但是,与对照相比,该剂量下多壁碳纳米管能够蓄积在线粒体,引起精母细胞线粒体DNA的损伤,线粒体氧耗率和细胞ATP水平也有不同程度下降。结论0.5μg/mL多壁碳纳米管可以特异性的导致生殖细胞线粒体DNA损伤具潜在生殖毒性。第二节纳米氧化锌对小鼠支持细胞和精母细胞DNA的影响目的纳米氧化锌作为近年来在生活领域应用广泛的纳米材料,虽然大多数研究者聚焦于纳米氧化锌的人体健康效应,然而涉及雄性生殖系统的数据非常有限。方法以鼠源性支持细胞(TM-4)和精母细胞(GC-2spd)作为体外模型,研究亚致死剂量纳米氧化锌的生殖效应及其分子机制。运用分子生物学方法阐明纳米氧化锌对支持细胞功能和精母细胞DNA的影响。结果两个细胞模型中8μg/mL纳米氧化锌是亚致死剂量,ROS均增加。支持细胞的纳米氧化锌处理组,谷胱甘肽水平下降,丙二醛水平上升,支持细胞中血睾屏障相关蛋白(ZO-1,occludin,claudin-5和connexin-43)均下调;纳米氧化锌处理后出现细胞膜通透性增加和肿瘤坏死因子α分泌增多。精母细胞中出现细胞S期阻滞和DNA损伤,抗氧化剂NAC处理可部分挽救这一表型。结论纳米氧化锌暴露可引起细胞ROS产生、生殖细胞DNA损伤,以及下调支持细胞的血睾屏障相关蛋白,进而使得血睾屏障完整性被破坏。这些影响可以被抗氧化剂部分逆转,提示纳米氧化锌通过氧化应激途径发挥上述毒效应。
[Abstract]:Infertility has always been a major scientific problem to be solved in the field of reproductive health. According to the WHO, infertility will become the third largest disease after cancer and cardiovascular disease in twenty-first Century. Research shows that semen quality has decreased significantly in more than half a century and the disorder of spermatogenesis has become a male. The most common cause of infertility is the most important threat to the reproductive health of adult males in China. The disturbance of spermatogenesis may be closely related to changes in genetic factors and exposure to environmental chemical pollutants. Spermatogenesis is a continuous cell division and differentiation process, including spermatogonial cell division, meiosis and sperm fines. In this complex process, the inherent DNA damage repair mechanism can repair the damage caused by the external environment or the organism itself in this complex process. In the early stage of the laboratory, the DNA damage repair gene X shot was found by the primary azoospermia case control study. Single nucleotide polymorphisms of line repair cross complementary protein 1 (X-Ray Repair Cross Complementing 1, XRCC1) are genetic predisposing factors of primary azoospermia, but it is unable to explain the molecular mechanism of spermatogenesis disorder. This study first constructed a Xrcc1 gene knockout mouse model to study its role in spermatogenesis; due to human genetic material. The quality is relatively stable, and the genetic variation of human in a short time is limited, so the disturbance of spermatogenesis may also be affected by environmental factors and there are many environmental factors. Then we choose nanomaterials that lack safety assessment in the reproductive system as the research object to evaluate the effect of nano materials on male reproduction. The study assumes that the 1.DNA damage repair gene Xrcc1 plays a vital role in spermatogenesis in mice; 2. representative nanomaterials may cause damage to the germ cell DNA and then affect the process of spermatogenesis. To verify the hypothesis: 1. we use the Cre-Loxp conditional gene based on the death of the Xrcc1 full knockout mouse embryo. The knockout strategy was used to construct the Xrcc1 gene knockout mouse model of primordial germ cells, and to evaluate the spermatogenesis and the state of spermatogenesis in this model. 2. through the culture of mouse spermatogonial stem cells in vitro, the role of Xrcc1 in its normal physiological function was explored. 3. the environmental factors were discussed by the evaluation of the in vitro germ cell line of nanomaterials. The role of DNA damage repair gene Xrcc1 in the reproductive function of male mice and its mechanism research purpose XRCC1 can be used as a recruitment protein in the process of DNA damage repair, interacting with the repair proteins in the process of DNA damage repair, and exerting the ability to repair DNA damage. The previous study showed that the DNA injury was repaired. Complex processes are of great significance in germ cells. However, the exact function of Xrcc1 in maintaining spermatogenesis and reproductive system is still unclear. Methods we constructed a mouse model of primitive germ cell knockout Xrcc1 by Cre-Loxp principle, combined with the evaluation of reproductive function, pathological examination, and molecular mechanism research. The effect of Xrcc1 in the process of spermatogenesis in mice was elucidated. Results the male sterile phenotype of Xrcc1 completely knockout mice was found through the mating fertility experiment. Compared with the control group, the sperm number and sperm vitality of the Xrcc1 knockout mice were significantly lower. The pathological results of the testicular histology suggest that the Xrcc1 knockout group of seminiferous tubules has obvious pathology. The spermatogonial cells in the.Xrcc1 knockout mice found the occurrence of apoptosis, the increase of oxidative stress in the testicular tissue and the dysfunction of mitochondria. In addition, the spermatogonial factor of the spermatogonial cells in the Xrcc1 knockout mice was damaged. After the deletion of Xrcc1, the spermatogonial stem of the mice was disordered, the mitochondrial function of the mice was damaged and the loss of the spermatogonial function of the mice was damaged. The death phenomenon.Xrcc1 participates in the normal physiological process of spermatogenesis in mice. Second the role and mechanism of DNA damage repair gene Xrcc1 in mouse spermatogonial stem cells in order to further clarify the deletion of Xrcc1 in the primitive germ cells of mice, the abnormal function of mitochondria, and the relationship between the oxidative stress in cells and the use of spermatogonial stem in vitro. The Spermatogonia Stem Cells (SSCs) model is used to verify this hypothesis. By establishing a SSCs model, the adenovirus interfering with RNA packaging is constructed, the Xrcc1 in SSCs is knocked down by adenovirus and the antioxidant N- acetylcysteine (N-acety1-L-cysteine, NAC) is also treated to evaluate the oxidative stress state and mitochondrial function in the SSCs. Results Xrcc1 deficiency could cause the self-renewal of SSCs and the decrease of self differentiation factor and the increase of the related indexes of oxidative stress, which resulted in the decrease of the transcriptional book of the respiratory chain complex in the mitochondrial genome, the decrease of the cell ATP level and the reduction of the expression of.NAC related to the spermatogenesis related genes, which could relieve the decrease of Xrcc1. Oxidative stress and the decrease of transcriptional transcript of the respiratory chain complex encoded by the mitochondrial genome and the decrease of cell ATP levels, but no effect on self renewal and the expression of self differentiation and spermatogenesis related genes. Conclusion in SSCs, the decrease of Xrcc1 can affect cell mitochondrial function through oxidative stress and through it His approach affects the dry maintenance of SSCs and spermatogenesis. Third the effects of the representation of nanomaterials on mouse spermatocyte DNA: the first section of the effect of multi wall carbon nanotubes on mouse spermatocytes; multi wall carbon nanotubes are widely used in many fields. However, the reproductive effect of low dose multi wall carbon nanotubes was limited. The reproductive toxicity of multi wall carbon nanotubes was evaluated by the mouse derived spermatogonial model (GC-2spd). The cell viability experiment was used to select the dose which did not affect the cell viability, and the multi wall carbon nanotubes were observed in the cells by transmission electron microscope. The effect of multi wall carbon nanotubes on the reproductive cell DNA was detected by the PCR method. The results showed that the 0.5 u g/mL multi walled carbon nanotubes did not decrease the cell viability in the spermatocyte line. At this dose, the cell cycle and the level of Reactive Oxygen Species, ROS were not affected. The lower multi wall carbon nanotubes can accumulate in the mitochondria, cause the damage of the mitochondrial DNA of spermatocyte, the oxygen consumption rate of mitochondria and the ATP level of the cell also decrease in varying degrees. Conclusion 0.5 mu g/mL multi wall carbon nanotubes can specifically lead to the potential reproductive toxicity of the mitochondrial DNA damage in the germ cells. The second section of nano Zinc Oxide supports the mice. The effect of cellular and spermatocyte DNA on nano Zinc Oxide as a widely used nano material in the field of life in recent years. Although most researchers focus on the human health effects of nanoscale Zinc Oxide, the data involved in the male reproductive system are very limited. Methods using mouse derived support cells (TM-4) and spermatocyte (GC-2spd) are used as a method. In vitro model, the reproductive effect and molecular mechanism of sublethal dose of nano Zinc Oxide were studied. The effect of nanoscale Zinc Oxide on the function of support cell and DNA of spermatocyte was elucidated by molecular biological methods. Results in two cell models, 8 g/mL nanoscale Zinc Oxide was sublethal dose and ROS increased. The nano Zinc Oxide treatment of support cells In the group, the level of glutathione decreased, the level of malondialdehyde increased, and the blood testis barrier related proteins (ZO-1, occludin, claudin-5 and connexin-43) in the supporting cells were all downregulated. The membrane permeability and tumor necrosis factor alpha secretion increased after the nano Zinc Oxide treatment. The S phase block and DNA damage in the spermatocyte and the antioxidant NAC were found in the spermatocyte. Conclusion the nano Zinc Oxide exposure can cause ROS production, DNA damage of germ cells, and down regulation of blood testis barrier related proteins in the supporting cells, which can cause the integrity of the blood testis barrier to be destroyed. These effects can be reversed by the antioxidant part, suggesting that nanoscale Zinc Oxide plays a role in the oxidative stress pathway. The toxic effects mentioned above.

【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R114

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