出生前暴露于甲基对硫磷和氯氰菊酯对F1代大鼠 NMDAR1受体表达变化及DNA损伤的探讨

出生前暴露于甲基对硫磷和氯氰菊酯对F1代大鼠NMDAR1受体表达变化及DNA损伤的探讨研究生：王静林导师：刘苹教授吴锡南教授【摘要】目的：探讨出生前暴露甲基对硫磷和氯氰菊酯混配对子鼠出生后NMDAR1受体表达变化及神经细胞DNA损伤的影响。

方法：47只孕鼠随机分为4组，甲基对硫磷和氯氰菊酯的剂量分为0、1/300 LD50、1/95 LD50、1/30LD50 4个水平，即为0 ，0.0230+0.8000mg/kg BW，0.0725+2.5265 mg/kg BW，8.0000+0.2300 mg/kg BW。

在大鼠怀孕第1天到第15天每日一次对孕鼠经口灌胃染毒。

分别在子鼠出生后第7、14、21、28天每组随机抽取5只子鼠，免疫组化染色测定子鼠大脑皮层、中脑、海马NMDAR1受体蛋白的表达。

并在怀孕16天取出胎鼠和出生后30天取出子鼠脑用胰蛋白酶消化，进行单细胞凝胶电泳，吖啶橙染色分析神经细胞DNA损伤。

结果：出生后7天阳性锥体细胞多集中在大脑皮层的两侧底部，其它部位也有少量存在，免疫阳性反应细胞着色较浅。

14天、21天、28天子鼠脑NMDAR1受体阳性细胞广泛分布于整个大脑皮层和海马，但以顶叶皮层内侧外侧、梨状区皮层、海马、中脑黑质网状结构阳性细胞数多，细胞形态清晰，着色深。

在子鼠大脑皮层外侧，仅中剂量组NMDA受体蛋白表达较对照组降低(P<0.05)，表明在中剂量水平下调了NMDAR1受体蛋白的表达，在海马CA1区，出生后14天的子鼠海马CA1区NMDAR1受体在高剂量组的表达低于低剂量组的受体表达（P<0.01），出生后21天的子鼠脑海马CA1区NMDAR1受体在高剂量组的表达低于对照组和低剂量组的受体表达（P<0.01），随着甲基对硫磷和氯氰菊酯剂量的升高，在海马CA1区的NMDA受体阳性细胞数量减少（剂量因素回归系数β1＝-0.377，P<0.01），表明甲基对硫磷和氯氰菊酯混配对海马CA1区NMDAR1受体蛋白的表达有显著影响，下调了NMDA 受体蛋白的表达，并存在明显的剂量反应关系。

在对照组和低剂量组中，出生后7天的子鼠脑海马CA1区NMDAR1受体表达均低于14、21、28天的表达，在中剂量组和高剂量组中，出生后7天的子鼠脑海马CA1区NMDAR1受体表达均低于21、28天的表达（P<0.01）。

与对照组相比较，低剂量组和中剂量组出生后21天子鼠海马CA1区受体蛋白表达高峰不同程度的降低，而在高剂量组受体表达高峰后移至出生后28天。

在海马CA3区，14天子鼠脑NMDA受体阳性细胞表达数在对照组低于低剂量组(P<0.01)，高剂量组低于低剂量组(P<0.05)，表明在低剂量水平，甲基对硫磷和氯氰菊酯混配可能上调了出生后14天子鼠海马CA3区NMDAR1受体蛋白的表达，随着剂量水平的升高NMDA受体蛋白表达反而下降；其它部位各剂量水平间无统计学意义（P＞0.05）。

子鼠大脑皮层外侧、内侧、海马CA1、CA3在不同的时间NMDAR1受体蛋白表达不同（P＜0.05），对照组出生后14天NMDA受体蛋白表达水平最低，21天到高峰，28天有所下降，在三个剂量组蛋白表达高峰后移，并随着剂量水平的升高NMDA受体蛋白表达高峰后移特别明显。

在1/95LD50~1/30LD50剂量水平的甲基对硫磷和氯氰菊酯混配可明显引起孕16天胎鼠神经细胞DNA的断裂(P<0.05)，在高剂量水平可引起出生后30天胎鼠神经细胞DNA的断裂(P<0.05)，DNA损伤程度和彗星样细胞出现率与甲基对硫磷和氯氰菊酯剂量之间存在正相关关系（相关分析:孕16天胎鼠脑DNA损伤程度r1=0.836,P=0.000; 彗星样细胞出现率r2=0.849,P=0.000。

出生后30天子鼠脑DNA损伤程度r3=0.865,P=0.000; 彗星样细胞出现率r4=0.969,P=0.000），即随着甲基对硫磷和氯氰菊酯混配剂量的增加，DNA损伤程度加重以及彗星样细胞出现比例也增加。

结论：大鼠出生前暴露于中、高剂量水平的甲基对硫磷和氯氰菊酯混配下调了子鼠脑皮层外侧、海马CA1、CA3 NMDAR1受体蛋白表达，而在低剂量水平上调了海马CA3区NMDAR1受体蛋白表达，并干扰了子鼠NMDA受体的正常表达过程，延迟了受体蛋白的表达高峰；大鼠出生前暴露于甲基对硫磷和氯氰菊酯混配引起脑细胞DNA链断裂损伤，呈剂量－反应关系,这种效应在子鼠可能持续一段较长的时间。

关键词：甲基对硫磷氯氰菊酯混配农药NMDAR1受体DNA损伤单细胞凝胶电泳技术（SCGE）The Expression of NMDAR and The DNA Damage of Nerve System in offsprings of rats after prenatal exposured to Combined Cypermethrion and MythylparathionPostgraduate: WANG JinglinSuperviser: Prof. LIU Ping,Prof. WU XinanAbstract: Object: To detect the expression of NMDAR1 and the DNA damage of nerve system in offsprings of rats after prenatal exposured to combined Cypermethrion and Mythylparathion.Methods: 47 pregnant rats were devided into four groups randomly, i.e, control group and three combined pesticides treated groups 1/300LD50, 1/95LD50 and 1/30LD50 which corresponding to four level of doses: 0, 0.0230+0.8000mg/kg BW, 0.0725+2.5265mg/kg BW and 8.0000+0.2300mg/kg BW. From the 0-day to the 15-day, each day the rats were exposed to the combined pesticides of Mythylparathion and Cyperthrion orally. Take 5 off-springs rats in each group randomly after birth on 7, 14, 21 and 28 day respectively and by using immunohistochemistry(IHC), detect the NMDAR1 protein expression levels in the regions of pallium, diencephalon and hippocampus. Also Single Cell Gel Electrophoresis (SCGE or Comet Assay) and acrodine orange stain were used to analyse the DNA damage of the neuron cells of the 16th-day pregnant and the 30th-day off-spring rats after the cells were trypsinized and made into single-cell suspension.Results: In the 7th-day offspring sample, positive cells mostly focus in the bottom of the pallium and little cells scatter in other regions of the pallium CA1～4 regions. In the 14th, 21th and 28th-day samples, the positive NMDAR1 cells distribute abroad in the whole region of pallium and hippocampus, while relatively centralized in the regions of inner and outer sides of coping pallium, pear-like pallium, hippocampus and reticular-region of diencephalon. The positive cells in such regions were stained deeply and clearly. In the regions of outer side of pallium, the protein expression level of NMDAR1of the 14th and 21th-day samples in high-dose level was lower compared with the low-dose level(P<0.01) and the positive cells decrease with the increase of the doses(β1=－0.377, P<0.01) which indicates that combined pesticides of Mythylparathion and Cyperthrion may have significant effect on the expression of NMDAR1 in CA1 region and the relation of level-effect may exists.In the control and low-dose level groups, the NMDAR1 expression of the 7th group was lowerthan14th, 21th and 28th-day. In the middle and high-dose level groups, the NMDAR1 expression ofthe 7th group was lower than21th and 28th-day . Compared with the control, the protein expression acme in 21th-day in low and middle reduced incoordinately. In the region of CA3, 14th-day samples, the positive cells in the control and high-dose level groups are both lower than thelow-dose level group(P<0.01, P<0.05), which shows that low-dose level combined pesticides may up-regulate the protein expression but with the doses increases, the expression may decrease. In other regions there is not statistical significance among the different groups (P>0.05). The NMDAR1 level in the regions of pallium、hippocampus CA1 and CA3 are different in different time points (P<0.05). In control group, the protein level was lowest in and highest in 21th-day and decreased in 28th-day. The expression acmes of three groups are all retrusive and the retrusive tendency was consistent with the increase of the doses. In the range of 1/300LD50 to 1/30LD50, the combined pesticides of Mythylparathion and Cyperthrion can induce the neuron cell DNA strain rupture remarkably of the neuron cell in the 16th-day fetal sample(P<0.05) while the high-dose level group can induce the damage of the 30th-day off-spring sample (P<0.05) and the neuron, DNA damage(arbitrary unit) increased with the rising concentration of the combinedpesticides.(corelation analysis:DNA damage of the 16th-day fetal sample r1=0.836,P=0.000; comet cell s appearance rate r2=0.849,P=0.000; DNA damage of the 30th-day off-spring sampler3=0.865,P=0.000; comet cell s appearance rate r4=0.969,P=0.000).Conclusions: The exposure to high-level combined pesticides of Mythylparathion and Cyperthrion down-regulated the expression of NMDAR1 in the regions of outer side of pallium,CA1 and CA3 of hippocampus while the low-level exposure up-regulated the expression of NMDAR1 in the regions of CA3 and delayed the acme of the protein expression in the prenatal rats.Also the exposure induced the neuron cell’s DNA chain to rupture and the damage displayed an dose-effect relation and may persist for a long time.Key Words: Mythylparathion, Cyperthrion, combined pesticides, NMDAR1, DNA damage, Single Cell Gel Electrophoresis(SCGE).。