【摘要】 目的：观察除草剂西玛津对小鼠子代胚胎期及出生后第21天(PND21)多巴胺能神经元发育的影响，并探讨Wnt1-Ngn2-En1/2信号通路在神经元损伤中的作用。方法：健康SPF级昆明小鼠雌性30只，雄性15只，按照2∶1进行合笼。将孕鼠随机分为对照组(0.3%甲基纤维素溶液)和西玛津低剂量(50μg/kg)、高剂量组(200μg/kg)进行灌胃染毒，收集胚胎第8.5天(E8.5)、第12.5天(E12.5)和PND21仔鼠的中脑组织，采用免疫荧光杂交技术(FISH)、实时荧光定量PCR(qPCR)和Western blot分别检测Wnt1-Ngn2-En1/2信号通路相关基因(Wnt1、Ngn2、En1、En2、Nr4a2与Th)mRNA和蛋白的表达；通过旷场实验检测孕鼠染毒西玛津后对PND21雌、雄仔鼠自主行为能力的影响。结果：qPCR与Western blot检测结果显示，与对照组相比，在E8.5胚胎组织中，西玛津低剂量染毒组Wnt1和Ngn2以及高剂量组Wnt1、Ngn2、En1和En2的m RNA与蛋白表达均明显下降(P<0.05)；在E12.5胚胎组织中，低剂量染毒组Nr4a2和Th以及高剂量组Nr4a2、Th、Wnt1、Ngn2、En1、En2的mRNA和蛋白表达均显著下降(P<0.05)。FISH检测结果显示，与对照组相比，西玛津低、高剂量染毒组E8.5与E12.5胚胎组织中Wnt1的mRNA表达均明显下降(P<0.05)；西玛津高剂量组PND21雄性仔鼠的自主探索能力与运动能力显著减弱(P<0.05)，且PND21仔鼠组织中Wnt1与Th的蛋白表达也明显下调(P<0.05)。结论：西玛津能够通过Wnt1-Ngn2-En1/2信号通路影响子代小鼠多巴胺能神经元发育，并损伤雄性仔鼠的自主行为能力。更多还原

【Abstract】 OBJECTIVE: To investigate the role of the Wnt1-Ngn2-En1/2 signaling pathway in the herbicide simazine-induced damage to the dopaminergic neurons in embryonic and weaned mice. METHODS:Fifteen female and fifteen male healthy SPF Kunming mice were kept in containers in a 2∶1 ratio. During pregnancy, three groups of maternal mice were randomized∶ control group(0.3% methyl cellulose solution),low-dose group(50 μg/kg) and high-dose group(200 μg/kg). The tissues of the offspring on the day 8.5(E8.5), day 12.5(E12.5), and postnatal day 21(PND21) of development were obtained. mRNA and protein expression of the Wnt1-Ngn2-En1/2 signaling pathway-related genes(Wnt1,Ngn2,En1,En2,Nr4a2 and Th)were detected using immunofluorescence hybridization(FISH), real-time fluorescence quantitative PCR(qPCR)and Western blot. Through an open field experiment, effect of the treated pregnant mice on the autonomic behavior of PND21 female and male mice were identified. RESULTS:Western blot and qPCR data in E8.5demonstrated that in comparison to the control group, low simazine-exposed groups had a decrease in the mRNA and protein expression of Wnt1 and Ngn2(P<0.05),and the high-dose group had a substantial decline in the expressions of Wnt1,Ngn2,En1 and En2(P<0.05). The results of E12.5 embryo show the mRNA and protein expression of Nr4a2 and Th in the low-dose group declined as compared with the control group,(P<0.05), and the expression of Nr4a2, Th, Wnt1, Ngn2, En1 and En2 in high-dose group decreased significantly compared with that in control group(P<0.05). The FISH data verified that Wnt1 mRNA expression was substantially reduced in the low-dose and high-dose sections of E8.5 and E12.5 as opposed to that of the control group(P<0.05). The high-dose group of PND21 offspring exhibited significantly reduced motor and self-exploration abilities in comparison to the male offspring of the control group(P<0.05),and there was a notable down-regulation of Wnt1 and Th protein expressions(P<0.05). CONCLUSION:Simazine affected the development of dopaminergic neurons in male offspring through the Wnt1-Ngn2-En1/2 signaling pathway,and damaged the ability of independent behavior of male mice.更多还原