低铅暴露对睾丸细胞Nrf2与Mrp1表达的影响及铅对我国男性生殖健康影响的Meta分析

发布时间：2018-04-15 05:36

本文选题：铅 + 男(雄)性　；参考：《武汉大学》2013年博士论文

【摘要】：研究背景铅存在于人们生活与工作的环境中,由于它广泛分布,并能产生男(雄)性生殖毒性,在现代优生优育与男性生殖健康问题中极为重视。铅严重地影响了男性的生殖健康,它能通过血-睾屏障储留在睾丸组织中,并对睾丸组织产生直接的毒性损害。Nrf2是转录因子中CNC家族的成员,被认为是细胞内重要的氧化应激反应调节子,与抗氧化反应元件密切相关。Nrf2可通过激活Keapl-Nrf2-ARE信号通路对其下游靶基因进行调控,这包括一些抗氧化酶、Ⅱ相解毒酶、药物外排泵、清道夫受体及其转录调节子等的调节。Mrp1是一个ATP依赖的外排泵,被视为是有代表性的谷胱甘肽-S-共轭外排泵,能够向细胞外输送多种物质。现代研究表明,每一个有机体都有其自身的防御系统,某些重金属,诸如汞、镉等可以通过Mrpl实现自身机体的自我解毒防御作用,与Nrf2的激活有一定关系。Long等人研究也显示包括铅在内的一些有毒重金属能够诱导斑马鱼MRP1基因的转录表达。关于铅对男性生殖系统睾丸毒性作用机制至今仍不完全明了,目前在铅中毒防治过程中常规使用药物又因其副作用而受到不少质疑,进一步搞清铅的毒性作用机制并积极寻找有效的排铅等有效治疗方法意义重大。本研究通过开展体内实验和体外实验,探讨低铅环境中铅对雄性大鼠睾丸和小鼠睾丸支持细胞(TM4细胞)的Nrf2和Mrp1的影响,寻求可能的自身防御性排铅解毒机制；同时运用Meta分析方法对铅致我国男性生殖健康影响进行定量综合分析,为探寻新的、更有效的重金属铅的解毒途径提供理论依据。第一部分铅对我国男性生殖健康影响Meta分析目的研究铅对男性生殖健康的影响。材料与方法采用Meta分析方法对已在正式刊物上发表的有关铅对男性生殖健康影响的文献进行综合定量分析。结果铅接触男性阳痿、早泄的合并相对危险度(Relative risk,RR)分别为2.55(95%C1=1.39-4.70)、2.23(95%CI=1.38-3.61),而精子总数、精子畸形率和精子活动率的合并加权均数差(weighted mean difference,WMD)则分别为-13.39×109/L(95%CI=-21.49×109/L～5.30×109/L)、8.49%(95%CI=6.0l%～10.97%)和-8.29%(95%CI=-12.94%～-3.64%),差异均有统计学意义(P0.05)。结论铅可对男性的生殖健康造成损伤,引起阳痿率和早泄率的升高,精子总数的减少,精子畸形率的上升和精于活动率的下降。第二部分长期低铅作用对大鼠睾丸Nrf2与Mrpl表达的影响及其相关指标研究目的本研究旨在探讨低浓度铅长期慢性作用对大鼠睾丸Nrf2和MRP1表达的影响。材料与方法适应性喂养后,雌性SD大鼠随机分为3组,通过自由饮水染毒,从怀孕前10天至断乳各组分别用0,0.8和1.5g/L的醋酸铅喂养；然后取各组15只雄性子代也通过自由饮水染毒,从断奶到6月龄各组分别用0,0.3和0.9g/L的醋酸铅喂养。应用RT-PCR,蛋白质印迹法,免疫组化和组织免疫荧光法检测大鼠睾丸Nrf2与MRP1(?)勺表达水平及Nrf2的分布定位。采用原子吸收光谱法,分光光度法、酶标法分别测定睾丸组织铅含量、GST活力及GSH含量等。结果随着铅染毒剂量的增加,大鼠睾丸组织铅含量增高,GST活力与GSH含量下降,Nrf2和MRP1(?)勺表达均呈现上升趋势。Nrf2与MRP1在蛋白水平的表达与其在mRNA水平的表达基本一致。MRP1的相对表达量在各组间的差异都有显著性(P0.05),Nrf2的显著性差异存在于两低铅剂量组与对照组间(P0.05)。免疫荧光结果显示,染铅剂量越高,Nrf2核移位发生明显。结论MRP1在低铅染毒环境中可能通过激活Nrf2而促进了细胞内铅的外排来实现机体的去毒防御功能。第三部分低铅暴露下TM4细胞Nrf2对Mrp1表达的调节作用目的本部分旨在通过体外实验(细胞实验)对Nrf2分别应用激活剂tBHQ(?)口抑制剂ATRA处理,进一步探讨铅染毒生殖细胞(TM4细胞)可能存在的自身解毒防御机制。材料与方法利用CCK-8比色法检测不同浓度tBHQ和ATRA处理TM4细胞的存活率。建立空白对照组、20μM醋酸铅处理组、20μM醋酸铅tBHQ处理组及20pM醋酸铅+ATRA处理组4个细胞实验模型组,利用原子吸收光谱法测定各组细胞内的铅含量,qRT-PCR检测各组Nrf2与Mrp1的表达水平,同时应用细胞免疫荧光法观察Nrf2在细胞中TM4细胞内Nrf2的定位及变化。结果加入40μM tBHQ组的TM4细胞存活率与正常对照组比较无差异,不存在统计学差别(P0.05)；加入2μM ATRA组的TM4细胞存活率与对照组存活率比,稍有增加,但也无统计学意义(P0.05)。Nrf2与Mrp1两者的相对表达量在总的升降趋势上是基本一致的；与空白对照比,其它染铅毒组的Nrf2与Mrpl(?)分量均有上调,且都有统计学差异(P0.05)；与单纯低铅处理组比,同时加入tBHQ的组Nrf2与Mrpl表达都增加,其中Nrf2的表达在两者间有显著性差异(P0.05),而同时加入ATRA的组Nrf2与Mrpl表达都下降,其中Mrpl的表达在两者间有显著性差异(P0.05)。细胞免疫荧光检测中未见明显核转位,但同时加了tBHQ的组中Nrf2荧光强度感观上较其它3组有所增加。空白对照组细胞的铅含量很低；与对照组比,其它3组的细胞铅含量均有显著性的增高(P0.05)；与单纯低铅处理组比,同时加了tBHQ组的细胞铅含量减少,同时加了ATRA组的细胞铅含量增高,且有显著性变化(P0.05)。结论铅染毒TM4细胞可以通过Nrf2的激活上调Mrpl的表达,增强Mrpl的外排泵功能,从而起到细胞解毒作用。
[Abstract]:Background of the study

Nrf2 is a member of the CNC family in the transcription factor . It is considered to be a representative glutathione - S - conjugated efflux pump , which is closely related to the anti - oxidation reaction element . Nrf2 is a representative glutathione - S - conjugated efflux pump , which is closely related to the activation of the Nrf2 . Long et al studies also show that some toxic heavy metals including lead can induce the transcriptional expression of the MRP1 gene of zebrafish .

In this study , the effects of lead on the testis and mouse testis supporting cells ( TM4 cells ) of male rat testis and mouse testis supporting cells ( TM4 cells ) were investigated by in vivo and in vitro experiments .
At the same time , Meta - analysis is applied to the quantitative analysis of the effect of lead - induced male reproductive health , which provides the theoretical basis for exploring new and more effective ways of detoxification of heavy metal lead .

Meta - analysis on the Effect of Lead on Reproductive Health in China

Objective To study the effects of lead on male reproductive health . Meta - analysis was used to quantify the effects of lead on reproductive health of male . Results Lead exposure to male impotence , premature ejaculation and relative risk ( RR ) were 2.55 ( 95 % CI = 1.39 - 4.70 ) and - 8.29 % ( 95 % CI = - 12.94 % ~ - 3.64 % ) , respectively .

The Effect of Long - term and Low - Lead on the Expression of Nrf2 in Rat Testis and Its Related Indexes

Objective To investigate the effects of low concentration lead on the expression of Nrf2 and MRP1 in rat testis . After adaptive feeding , female SD rats were randomly divided into 3 groups .
The expression level of Nrf2 and MRP1 ( ? ) and the distribution of Nrf2 were determined by AAS , Western blotting , immunohistochemistry and immunofluorescence staining . The results showed that the higher the dose of lead exposed to lead , the higher the content of GST and GSH in the testis , the higher the expression of Nrf2 and MRP1 , the higher the expression of Nrf2 and MRP1 ( P0.05 ) . Conclusion The higher the dose of lead exposure , the higher the expression of Nrf2 and MRP1 ( ? ) , the higher the level of Nrf2 and MRP1 , the higher the Nrf2 nuclear translocation . Conclusion MRP1 may promote the external discharge of lead in the cells by activating Nrf2 .

Regulatory Effects of TM4 Cells Nrf2 on the Expression of Mr p1 in the Third Part of Low Lead Exposure

Objective To investigate the effects of different concentrations of tBHQ and ATRA on the survival rate of TM4 cells ( TM4 cells ) .
The survival rate of TM4 cells in 2渭M ATRA group was significantly higher than that in control group , but there was no statistical significance ( P0.05 ) .
Compared with the control of blank control , the components of Nrf2 in other lead - exposed groups were up - regulated and that of the other groups were statistically different ( P0.05 ) .
Compared with the simple low - lead treatment group , the expression of Nrf2 in the group of tBHQ was significantly different from that in the other three groups ( P0.05 ) , but the expression of Nrf2 was significantly different between the two groups ( P0.05 ) . However , there was no significant nuclear translocation in the expression of Nrf2 in the cell immunofluorescence assay , but the fluorescence intensity of Nrf2 in the group with tBHQ was higher than that in the other 3 groups .
Compared with the control group , the lead content of other three groups increased significantly ( P0.05 ) .
Compared with the simple low - lead treatment group , the lead content of the cells in the tBHQ group was decreased , and the lead content of the cells in the ATRA group was increased , and there was a significant change ( P0.05 ) .

【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R114

【参考文献】