【作者】 杨雯雯；

【导师】 陈小玉；

【作者基本信息】 郑州大学 ， 卫生毒理学， 2009， 硕士

【摘要】 近年来由于日益严重的环境污染,巨大的社会压力以及不良的生活习惯,不孕症的发病率明显上升,尤其是男性不孕症的发病率有逐年增加的趋势,影响了人类的健康与繁衍。其中环境污染是导致此现象的重要原因。能引起生殖障碍的环境污染物种类繁多,重金属是其中的一种。锰是动植物和人类所必需的微量元素之一,参与机体许多重要的生化反应,对维持机体的生命活动发挥着重要的作用,但同时它也是一种金属毒物。锰及其化合物用途非常广泛,多用于钢铁工业、装饰工程及地下工程的防腐支护,其化合物也是医药、化学制剂、油漆、焊接、合成工业等的重要原料。由于三羟基甲基锰燃料抗爆剂、杀真菌剂代森锰（MANEB）和核磁共振断层摄影术中的显影剂MnDPDP的应用,使人们在生活中大范围长期接触锰及其化合物的机会增多,锰的毒性作用受到人们的广泛关注。近年来,人们在对锰的神经和免疫毒作用认识的基础上,愈来愈关注锰的生殖毒性,并做了大量的研究。目前研究认为锰对雄性生殖系统存在明显的毒作用,若男性摄入过量锰可致睾丸组织发生病理学及生化酶学的改变。锰可通过降低睾丸抗氧化酶的活性,造成ROS（活性氧）在睾丸组织中蓄积而损伤生精细胞,从而诱导其凋亡。锰还可引起细胞内Ca²⁺稳态失衡和线粒体功能障碍,使细胞周期停滞,从而诱导睾丸间质细胞凋亡和类固醇激素合成的减少。本课题组以往的研究发现,锰可能通过减少睾丸间质细胞的数量而使睾酮分泌量减少,但是睾丸间质细胞数量减少的原因及其具体的机制还不清楚。本研究利用MA-10小鼠睾丸间质瘤细胞（MA-10 mouse Leydig tumor cells,mLTC-1）作为模型,观察MnCl₂对睾丸间质细胞增殖的影响,并通过细胞凋亡及参与凋亡调控的Bcl-2、Bax和Caspase-3基因表达水平的检测,初步了解其作用机制,为进一步研究其雄性生殖和发育毒性机制提供依据。方法1.MnCl₂对mLTC-1细胞活性的影响:采用MTT比色法将对数生长期的mLTC-1细胞以4×10⁴/ml接种于96孔板上。A:分别加入含不同浓度MnCl₂(0、10^-7、10^-6、10^-5、10^-4和10^-3mol/L)的培养液对细胞染毒,培养24h;B:分别加入含不同浓度MnCl₂（0、0.1、0.3、0.5、0.7和0.9 mmol/L）的培养液对细胞染毒,分别培养12h、24h、36h和48h。每个浓度设8个复孔,在酶标仪上测各孔OD值,确定染毒剂量和时间。2.TUNEL凋亡细胞原位检测法检测细胞凋亡:将对数生长期的mLTC-1细胞以4.0×10⁴/ml、每孔2ml接种于内含盖玻片的6孔培养板中,分别加入含不同浓度MnCl₂（0、0.3、0.5和0.7 mmol/L）的培养液染毒,24h取出爬片,TUNEL法检测细胞凋亡。3.mLTC-1细胞中Bcl-2、Bax和Caspase-3 mRNA表达水平:采用RT-PCR法将铺满瓶底80%左右的mLTC-1细胞分别加入含不同浓度MnCl₂（0、0.3、0.5和0.7 mmol/L）的无FBS的培养基,培养24h后,提取RNA,以β-actin基因为内参照,运用半定量RT-PCR技术检测各个染毒组MnCl₂对mLTC-1细胞Bcl-2、Bax和Caspase-3 mRNA的表达。结果1.MTT结果显示:A:10^-5mol/L～10^-7mol/L的MnCl₂处理组对mLTC-1细胞活性没有明显的影响;而10^-4mol/L和10^-3mol/L能明显抑制细胞的增殖,且与对照组相比,差异有统计学意义（P＜0.05）。B:不同浓度的MnCl₂作用24h后细胞的抑制率明显大于12h,差异有统计学意义（P＜0.05）;24h、36h和48h细胞抑制率的差异无统计学意义（P＞0.05）。2.TUNEL细胞原位检测结果显示:0.3、0.5和0.7 mmol/L MnCl₂作用24h,各个染毒组凋亡指数与对照组相比,差异均有统计学意义（P＜0.05）。0.5和0.7mmol/L染毒组凋亡指数均高于0.3mmol/L染毒组,差异均有统计学意义（P＜0.05）;0.5和0.7mmol/L染毒组的凋亡指数相比,差异有统计学意义（P＜0.05）。3.半定量RT-PCR结果显示:MnCl₂染毒组的Bcl-2 mRNA的表达量低于对照组表达量,且差异有统计学意义（P＜0.05）。MnCl₂染毒组的Bax和Caspase-3mRNA的表达量显著高于对照组,差异有统计学意义（P＜0.05）。结论本研究通过体外实验证实,MnCl₂在10^-4mol/L剂量以上即可对细胞产生毒性作用,抑制mLTC-1细胞增殖,并可有效的下调Bcl-2 mRNA的表达和上调Bax和Caspase-3 mRNA的表达。因而推测MnCl₂可能通过影响Bcl-2、Bax和Caspase-3基因的表达而调控mLTC-1细胞凋亡。更多还原

【Abstract】 ObjectiveIn recent years,because of the serious environment pollution,huge social pressure and improper living habits the incidence of infertility patients has raised significantly,and especially male infertility rate is increasing year by year which influences human health and procreation.Environmental pollution is an important reason for this phenomenon.A range of environmental pollutants can lead to reproductive disorders and heavy metal is one of them.Manganese is one of trace elements which are necessary for flora,fauna and humanity.It participates in many important biochemical reactions and plays an important role in maintaining life activity of human.But simultaneously it is also a kind of metallic toxicant. Manganese and its compounds are in wide use,which are extensively used in iron and steel industry,decorative works and underground engineering support of the anti-corrosion.Its compounds are important raw materials in chemical,medicine, welding,painting,synthetic industry and so on.Nowadays,the wide application of antiknock additive Methylcyclopentadienyl Manganese Tricarbonyl,fungicide Maneb and eikonogen MnDPDP in the tomography of nuclear magnetic resonance increase opportunities of long-term and wide range exposure of Manganese and its compounds in daily life.Therefore,people begin to pay more attention to manganese toxicity. Having a certain understanding of manganese toxicity on nerve and immune system, in recent years people become to pay more and more attention to reproductive toxicity and have done a lot of work.Current studies suggest that manganese can cause toxicological effects in male reproductive system obviously.Excessive ingestion of manganese would lead some changes of pathology and enzymology in testicle organization.Research showed that manganese can result in ROS accumulation in testicular tissue by lowering the activity of testicular antioxidant enzyme.Study also showed that manganese may induce rat spermatogenic cells apoptosis with relation to down-regulation of mRNA expression of Bcl-2.Studies showed that manganese can cause disturbance of calcium homeostasis,mitochondrial dysfunction and arresting of the cell cycle,thus induce apoptosis of primary Leydig cells and reduce steroidogenesis.Whether or not apoptosis of Leydig cells induced by manganese is concerned with apoptosis-related genes is still unknown.This study will use MA-10 mouse Leydig tumor cells （mLTC-1） as a model,and observation the adverse effect of manganese chloride on the mouse Leydig Tumor cells proliferation and apoptosis through measuring the level of Bcl-2,Bax and Caspase-3 expression,it will provide a theoretical basis for the mechanisms of reproductive and developmental toxicity in the future.Methods1.The cell viability of mLTC-1 affected by MnCl₂:The mLTC-1 cells of logarithmic phase were plated in 96-well plates.A:The cells were treated with manganese chloride at concentrations of 10^-7,10^-6,10^-5,10^-4 and 10^-3 mol/L for 24h;B: The cell was treated with manganese chloride at concentrations of 0.1,0.3,0.5,0.7and 0.9 mM for 12h,24h,36h and 48h.The control group was left untreated.Each concentration for six parallel way,the OD value was measured by MTT.2.The situ detection of apoptotic mLTC-1 cells with TUNEL assay:The mLTC-1 cells of logarithmic phase were cultured on 6-well plate which contains coverslips at a density of 8×10⁴ cells/well.After a 24h exposure to manganese chloride at concentrations of 0.3,0.5 and 0.7 mM,apoptotic cells were detected via TUNEL assay. 3.The mRNA level of Bcl-2,Bax and Caspase-3 in mLTC-1 cells was determined by semi-quantitative RT-PCR:The mLTC-1 cells which covered around 80%of the culture flask were treated with manganese chloride at concentrations of 0.3,0.5 and 0.7 mM for 24h.Total RNA was isolated from mLTC-1 cells,then the mRNA level of Bcl-2,Bax and Caspase-3 was measured by RT-PCR according to the manufacturer’s instruction.Results1.The results of MTT assay showed that:A:manganese chloride had no remarked effect on the viability of mLTC-1 cells at the concentrations of 10^-5,10^-6 and 10^-7mol/L,but could inhibit cells growth at the concentrations of 10^-3 and 10^-4mol/L and the differences was statistically significant compared with the control group（P＜0.05）.B:After exposure to different concentrations of manganese chloride at 24h inhibition ratios of mLTC-1 cells were greater than them at 12h significantly, the differences had statistically significant（P＜0.05）.But there had no statistically significant differences among 24h,36h and 48h（P＞0.05）.2.The result of TUNEL assay:TUNEL-positive cells were shown to be stained dark brown under the light microscope and nuclear condensation was observed. Apoptotic indexs were high after treated with 0.3,0.5 and 0.7 mM manganese chloride and the differences were statistically significant compared with the control group（P＜0.05）.At the concentrations of 0.5 and 0.7 mM apoptosis indexs were higher than at 0.3 mM and the differences were statistically significant（P＜0.05）;At the concentration of 0.7 mM apoptosis indexs were higher than at 0.5 mM and the differences were statistically significant（P＜0.05）.3.Effect of manganese chloride on mRNA Levels of Bcl-2,Bax and Caspase-3: Semi-quantitative RT-PCR results showed that the level of Bcl-2 mRNA in the mLTC-1 cells was decreased after treated with 0.3,0.5 and 0.7 mM manganese chloride and the differences are statistically significant compared with the control group（P＜0.05）.The level of Bax and Caspase-3 mRNA was increased and while the differences are statistically significant compared with the control group（P＜0.05）. ConclusionThis study demonstrated that manganese chloride had significant toxicity effect and can inhibit mLTC-1 cells growth and proliferation at the concentration of 10^-4 mol/L,and effectively inhibit the mRNA expression of Bcl-2 and up-regulate the mRNA expression of Bax and Caspase-3.Therefore manganese chloride-induced mLTC-1 cells apoptosis may be responsible for the down-regulated expression of Bcl-2 and the up-regulated expression of Bax and Caspase-3.更多还原