摘要
二硫化碳(Carbon disulfide, CS2)是一种应用广泛的有机溶剂,主要用于橡胶的硫化、谷物蒸熏、石蜡、石油的精炼以及粘胶纤维的制造等。CS2为多系统毒物,其对神经系统、心血管系统、视觉系统和生殖系统的影响已有报道。CS2对男(雄)性生殖系统的影响主要表现为性功能由亢进到减退,精子数减少,活力下降和畸形精子数增多等。本实验室在前期的动物实验中发现,CS2可引起雄性大鼠血清性激素水平发生改变,导致雄性大鼠下丘脑-垂体-性腺轴(Hypothalamic-pituitary-gonad axis, HPGA)功能紊乱。
性激素的分泌主要受HPGA的调节,通过一系列正反馈及负反馈作用来调节激素的分泌,使外周血激素的水平保持相对稳定,对维持生殖系统脏器发育、生殖功能及第二性征具有重要作用。一氧化氮(Nitric Oxide,NO)是一种兼有第二信使、神经递质和效应分子等多种生理功能的生物信号分子,在生物体内发挥着十分重要的作用。NO广泛分布于男性生殖系统的各个器官,对生殖的调节作用具有双重性。研究表明,NO参与了整个HPGA的平衡调节,NO合成的多寡是机体生殖内分泌功能紊乱的重要步骤。本实验室前期研究发现CS2可造成雄性大鼠睾丸组织的氧化损伤,导致睾丸组织中NO含量、总一氧化氮合酶(NOS)活力和诱导型NOS(iNOS)活力下降,因而推测,NO可能在CS2导致的HPGA功能紊乱的过程中发挥重要作用。
目前,有关CS2对男(雄)性HPGA影响的研究鲜有报道,其具体的分子调控机制尚不清楚。本项目以CS2为受试物,以HPGA为研究对象,将职业人群调查、整体动物实验和体外细胞培养相结合,探讨CS2对男(雄)性HPGA功能的影响及其机制,并探讨NO供体硝普钠(Sodium Nitroprusside, SNP)和总NOS抑制剂N-甲基-L-精氨酸(NG-Monomethyl-L-arginine,L-NMMA)的干预作用,为进一步阐明NO对生殖内分泌的作用,丰富对CS2致男(雄)性生殖损伤分子机制的认识提供科学依据。
第一部分CS2职业接触人群调查研究
一、CS2职业暴露对男工性功能和生殖结局的影响
目的：探讨CS2对接触男工性功能及生殖结局的影响。
方法：对276名CS2暴露男工和126名非CS2暴露男工的基本情况、性功能及其配偶生殖结局进行调查研究。
结果：性功能调查结果显示,CS2暴露组男工性生活和谐比例及性生活频度降低,性生活厌恶感增加(P<0.05)；各组间男工妻子妊娠并发症发生率无统计学差异(P>0.05)。生殖结局调查结果表明,混合组(夫妻双方均接触CS2)的自然流产率与单纯组(只有男工接触CS2而其妻子不接触)和对照组比较均具有统计学差异(P<0.05)；混合组与对照组比较,子代低出生体重发生比例增高(P<0.05),子代智力发育及生长发育无统计学差异(P>0.05)。
结论：CS2职业暴露可导致男性性功能障碍和不良生殖结局,但对子代的智力和生长发育影响不明显。
二、CS2职业暴露对男工血清性激素水平的影响
目的：探讨CS2对职业接触男工血清性激素水平的影响。
方法：选择工龄、文化程度、生活条件等基本情况相似的CS2暴露男工63名和非CS2暴露男工69名,对其血清GnRH、FSH、LH和T水平进行检测。
结果：工龄2～8年的CS2暴露组男工,血清T和LH水平明显高于对照组(P<0.05);工龄8年以上的CS2暴露组男工,血清T、FSH、GnRH水平明显高于对照组,具有统计学差异(P<0.05)。
结论：职业接触CS2可导致男性生殖内分泌功能紊乱,血清LH水平首先发生改变,可作为反映CS2生殖损伤的早期指标。
第二部分CS2对大鼠下丘脑-垂体-性腺轴超微病理结构的影响以及NO的干预作用
目的：探讨CS2对雄性大鼠下丘脑-垂体-性腺轴超微病理结构的影响及NO的干预作用。
方法：36只雄性SD大鼠随机分为6组,以不同浓度CS2(0、50、250、1250 mg/m3)静式吸入染毒,共10周,另设CS2(1250 mg/m3)+SNP(5mg/kg)和CS2(1250 mg/m3)+L-NMMA(2mg/kg)干预组,SNP和L-NMMA从动物染毒结束前10 d开始腹腔注射,1次/d。染毒结束后,采用透射电镜观察大鼠下丘脑、垂体和睾丸组织超微病理结构的改变。
结果：CS2染毒可造成下丘脑神经元、垂体促性腺激素细胞、生长激素细胞和睾丸支持细胞线粒体肿胀,内质网扩张,NO供体对CS2引起的下丘脑、垂体、睾丸组织的损伤具有拮抗作用,而NOS抑制剂则进一步导致病变的发生。
结论：CS2可造成下丘脑、垂体和睾丸组织超微病理结构的改变,NO在这过程中发挥重要作用。
第三部分大鼠下丘脑神经元和垂体前叶细胞体外培养方法的建立
一、新生大鼠下丘脑神经元原代培养和鉴定
目的：探讨大鼠下丘脑神经元体外培养技术。
方法：选用新生SD大鼠,取下丘脑组织进行单细胞原代培养,倒置显微镜下动态观察培养细胞在体外生长的情况,并采用尼氏染色法对体外培养的神经元进行鉴定。
结果：培养3d时观察发现下丘脑神经元具有双极突起,突起较短；培养7d时神经元突起长度达到最高峰；尼氏染色可见胞质内有颗粒状的尼氏体,呈深蓝色。
结论：本实验室采用的大鼠下丘脑神经元原代培养方法能够为开展神经内分泌的分子生物学研究提供理想的体外实验模型。
二、大鼠垂体前叶细胞体外培养和鉴定
目的：探讨大鼠垂体前叶细胞体外培养技术。
方法：选用成年雄性SD大鼠,取垂体前叶进行单细胞原代培养,倒置显微镜下动态观察培养细胞在体外生长的情况,并采用免疫细胞化学染色技术对垂体前叶细胞LH和FSH分泌细胞进行鉴定。
结果：垂体前叶细胞呈圆形,折光性较强,成纤维细胞生长明显,原代培养后期成纤维细胞已为主要细胞。免疫细胞化学染色结果显示,LH和FSH免疫阳性细胞胞浆呈棕黄色。
结论：体外培养的垂体细胞中有多种细胞的生长,反复差速贴壁法能较好的去除成纤维细胞,提高垂体前叶细胞的纯度,但LH和FSH免疫反应阳性细胞比例较低。
第四部分CS2致雄性大鼠下丘脑-垂体-性腺轴功能紊乱机制的研究
一、CS2对大鼠下丘脑-垂体-性腺轴激素和激素受体mRNA表达水平的影响以及NO的干预作用
目的：探讨CS2对大鼠下丘脑-垂体-性腺轴激素和激素受体mRNA表达水平的影响以及NO的干预作用
方法：对体外培养的大鼠垂体前叶细胞进行染毒,共设6个染毒组,分别为对照组、3个不同浓度CS2染毒组(2.5、5.0、10.0μmol/ml CS2)、SNP干预组(10.0μmol/ml CS2+20μmol/ml SNP)和L-NMMA干预组(10.0μmol/ml CS2+50μmol/ml L-NMMA),支持细胞的6个染毒组分别为对照组、3个不同浓度CS2染毒组(1.25、2.50、5.00μmol/ml CS2)、SNP干预组(5.00μmol/ml CS2+10μmol/ml SNP)和L-NMMA干预组(5.00μmol/ml CS2+25μmol/ml L-NMMA),采用Real-time PCR对垂体前叶细胞中GnRHR mRNA和睾丸支持细胞中FSHR、ABP、INH mRNA表达水平进行检测。
结果：垂体前叶细胞中GnRHR mRNA表达水平在10.0μmol/ml CS2染毒时显著降低(P<0.05),NOS抑制剂L-NMMA可拮抗CS2的作用,使GnRHR mRNA表达水平显著增高(P<0.05)；在睾丸支持细胞中,不同浓度CS2染毒组与对照组比较,ABP和INHαmRNA表达水平没有明显变化(P>0.05),5.0μmol/ml CS2染毒组FSHR和INHpb mRNA表达水平显著降低,INHpa mRNA表达水平显著增高(P<0.05)；NO供体SNP干预组与5.0μmol/ml CS2染毒组比较,INHa mRNA表达水平显著增高(P<0.05),ABP、FSHR、INHβa和INHβb mRNA表达水平改变均不明显(P>0.05)；NOS抑制剂L-NMMA干预组与5.0μmol/ml CS2染毒组比较,ABP mRNA表达水平显著增高(P<0.05),FSHR、INHα、INHβa和INHβb mRNA表达水平改变均不明显(P>0.05)。
结论：CS2可抑制垂体前叶细胞中GnRHR、支持细胞中FSHR和INHβb的表达,通过抑制HPGA中多种激素和激素受体的合成,导致HPGA功能紊乱,CS2对垂体GnRHR的抑制作用与NOS活性有关。
二、CS2对大鼠下丘脑神经元、垂体前叶细胞和睾丸支持细胞内cAMP/cGMP的影响以及NO的干预作用
目的：探讨CS2对大鼠下丘脑神经元、垂体前叶细胞和睾丸支持细胞内cAMP/cGMP的影响以及NO的干预作用。
方法：对体外培养的大鼠下丘脑神经元和垂体前叶细胞进行染毒,共设6个染毒组,分别为对照组、3个不同浓度CS2染毒组(2.5、5.0、10.0μmol/ml CS2)、SNP干预组(10.0μmol/ml CS2+20μmol/ml SNP)和L-NMMA干预组(10.0μmol/ml CS2+50μmol/ml L-NMMA),支持细胞的6个染毒组分别为对照组、3个不同浓度CS2染毒组(1.25、2.50、5.00μmol/ml CS2)、SNP干预组(5.00μmol/ml CS2+10μmol/ml SNP)和L-NMMA干预组(5.00μmol/ml CS2+25μmol/ml L-NMMA),采用酶联免疫吸附试验对下丘脑神经元、垂体前叶细胞和睾丸支持细胞中cAMP和cGMP水平进行检测。
结果：下丘脑神经元中,各染毒组cAMP水平变化不明显(P>0.05),cGMP水平在10.0μmol/ml CS2染毒组显著降低,SNP可拮抗CS2的作用,使cGMP水平显著增高(P<0.05)；垂体前叶细胞中cAMP和cGMP水平不随CS2染毒浓度的增加而发生改变(P>0.05),但L-NMMA干预组与10.0μmol/ml CS2染毒组比较,细胞中cAMP和cGMP水平均显著增高(P<0.05)；睾丸支持细胞中各染毒组cAMP和cGMP水平均无明显改变(P>0.05)。
结论：CS2可明显降低大鼠下丘脑神经元中cGMP水平,并能被SNP拮抗,但对垂体和睾丸cAMP和cGMP没有影响,提示CS2可能通过NO-cGMP途径影响下丘脑分泌功能,而CS2对垂体和支持细胞分泌功能的影响则不依赖于cAMP和cGMP。
Carbon disulfide (CS2), a volatile organic solvent, is often used in various industrial processes, such as vulcanizing rubber, fumigating grain, extracting oil, and manufacturing viscose rayon fibers. CS2 adversely affects nearly all organ systems. There are many reports concerning CS2 toxicity particularly in peripheral and central nervous systems, cardiovascular system, ophthalmological system, and even reproductive system. Many epidemiological studies showed that CS2 could affect male sexual function, such as change of sexual function from sthenic to decrease, decrease of sperm count and sperm motility and increase of the ratio of sperm deformity. Preliminary study in our laboratory showed that CS2 can lead to secretion disorders of hypothalamus-pituitary-gonadal axis (HPGA) of male rat.
Sex hormones secretion is mainly affected by the regulation of HPGA which makes peripheral hormone levels remain relatively stable through positive feedback and negative feedback effect of HPGA to regulate endocrine hormone. HPGA plays an important role in reproductive system viscera development, reproductive function and secondary sex characteristics. Nitric oxide (NO) is a kind of signal molecule which may have muhiplicate physiological functionsuch as secondary messenger, neurotransmitter and effect molecule. NO may distribute in almost all kind of organs of male reproductive system, which may have the function of bifunctional regulation for reproduction. There are many reports shows that, NO participated in the whole HPGA adjustment, the amount of NO is an important step of the reproductive endocrine dysfunction. In our previous studies, CS2 exposure could lead to decrease of the NO concentration in testicular tissue, total nitric oxide synthase (NOS) activity and inducible NOS (iNOS) activity. Therefore, we speculated that NO plays an important roal in the effects of CS2 on the disorders of the HPGA in male.
However, the mechanism by which CS2 exposure causes disorders of HPGA in male remains unknown. The aim of the present study was to determine the effects of CS2 on HPGA in male, and the intervention of NO donor (sodium nitroprusside, SNP) and NOS inhibitor (N-methyl-L-arginine, L-NMMA), in order to further clarify the role of NO in reproductive endocrine system and pave the way for further research on mechanisms of CS2 toxicity in male reproductive system.
Part I Research of workers exposed to carbon disulfide
1、Effect of carbon disulfide on male sexual function and reproductive outcomes
Objective:To investigate the sexual function and reproductive outcomes of male workers exposed to CS2.
Methods:In a retrospective study, the basic information, sexual function and reproductive outcomes of 276 workers exposed to CS2 were collected through a self-administered questionnaire. Another 126 unexposed male workers set as control.
Results:The sexual function investigation showed that male workers exposed to CS2 had sexual unsatisfactory, decreased sexual frequency, and increased sex aversion (P<0.05). The rates of pregnancy complications of the wives in all groups had no statistical significance (P>0.05).The rate of spontaneous abortion of the mixed exposure group (both of male workers and their wives exposed to CS2) was significantly increased than that of the single exposure (male workers exposed to CS2 but their wives did not) and control group (P<0.05). The rate of low birth weight infants of the mixed exposure group had significant increased than that of the control group (P<0.05). Offsprings'intelligence and physical development had no significant difference in all groups (P>0.05).
Conclusion:Occupational exposure to CS2 had an effect on the attitude and frequency of sex life of male workers, and may result in bad reproductive outcomes as well. But there were no significant changes of development in offsprings.
2、Levels of sex hormones in male workers exposed to carbon disulfide
Objective:To examine the effects of CS2 on the levels of sex hormones in male workers.
Methods:The study subjects were 63 male workers exposed to CS2 and 69 non-exposed workers. The levels of gonadotropin-releasing hormone (GnRH), follicle stimulating hormone (FSH), luteinizing hormone (LH) and testosterone (T) in serum were determined to assess endocrine function. Potential confounding factors were adjusted for.
Results:The levels of serum LH and T in the exposed workers who exposed to CS2 for 2-8 years were significantly higher than that of the control group (P<0.05). The levels of serum GnRH, FSH and T in the exposed workers who exposed more than 8 years were significantly higher than that of the control group (P<0.05).
Conclusion:Occupational exposure to CS2 could induce endocrine dysfunction in male workers. The level of serum LH which changes primarily can be used as the early indicators reflect injury of reproductive function induced by CS2.
PartⅡUltrastructure of hypothalamus-pituitary-gonad axis in carbon disulfide-treated rats and the intervention of NO
Objective:To observe the ultrastructural changes of HPGA in carbon disulfide-treated rats and the intervention of NO.
Methods:36 Sprague-Dawley male rats were randomly divided into 6 groups. Four groups were treated with CS2 at doses of 0,50,250 and 1250 mg/m3 by inhalation for 10 weeks. The rest two groups were intraperitoneal injection with SNP (5 mg/kg) and L-NMMA (2 mg/kg) once a day for 10 days after exposed to CS2 at a concentration of 1250 mg/m3 for 8 weeks. Ultramicropathology technique was used to detect ultrastructure of hypothalami, pituitaries and testes in rats.
Results:Ultramicropathology technique analysis showed that CS2 caused ultrastructural changes of hypothalamic neurons, gonadotropin cells, growth hormone cells and Sertoli cells in male rats. Mitochondria became sweller and endoplasmic reticulum widened. SNP could decrease the effects of CS2 on ultrastructural changes. The effects were adverse while injected with L-NMMA.
Conclusion:CS2 could induce ultrastructural changes of hypothalamus, pituitary and testis in male rat, which is mediated by NO.
PartⅢCulture of rat hypothalamus neurons and anterior pituitary cells in vitro
1、Culture and identification of newborn rat hypothalamus neurons in vitro
Objective:To find out the best techniques for culture of nerve cells from hypothalamus in vitro.
Methods:We conducted primary culture of Hypothalamus neurons of newborn SD rat and identified the neurons by Nissl staining. We made dynamic observation on cultured cells by inverted microscope examination.
Results:The processus of nerve cells isolated from the rat hypothalamus were found on the third day of culture, and then reached their highest levels on the 7th day. Nissl staining showed Nissl's body dyeing was thick blue and granule formed.
Conclusion:The method for culture of hypothalamal nerve cells were established successfully and could be used as an ideal experimental model on neuroendocrine molecular biology research.
2、Culture and identification of rat anterior pituitary cells in vitro
Objective:To find out the best techniques for culture of rat anterior pituitary cells in vitro.
Methods:Anterior pituitary of adult SD male rats were isolated for single cell primary culture. We made dynamic observation on cultured cells by inverted microscope examination. Immunocytochemistry method was used to identify expression of LH and FSH.
Results:Anterior pituitary cells were round and had strong refraction. Fibroblasts grew obviously and became the cell in majority in the late stage of the primary culture cells. Immunochemistry staining showed that LH and FSH positive product was brown in cytoplasm.
Conclusion:There were variety hormone cells in cultured pituitary cells. The repeated differential adhesion method could remove fibroblasts and sulted in better purification of anterior pituitary cells. LH and FSH positive cells accounted low proportion in cultured pituitary cells.
Part IV Mechanism research on secretion disorders of HPGA induced by CS2 in male rat
1、Effect of CS2 on hormone and hormone receptor mRNA in rat cells in vitro and the intervention of NO
Objective:To investigate effect of CS2 on GnRH, ABP, FSHR and INH mRNA in rat cells in vitro and the intervention of NO.
Methods:The cultured anterior pituitary cells were divided into six groups:control group; three various concentrations of CS2 groups (2.5,5.0,10.0μmol/ml);SNP group (10.0μmol/ml CS2+20μmol/ml SNP) and L-NMMA group (10.0μmol/ml CS2+50μmol/ml L-NMMA). The six groups of Sertoli cells were control group, three various concentrations of CS2 groups (1.25,2.50,5.00μmol/ml), SNP group (5.00μmol/ml CS2+10μmol/ml SNP) and L-NMMA group (5.00μmol/ml CS2+25μmol/ml L-NMMA). Real-time PCR was used to detect GnRHR mRNA in anterior pituitary cells and ABP, FSHR, INH mRNA in Sertoli cells of rat.
Results:The mRNA level of GnRHR in rat anterior pituitary cells was significantly lower in 10.0μmol/ml CS2 group than that of the control group, which could be attenuated by L-NMMA (P<0.05). In Sertoli cells of rat, no significant effects of ABP and INHαmRNA were observed in various concentrations of CS2 groups (P>0.05). In the 5.0μmol/ml CS2 group, FSHR and INHβb mRNA expression level were significantly lower and INHβa mRNA expression level was significantly higher than that of the control group (P<0.05). In the SNP group, INHa mRNA expression level was significantly higher than that of the 5.0μmol/ml CS2 group (P<0.05), but ABP, FSHR, INHβa and INHβb mRNA were no significant differences between these two groups (P>0.05). In the L-NMMA group, ABP mRNA expression level was significantly higher than that of the 5.0μmol/ml CS2 group (P<0.05), but no significant effects of FSHR, INHα, INHβa and INHβb mRNA were observed between these two groups (P>0.05).
Conclusion:CS2 could induce secretion disorders of HPGA in male rat by inhibit synthesis and secretion of various hormones and hormone receptors including GnRHR in pituitary and FSHR, INHβb in Sertoli cells. Inhibition of GnRHR in pituitary induced by CS2 related to activity of NOS.
2、Effect of CS2 on cAMP and cGMP in rat hypothalamus neurons, anterior pituitary cells and Sertoli cells, and the intervention of NO
Objective:To investigate effect of CS2 on cAMP and cGMP in rat hypothalamus neurons, anterior pituitary cells and Sertoli cells, and the intervention of NO.
Methods:The cultured hypothalamus neurons and anterior pituitary cells were divided into six groups:control group; three various concentrations of CS2 groups (2.5,5.0,10.0μmol/ml); SNP group (10.0μmol/ml CS2+20μmol/ml SNP) and L-NMMA group (10.0μmol/ml CS2+50μmol/ml L-NMMA). The six groups of Sertoli cells were control group, three various concentrations of CS2 groups (1.25,2.50,5.00μmol/ml), SNP group (5.00μmol/ml CS2+10μmol/ml SNP) and L-NMMA group (5.00μmol/ml CS2+25μmol/ml L-NMMA). Enzyme-linked immuno sorbent assay (ELISA) was used to detect cAMP and cGMP in rat hypothalamus neurons, anterior pituitary cells and Sertoli cells.
Results:In hypothalamus neurons of rat, no significant effect of cAMP was observed in various concentrations of CS2 groups (P>0.05). The level of cGMP in rat hypothalamus neurons was significantly lower in 10.0μmol/ml CS2 group than that of the control group, which could be attenuated by SNP (P<0.05). There were no significant differences in the levels of cAMP and cGMP in rat anterior pituitary cells between different doses of CS2 groups and control group (P>0.05). In the L-NMMA group, cAMP and cGMP levels were significantly higher than that of the 10.0μmol/ml CS2 group (P<0.05). In rat Sertoli cells, significant effects of cAMP and cGMP were observed neither in various concentrations of CS2 groups nor the SNP and L-NMMA group (P>0.05).
Conclusion:CS2 could significantly reduce the level of cGMP in hypothalamus, which could be attenuated by SNP, but no significant effect of cAMP and cGMP was observed in pituitary and Sertoli cells. It suggested that CS2 could induce secretion disorders of hypothalamus in male rat through NO-cGMP pathway, but the effect of CS2 on endocrine function of the pituitary and Sertoli cells were not dependent on cAMP and cGMP.
引文
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