白细胞介素-1β蛋白在孕哺期铅暴露仔鼠海马组织中的表达

为探讨母体铅染毒对其子一代海马组织中白细胞介素-1β（IL-1β）蛋白表达的影响,将雌性小鼠自妊娠第1天开始经饮水染铅（0.3,1.0,3.0g/L,对照组饮蒸馏水）至仔鼠出生后21d断乳为止。随机抽取各组仔鼠,于出生后7,14,21d分别测其血液和海马组织中铅的含量;于出生后21d,采用Western blot方法测定各组海马组织中IL-1β的表达情况。结果表明,孕哺期不同剂量铅暴露后,出生后7,14,21d的仔鼠血铅、海马铅水平均明显高于对照组（P〈0.05）。Western blot结果显示,IL-1β蛋白在不同剂量铅暴露组海马组织中的表达明显高于对照组（P〈0.05）。相关分析表明,IL-1β蛋白的表达与血铅、海马铅呈显著正相关（P〈0.05）。母体铅暴露使铅在仔鼠体内蓄积,提示仔鼠血铅和海马铅的升高,引起海马组织中IL-1β表达的上调,可能损伤了海马神经元,进而损伤了神经系统。     

母体铅暴露对仔鼠大脑皮层组织中IL-1β和TNF-α表达的影响

探讨母体铅染毒对其仔鼠大脑皮层组织中IL-1β和TNF-α表达量的影响,揭示铅神经毒性的潜在机制。母鼠采用自由饮水的方式自妊娠1d开始经饮水染铅(0.1%,0.5%和1.0%的浓度溶解在去离子水中,对照组饮蒸馏水)至仔鼠出生后21d断乳为止,每组10只。于出生后21d,用石墨炉原子吸收光谱仪测定仔鼠血液和大脑皮层组织中铅含量。用免疫组织化学染色法和Western blot方法测定大脑皮层组织中IL-1β和TNF-α蛋白表达情况。结果表明,各剂量染铅组血铅和脑铅含量与对照组比较明显升高(P<0.05);与对照组相比,随着试验组染铅浓度的升高,IL-1β和TNF-α的表达量显著增加(P<0.05)。铅可能通过诱导大脑皮层中IL-1β和TNF-α的高表达,影响学习记忆从而造成神经系统损害。     

生命早期铅暴露对小鼠海马内IGF1表达的影响

雌性小鼠自妊娠第1天开始经饮水染铅(0.1%、0.5%、1%浓度溶解在去离子水中,对照组饮蒸馏水)至仔鼠出生后21d断乳为止。在出生后的第21天,分别采用石墨炉原子吸收光谱法测定血铅和海马组织中铅的水平,免疫组织化学和免疫印迹法检测海马组织中胰岛素样生长因子1(insulin-like growth factor 1,IGF1)的表达。结果显示:与对照组相比,铅暴露组血铅水平和海马组织中铅的水平显著增高(P0.05);铅暴露组IGF1的表达比对照组明显下降(P0.05)。结果表明:母体铅暴露使铅在仔鼠体内蓄积,使仔鼠海马组织中IGF1蛋白的表达下调,从而造成海马神经元损伤,引起神经毒性。     

母体铅暴露对仔鼠肝脏MMP-2和MMP-9 mRNA表达的影响

为探讨母体铅暴露对仔鼠肝脏MMP-2和MMP-9 mRNA表达的影响,采用自由饮水模式建立铅暴露动物模型,将40只雌性小鼠自妊娠第1天开始经饮水染铅(1.0,5.0,10.0g/L,对照组饮蒸馏水)至仔鼠出生后21d,随机分为低、中、高剂量染毒组和对照组,仔鼠21日龄,分别测其血液和肝脏中铅的含量,然后取其肝脏组织,通过实时荧光定量PCR(Real-time PCR)技术检测各组仔鼠肝脏中MMP-2和MMP-9mRNA的表达情况。结果显示,21d后,铅暴露组仔鼠血液和肝脏中铅水平均明显高于对照组(P<0.05);与对照组相比,低、中和高剂量铅暴露组仔鼠肝脏内MMP-2mRNA的表达量明显增高,且差异具有统计学意义(P<0.05)。中、高剂量铅暴露组仔鼠肝脏组织中MMP-9mRNA的表达明显高于对照组(P<0.05),但低剂量铅暴露组仔鼠肝脏组织中MMP-9mRNA的表达与对照组相比差异不显著(P>0.05)。结果表明,母体铅暴露可能通过改变仔鼠肝脏中MMP-2 mRNA的表达进而造成肝脏损伤。母体铅暴露使铅在仔鼠体内蓄积,铅暴露可能通过增强仔鼠肝脏中MMP-9mRNA的表达进而造成肝脏损伤,从而引起组织发生病变。     

成年摇晃小鼠海马组织中小清蛋白的表达

为探讨小清蛋白(PV)在成年摇晃小鼠海马中的表达情况,用免疫荧光组织化学法对野生型小鼠(R^+/+)、杂合子摇晃小鼠(R^+/-)、纯合子摇晃小鼠(R^-/-)海马CA1区、CA3区、齿状回中PV的分布和表达进行研究。结果表明,PV在摇晃小鼠海马CA1区、CA3区和齿状回中均有分布,主要表达于CA1区和CA3区的锥体细胞层及齿状回的颗粒细胞层。与野生型相比,杂合子摇晃小鼠CA1区PV阳性细胞数显著降低(P<0.01),而纯合子摇晃小鼠增加;CA3区和齿状回中的PV阳性细胞数均降低,但纯合子摇晃小鼠高于杂合子摇晃小鼠。提示摇晃蛋白(Reelin)影响PV的表达,且PV的表达异常可能与摇晃小鼠的表现型有关。     

铅对仔鼠学习记忆及海马组织中钙结合蛋白-2mRNA表达的影响

采用自由饮水的方式于母鼠孕哺期染毒,染毒剂量为0.3、1.0、3.0 g/L,每组10只.用石墨炉原子吸收光谱仪测定仔鼠出生后第21天时血液和海马组织中铅含量.用RT-PCR方法测定21 d不同剂量组仔鼠海马组织中钙结合蛋白-2 (Calsyntenin-2) mRNA的表达、结果显示,各剂量染铅组血液、海马组织中铅含量与对照组相比,明显升高(P＜0.05).Y型迷宫试验结果显示,低、中、高剂量铅暴露组21 d的仔鼠,获取记忆的能力(T1)、保持记忆的能力(T2)和记忆保持率(T3)均低于同期对照组仔鼠,差异均有统计学意义(P＜0.05).与对照组相比,中、高剂量铅暴露组海马组织中Calsyntenin-2 mRNA的表达明显低于对照组(P＜0.05),但是低剂量组与对照组相比,差异无统计学意义(P＞0.05).结果表明,孕哺期母体铅暴露使铅在仔鼠体内蓄积,引起仔鼠学习记忆功能损害;铅的神经毒性可能与下调海马组织中Calsyntenin-2 mRNA的表达、影响突触的信息传递有关.     

孕鼠暴露双酚A对仔鼠存活率、生殖激素及相关基因的影响

为了研究孕鼠在孕期暴露双酚A（bisphenol A,BPA）对仔鼠生殖激素及相关基因的影响,试验将40只昆明孕鼠随机分为A、B、C、D共4组,每组10只。其中A组为对照组,饲喂普通鼠粮;B、C、D组孕鼠整个妊娠期（妊娠1 d至分娩）分别按每只鼠每天50、500、2 500 mg/kg体重给予BPA,待孕鼠自然分娩,观察记录仔鼠死亡情况。至仔鼠性成熟（56日龄）剖杀仔鼠,摘取睾丸或卵巢称重并计算脏器指数,HE染色观察卵巢或睾丸组织结构的变化,ELISA试剂盒分析仔鼠血清睾酮（T）、促卵泡素（FSH）及雌二醇（E₂）水平,免疫组织化学方法检测仔鼠睾丸或卵巢Bax、Bcl-2蛋白的表达,实时荧光定量PCR检测仔鼠睾丸StAR、CYP11a或卵巢AMH、Kitlg mRNA表达。结果显示,孕鼠暴露BPA极显著增加了仔鼠死亡率（P<0.01）,显著降低了仔鼠睾丸重（P<0.05）。ELISA检测结果表明,孕鼠暴露BPA极显著降低了仔鼠T（♂）及FSH（♀）含量（P<0.01）,极显著升高了仔鼠（♀） E₂水平（P<0.01）。HE染色结果显示,随BPA剂量增加,仔鼠睾丸组织损伤严重,间质细胞减少;卵巢组织结构随BPA剂量增大,空泡逐渐增多,黄体颗粒数量逐渐减少。免疫组化结果显示,孕鼠暴露BPA增加了仔鼠睾丸或卵巢组织中Bax阳性蛋白表达,减少了Bcl-2阳性蛋白表达,显著降低了雄性仔鼠StAR mRNA表达量（P<0.05）;B、D组雄性仔鼠CYP11a mRNA表达量极显著降低（P<0.01）,而C组CYP11a mRNA表达量极显著升高（P<0.01）;C、D组雌鼠Kitlg mRNA表达极显著降低（P<0.01）,AMH mRNA表达量显著升高（P<0.05）。本试验结果表明,孕鼠妊娠期暴露不同剂量BPA增加了仔鼠死亡率,扰乱了生殖激素平衡和睾丸/卵巢中相关凋亡蛋白及生殖基因表达。     

叶酸对急性铅中毒大鼠海马和皮层组织中GSH及Nrf2、HO-1表达的影响

探讨叶酸对急性铅中毒大鼠海马和皮层组织中谷胱甘肽(GSH)及核因子E2相关因子(nuclea factor erythroid-2-related factor 2,Nrf2)、血红素氧合酶1(heme oxygenase 1,HO-1)蛋白表达的影响。采用自由饮水模式将24只雄性6周龄大鼠平均随机分为4组,建立铅暴露模型,对照组C组(去离子水),铅暴露组L组(0.2%醋酸铅溶液),铅暴露和叶酸治疗组T组(0.2%醋酸铅溶液+0.4 mg/kg叶酸灌胃),叶酸组F组(去离子水+0.4 mg/kg叶酸灌胃)。分别取海马和皮层组织用石墨炉原子吸收光谱法测定铅含量,用比色法测量其GSH质量分数,通过免疫荧光法检测其Nrf2以及HO-1蛋白的表达情况。结果显示:铅含量在L和T组显著升高(P0.01);海马和皮层中GSH含量L组中明显低于C组,T组显著高于L组,F组高于C组,具有显著差异(P0.05);免疫荧光结果显示,L组中Nrf2和HO-1免疫荧光阳性反应物平均积分光密度值(IOD/area,mean density)相比于C组明显增高,T组相比于L组明显降低,F组低于C组,差异极显著(P0.01)。叶酸可以调控Nrf2和HO-1蛋白的表达对铅暴露大鼠所诱导的氧化损伤具有修复作用。     

母鼠妊娠期铅镉联合暴露对仔鼠脑损伤病理学变化的影响

为探讨母鼠妊娠期铅镉联合暴露对新生鼠生长发育及脑组织病理学变化的影响,20只怀孕SD母鼠被随机分为4组,即A组为对照组(饮用蒸馏水)、B组为铅组(300mg/L)、C组为镉组(10mg/L)、D组为铅+镉组(300mg/L+10mg/L)。采用饮水染毒,期间记录母鼠体质量及临床症状,染毒结束后记录新生鼠体质量及脑质量,并采集新生鼠大脑皮质观察其病理组织学变化,染毒时间为21d。结果表明,与对照组比较,各染毒组母鼠体质量均低于对照组,但差异不显著(P>0.05);而新生鼠体质量均显著低于对照组(P<0.05),脑质量也明显下降,除C组无显著性差异外,其余各组差异显著(P<0.05);病理组织学变化显示,光学显微镜观察大脑皮质未见明显异常,透射电镜下可见各染毒组神经细胞核染色质浓缩,线粒体肿胀、嵴部分或完全消失。铅镉联合毒性明显强于铅镉单独作用,铅镉联合表现协同毒性效应,镉在铅镉联合毒性损伤中发挥主要作用。     

利用免疫印迹技术检测睾丸中几种20～50ku蛋白的表达

为了建立睾丸中20～50 ku蛋白的免疫印迹检测技术,我们从蛋白提取、电泳条件、转膜条件、免疫反应条件等多方面进行了摸索。结果表明:RIPA和蛋白酶抑制剂不仅能保证蛋白提取的效率,而且可以有效防止蛋白降解;电泳时采用10%的分离胶,可以达到较好的分离效果;采用蛋白预染分子标准结合DAB染色可以获得较好的结果。利用上述程序检测了StAR蛋白、P-ERK和ERK的表达,得到了比较满意的效果。     

Effect of lead exposure on the erythrocytic antioxidant levels in goats

Fifteen adult female goats were orally exposed to 5.46 mg lead (as lead acetate) per kg body weight daily for 2 weeks to study the antioxidant enzymes of the erythrocyte, lipid peroxide level, total thiol groups and total antioxidant status (TAS) in plasma. Ten goats served as unexposed control. Blood samples were collected before exposure (day 0) and on days 7 and 14. Ten per cent erythrocyte haemolysate was prepared and analysed for glutathione peroxidase (GPx), superoxide dismutase (SOD), catalase (CAT), total thiol groups and lipid peroxide. TAS was determined in plasma. There was a significant (P < 0.05) increase of erythrocytic GPx, SOD, CAT, total thiol groups and TAS on day 7 which was followed by a significant (P < 0.05) decrease of all these parameters on day 14. Lipid peroxide level increased significantly (P < 0.05) and the maximum level was attained by day 14. The results obtained indicate a possible role of free radicals in lead poisoning pathogenicity.     

Effect of chronic lead exposure on the canine bone marrow

The effect of chronic oral led acetate administration on canine bone marrow was studied. Two dogs (group 1) were used as controls, 4 dogs (group 2) were given 2 mg of lead/kg of body weight daily, and 4 dogs (group 3) were given 5 mg of lead/kg daily. After a 7-day stabilizaion period, lead dosing was conducted for 91 days (13 weeks), after which half of each group was treated with calcium ethylenediaminetetraacetic acid. All dogs were then observed for another 28 days (4 weeks). Blood lead values and bone marrow cellular changes were monitored once a week during the 126 days (18 weeks) of study. Lead-dosed dogs had lower weight gains than the controls. Clinical signs of toxicosis were observed after 6 weeks in one dog in group 3. Anorexia, body weight loss, CNS depression, muscular weakness, and trembling were seen. Blood lead concentrations increased in all group 2 and 3 dogs. Lead caused increases in bone marrow segmented neutrophils and myeloid series cells, and increased myeloid:erythroid ratios. Blood lead concentrations and myeloid:erythroid ratios decreased after cessation of lead administration.     

Time-course changes of hippocalcin expression in the mouse hippocampus following pilocarpine-induced status epilepticus

Hippocalcin participates in the maintenance of neuronal calcium homeostasis. In the present study, we examined the time-course changes of neuronal degeneration and hippocalcin protein level in the mouse hippocampus following pilocarpine-induced status epilepticus (SE). Marked neuronal degeneration was observed in the hippocampus after SE in a time-dependent manner, although neuronal degeneration differed according to the hippocampal subregions. Almost no hippocalcin immunoreactivity was detected in the pyramidal neurons of the cornu ammonis 1 (CA1) region from 6 h after SE. However, many pyramidal neurons in the CA2 region showed hippocalcin immunoreactivity until 24 h after SE. In the CA3 region, only a few hippocalcin immunoreactive cells were observed at 12 h after SE, and almost no hippocalcin immunoreactivity was observed in the pyramidal neurons from 24 h after SE. Hippocalcin immunoreactivity in the polymorphic cells of the dentate gyrus was markedly decreased from 6 h after SE. In addition, hippocalcin protein level in the hippocampus began to decrease from 6 h after SE, and was significantly decreased at 24 h and 48 h after pilocarpine-induced SE. These results indicate that marked reduction of hippocalcin level may be closely related to neuronal degeneration in the hippocampus following pilocarpine-induced SE.     

Effect of gestational exposure to nonylphenol on the development and fertility of mouse offspring

Nonylphenol (NP), a kind of environmental chemical, is thought to imitate endogenous hormones, inhibit the actions of hormones, and induce reproductive abnormalities. A number of experimental animals, usually rats, have been used to evaluate the potential reproductive toxicity of NP. However, the findings of previous studies were contradictory in some cases. Therefore, we used ICR mice as a biomodel for in utero study of NP. After mating, 8- to 12-week-old females were assigned to four groups (n=8) for subcutaneous injections from day 5 to 20 of gestation. Group I animals received corn oil alone as a control, while the mice of groups II, III and IV received NP at concentrations of 1/1000, 1/100 and 1/10 of the LD(50), respectively. A dose-dependent decrease was observed in terminal body weights of males of the F1 generation; however, a very small negative effect was only found in the females of the NP1/10 group. No significant effect was found on the liver weights of both sexes. The weights of the testis and epididymis were slightly decreased in the NP1/10 group. The NP1/100 treatment increased ovary weight considerably. The uterus weight tended to be increased in the NP treatment groups; however, there were large variations. The gestational exposure of the groups had no significant effect on the rate of pregnancy (94.4-100%) and the number of fetuses per litter (13.6-14.3 males, 12.3-13.7 females) compared with the control group. However, the overall mortality of fetuses/embryos was increased considerably in the NP1/100 (male: 13.9%) and NP1/10 (female: 9.8%) groups. These results suggest that exposure to NP in utero possibly affects the body weight and some reproductive organ weights, but does not influence the potential fertility of the F1 generation.     

醋酸铅对小鼠卵巢颗粒细胞凋亡基因p53、Bax、Bc1-2表达的影响

将40只未成年雌性昆明系小鼠随机分成3个处理组和1个对照组,每组10只.处理组小鼠分别连续2 d腹腔注射铅10、20、40 mg/kg(按体质量给药),对照组小鼠注射等体积的生理盐水.于注射后24、72 h分离卵巢,用原位末端标记法(TUNEL)测定卵巢颗粒细胞的凋亡率,同时用半定量RT-PCR方法检测凋亡基因p53、Bax、Bc1-2mRNA表达.结果表明,铅可促进颗粒细胞凋亡,且随剂量的增加和作用时间的延长,颗粒细胞中p53、Bax基因表达量逐渐增加,与对照组相比差异显著或极显著(P<0.05或P<0.01);而Bc1-2基因表达量则逐渐减少(P<0.05或P<0.01);表明铅可通过上调促细胞凋亡基因p53、Bax基因表达量,下调Bc1-2基因表达量,诱导卵巢颗粒细胞的凋亡.