【作者】 路浩；

【导师】 刘宗平；

【作者基本信息】 扬州大学 ， 临床兽医学， 2008， 博士

【摘要】 近年来,对环境化学物毒性危害的研究已由原来单一因素为主向因素间联合作用方向发展。铅、镉均是生产和生活中接触频率大、暴露范围广的蓄积性毒物,能对发育中的中枢神经系统产生重要影响。铅、镉单独作用的报道较多,而对两者的联合毒性研究则相对较少,尤其是铅镉联合对处于发育早期的动物中枢神经系统毒性损伤的系统研究较为罕见。本研究选用新生大鼠作为试验动物,通过体外和体内实验相结合的方法较系统的探讨了铅、镉及其联合对新生大鼠中枢神经系统的毒性损伤效应及其可能的作用机理,为进一步认识铅、镉及其联合毒性作用提供理论依据。一、体外试验选用新生24h内SD大鼠作为试验用神经细胞的来源,采用尼氏染色对皮质神经细胞进行了形态鉴定和纯度分析,用CCK-8法检测了一个生长周期内神经细胞的活性分布,成功建立了大脑皮质神经细胞毒性试验的体外培养模型:在此基础上,在培养液中添加不同浓度的Pb、Cd,共分9组,分别为对照组、P₁（Pb100μmol/L）、P₂（Pb 200μmol/L）、C₁（Cd 5μmol/L）、C₂（Cd 10μmol/L）、P₁C₁（Pb100μmol/L+Cd 5μmol/L）、P₁C₂（Pb 100μmol/L+Cd 10μmol/L）、P₂C₁（Pb 200μmol/L+Cd 5μmol/L）、P₂C₂（Pb 200μmol/L+Cd 10μmol/L）组。分别从神经细胞形态学、神经细胞存活率、神经细胞凋亡率以及神经细胞GSH-Px、SOD、CAT、TChE活性和MDA含量等方面对铅、镉损伤新生大鼠大脑皮质神经细胞进行了研究;同时,添加N-乙酰半胱氨酸（NAC）,观察其在铅镉损伤神经细胞过程中的保护效果。结果表明,①本试验培养的大脑皮质神经细胞,经尼氏染色鉴定并计数神经细胞纯度在92%以上,且在一个生长周期内培养至第6d时神经细胞的OD值最高,因此,本试验将第6d确定为铅镉处理神经细胞的时间;②与对照组相比,铅、镉及其联合均能导致神经细胞数量减少、网络减少或消失;能使胞体短直径和突起长度显著变小和缩短（P＜0.05）;使神经细胞的存活率显著下降（P＜0.05）,且具有明显的时间与剂量效应;各染毒组神经细胞核皱缩、呈新月形、染色质致密浓染,甚至出现核碎裂,细胞凋亡率显著升高（P＜0.05）;与相应单独染毒组比较,各联合染毒组上述指标变化更为严重,P₁C₂组较P₂C₁组改变明显,其中以P₂C₂组最为显著（P＜0.05）;③与对照组比较,各染毒组神经细胞GSH-Px、SOD、TChE活性均显著下降（P＜0.05）,而CAT活性和MDA含量则显著升高（P＜0.05）;④在NAC拮抗铅、镉及其联合毒性试验中,可以看出NAC能使神经细胞存活率升高、细胞凋亡率下降,能使GSH-Px、SOD、TChE、CAT活性升高及MDA含量下降,但无显著性差异（P＞0.05）。由上述试验结果可以得出以下结论:①铅、镉及其联合能使神经细胞胞体变小,神经细胞突起变短,神经细胞间网络连接减少;能使神经细胞存活率下降,神经细胞凋亡率增加,且具有明显的时间和剂量效应。铅镉联合表现协同毒性效应,其中镉在联合作用中发挥主导作用;②铅、镉及其联合能显著改变神经细胞GSH-Px、SOD、CAT、TChE活性和MDA含量,因此,铅、镉的神经系统毒性与其脂质过氧化损伤有关;③添加NAC可以提高神经细胞的存活率,减少凋亡细胞的数量,增加GSH-Px、SOD、CAT、TChE活性,减少MDA的生成,说明NAC具有一定的拮抗铅、镉毒性损伤的作用,但保护效果不明显。二、体内试验35只怀孕母鼠被随机分为7组,分别为对照组（饮用蒸馏水）、铅组（300mg/L）、铅+NAC组（300mg/L+20mmol/L）、镉组（10mg/L）、镉+NAC组（10mg/L+20mmol/L）、铅镉联合组（300mg/L+10mg/L）、铅镉联合+NAC组（300mg/L+10mg/L+20mmol/L）。经饮水染毒,染毒时间为整个妊娠期,待分娩后观察并记录:①铅、镉及其联合对新生鼠生长发育的影响;②用光学显微镜、透射电子显微镜观察新生鼠大脑皮质组织病理学及超微结构的变化;③测定新生鼠脑组织GSH-Px、SOD、CAT、TChE活性及MDA含量;④ICP-MS检测了新生鼠体内主要微量元素Cu、Fe、Zn、Mn及Se含量;⑤FQ-PCR法检测了凋亡基因bcl-2、Bax和c-fos在新生鼠大脑皮质中的表达;免疫组织化学和FQ-PCR法检测了MT-3在新生鼠大脑皮质中的表达研究;⑥同时进行了相关指标的NAC保护试验研究。结果表明,①与对照组比较,各染毒组新生鼠体重和脑重均明显下降（P＜0.05或P＞0.05）,其中以铅镉联合组降低最为明显;②新生鼠大脑皮质组织经HE染色未见明显异常的病理组织学变化,但超微结构变化明显,表现神经细胞核固缩、变形,染色质边聚、轻度溶解,线粒体肿胀、嵴部分或完全消失,铅镉联合对新生鼠大脑皮质超微结构的损伤较铅、镉单独时严重;③各染毒组新生鼠体内微量元素Cu、Fe、Zn、Mn和Se含量均显著低于对照组（P＜0.05）,以铅镉联合组降低最为明显;④与对照组相比,铅、镉及其联合均使新生鼠脑组织GSH-Px、SOD、TChE、CAT活性下降及MDA含量升高（P＜0.05或P＞0.05）;⑤各染毒组新生鼠大脑皮质bcl-2基因的表达均低于对照组（P＜0.05或P＞0.05）,而Bax、c-fos和MT-3表达则显著高于对照组（P＜0.05）;⑥NAC保护组与相应染毒组比较,新生鼠体内微量元素Cu、Fe、Zn、Mn和Se含量均有所升高（P＜0.05或P＞0.05）,脑组织GSH-Px、SOD、TChE、CAT活性均有不同程度升高但差异不显著（P＞0.05）,MDA含量则显著下降（P＜0.05）,大脑皮质bcl-2基因的表达量明显增加（P＜0.05或P＞0.05）,而Bax和c-fos表达则显著减少（P＜0.05）。通过对本部分试验结果的分析,可得出以下结论:①母鼠妊娠期铅、镉及其联合暴露能明显减轻新生鼠体重和脑重,导致新生鼠脑发育迟缓;②母鼠妊娠期铅、镉及其联合暴露对新生鼠大脑皮质超微结构有显著影响,导致细胞膜破裂,细胞核固缩、变形,线粒体肿胀、嵴部分或完全消失;③铅、镉及其联合导致机体抗氧化酶活性降低可能与其微量元素Cu、Fe、Zn、Mn和Se含量下降有关;④铅、镉及其联合诱导神经细胞凋亡与bcl-2、Bax、c-fos基因的异常表达有关,铅镉联合表现协同毒性效应;⑤母鼠妊娠期铅、镉及其联合暴露的同时添加NAC可以提高新生鼠体重和脑重,新生鼠体内微量元素的含量增加,使新生鼠脑组织内GSH-Px、SOD、CAT、TChE活性升高,MDA含量显著降低,能增加bcl-2的表达量、减少Bax、c-fos和MT-3的表达量,表明NAC对铅、镉毒性损伤具有一定的保护作用。更多还原

【Abstract】 In recent years,the trend of studies on environmental chemical toxic substances is from the previous single factor to the present combined ones.Lead（Pb） and cadmium（Cd） are known as toxicants which are accumulative,large-contact frequent,extensive exposure in daily life.Lead and cadmium have deep influences upon the developmenting central nervous system（CNS）.The studies on single toxicity of cadmium or lead are reported in many documents.However,systemic studies of toxic damage on combination of lead and cadmium are seldom seen,especially in developmenting CNS.This study aims to investigate the toxic effects and mechanism of lead or/and cadmium on CNS of neonatal rats（in 24h） in vitro and in vivo,which will probably offer some theoretic evidence of further acquiring neurotoxicity mechanism of lead or/and cadmium.1.Experiments in vitro To study lead and/or cadmium neurotoxicity mechanism on CNS,the experimental neurons are obtained from the neonatal Sprague-Dawley rats in 24h.The model of neurons in vitro is successfully established,which is proved by adopting the Nissl’s staining,CCK-8 assay methods.The toxic tests of lead and/or cadmium are divided into 9 groups:the control group,P₁ group（Pb 100μmol/L）、P₂ group （Pb 200μmol/L）、C₁ group（Cd 5μmol/L）、C₂ group（Cd 10μmol/L）、P₁C₁ group（Pb 100μmol/L+Cd 5μmol/L）、P₁C₂ group（Pb 100μmol/L+Cd 10μmol/L）、P₂C₁ group（Pb 200μmol/L+Cd 5μmol/L）、P₂C₂ group（Pb 200μmol/L+Cd 10μmol/L）.The toxic effect of lead and/or cadmium on neuron’s morphology,viability,apoptosis rate,the enzymatic activity changes of glutathione peroxidase（GSH-Px）,superoxide dismutase（SOD）, catalase（CAT） and True choline esterase（TChE） and the contents of Maleic dialdehyde （MDA） are evaluated by related kits.Meanwhile,N-Acetylcysteine（NAC） is added in each experimental group to study the protective effects from the damages of lead and/or cadmium.The results are as follows:①The results of Nissl’s staining shows that the purity of neurons is above 92%,and the cortical neuron’s viability（OD）is highest on the sixth day of post-beginning of culturing.So neurons are treated the with experimental factors on the sixth day;②In comparison with the control group,the neurons are exposed to different concentrations of lead or/and cadmium,which led to the decrease in the number of neurons and neural network.The results indicated a significant decrease in pericaryons and processes（p＜0.05）,and survival rate of neurons（p＜0.05）.And the inhibitory effect is gradually enhanced with the prolonging of the culturing time and increasing concentrations of lead or/and cadmium.The nucleus crimpled,crescent liked and chromatin condensed,even nucleus disintegrated by the fluorescence microscope. And apoptosis rate is higher than the control group（P＜0.05）;In comparison with the single factor treats groups respectively,the combined factors groups have more significant changes,such as between P₁C₂ and P₂C₁,especially in P₂C₂ group（P＜0.05）;③In comparison with the control group,the activities of GSH-Px,SOD and TChE in neurons which are treated by lead or/and cadmium of different concentrations decrease significantly（P＜0.05）.However,the activity of CAT and the content of MDA in neurons are more significantly increased than that of the control group（P＜0.05）;④To compare the NAC protective groups with the poisoning groups,the neuron’s viability was increased,and the apoptosis rate of neurons was degraded relative to the poisoning groups. Meanwhile,the activities of GSH-Px,SOD,TChE and CAT are increased accordingly, but the content of MDA is decreased compared with the poisoning groups.However, there are no significant changes among the relative groups（P＞0.05）.From what has been discussed above,it might be concluded that:①The cortical neuron differentiation can be inhibited by lead or/and cadmium,which led to the pericaryon diminutus and process decurtation,lower survival rate and higher apoptosis rate and displays a dose and time effect in comparison with the control group.It is clear that lead and cadmium combined treatment exerts an synergistic effect,and cadmium played a great role in combining lead with cadmium;②The antioxidative enzyme activity of GSH-Px,SOD,CAT,TChE and content of MDA are significantly changed in each of experimental groups.Therefore, Nervous system toxicity of lead and/or cadmium is connected with lipid peroxidation;③The toxicity of lead or/and cadmium could antagonize by adding NAC,which improve survival rate of neurons and reduce apoptosis rate of neurons and increase activity of antioxidase and reduce lipid peroxidation.It is not obvious that adding NAC has a protective effects on the damage of lead or/and cadmium.2.Experiments in vivo To set up experimental animal mode of the damage Pb or/and Cd exposed in neonatal SD rats,35 SD pregnancy rats are used from the center of experimental animal of Yangzhou University,and are randomly divided into 7 groups:the control group（drinking water）,lead acetate group（300mg/L）,lead acetate+NAC（300 mg/L+20mmol/L）,cadmium acetate group（10mg/L）,cadmium acetate+NAC （10mg/L+20mmol/L）,group of lead and cadmium（300mg/L+10mg/L）,group of lead and cadmium+NAC（300mg/L+10mg/L+20mmol/L）,which are given by drinking water during gestational periods.After delivery,neonatal SD rats are used to examine the toxic effects of lead or/and cadmium and appraise protective effect of NAC.Experimental examination index was that①Toxic effect of lead and/or cadmium on physical growth （such as body weight,brain weight） and development of brain;②The studies on the changes in histopathology and ultrastructure of cerebral cortex of the neonatal SD rats;③Toxic effects of lead or/and cadmium on the biochemistry index of neonatal rats brain, such as the activity of GSH-Px,SOD,CAT and TChE and the content of MDA;④Effect of lead or/and cadmium on contents of trace element（Cu,Fe,Zn,Mn and Se） in neonatal rats;⑤The expression of bcl-2,Bax,c-fos in the cerebral cortex by FQ-PCR and MT-3 by FQ-PCR and immunohistochemistry method;⑥The protective effects of NAC on damage of lead or/and cadmium in developing brain of neonatal rats.The results are as follows:①In comparison with the control group,the body weight and brain weight are obviously decreased in lead or/and cadmium group（P＜0.05 or P＞0.05）, especially in the combination group;②The changes in histopathology of cerebral cortex is not observed under light microscope.However,the changes in ultrastructure of cerebral cortex is obvious that pyknosis of neuron nucleus,chromatin assemble,mitochondrial swelling and partial cristae under transmission electronic microscope;③In each of experimental groups,the activities of GSH-Px,SOD,CAT and TChE are significantly decreased than those in the normal group（P＜0.05）,however,the content of MDA is significantly increased in the experimental group than that in the normal group（P＜0.05）;④In the poisoning groups,the contents of trace element（Cu、Fe、Zn、Mn and Se） descend more significantly than the normal group（P＜0.05）;⑤In comparison with control group,the expression of bcl-2 in the cerebral cortex decrease（P＜0.05 or P＞0.05）,but the expression of Bax,c-fos and MT-3 increase in the experimental groups（P＜0.05 or P＞0.05）;⑥In comparison with the poisoning groups respectively,the protective groups of NAC are not only contents of trace element（Cu,Fe,Zn,Mn and Se） are improved,but also the activity of GSH-Px,SOD,CAT and TChE are higher（P＞0.05）and the content of MDA is significantly lower（P＜0.05）.The expression levels of bcl-2 in the cerebral cortex is obviously increased（P＜0.05 or P＞0.05）,but the expression levels of Bax and c-fos are significantly decreased in the experimental group（P＜0.05）.The conclusions drawn by analyzing the test results are as following:①The body weight and brain weight of neonatal rats are significantly decreased by lead or/and cadmium exposed to gestational rats;②There are obvious changes in ultrastructure of the cerebral cortex which displayed as cytomembrane disappearance and neuron nucelus concentration,mitochondrial swelling,disruption and total cristae loss;③The reducing activity of antioxidase led to lead or/and cadmium could be related to the decreasing contents of trace element in neonatal rats;④The apoptosis of neurons induced by lead or/and cadmium are in connection with the exceptional expression of bcl-2、Bax and c-fos genes.It is clear that lead and cadmium combined treatment exerts an synergistic effect;⑤NAC,as a new-style antioxidant,could antagonize toxicity of lead or/and cadmium, which improves activity of antioxidase and reducing lipid peroxidation and increasing content of trace element and increasing gene expression of bcl-2 and decreasing gene expression of Bax、c-fos and MT-3,while its protective effect was limited.更多还原