持续氟铝联合暴露对二代仔鼠空间学习记忆能力及cAMP-PKA-CREB信号通路的影响
发布时间：2018-08-24 16:34
【摘要】：目的:探讨妊娠期和哺乳期持续染毒对二代仔鼠空间学习记忆能力和海马组织内环磷酸腺苷(cAMP)-蛋白激酶A(PKA)-环磷酸腺苷反应元件结合蛋白(CREB)信号通路的影响。方法:将36只健康成年清洁级SD孕鼠随机分为9组,每组4只。各组饮水中氟化钠、三氯化铝浓度分别为(0,0)、(100,0)、(200,0)、(0,500)、(0,1 000)、(100,500)、(100,1 000)、(200,500)、(200,1 000)mg/L。采用自由饮水方式进行染毒,母鼠从妊娠第0天至一代仔鼠出生第21天(postnatal day 21,PND21)染毒;一代仔鼠于PND90(性成熟)每组随机选取6只(雌∶雄=2∶1)并进行合笼,从其PND22(断乳后)至二代仔鼠PND21继续染毒;每组随机选取二代仔鼠8只(雌雄各半),自PND22(断乳后)~PND60(成年)继续染毒。隔1周称1次体重。仔鼠处死前,观察仔鼠生理发育及神经反射行为指标,Morris水迷宫测试空间学习记忆能力,收集12h尿液。仔鼠处死后,留取大脑组织。采用氟离子选择电极法测定尿氟、脑氟浓度,用电感耦合等离子体发射光谱法测定尿铝、脑铝浓度,用酶联免疫吸附法检测海马组织中cAMP蛋白的含量,用实时荧光定量聚合酶链式反应(qRT-PCR)方法检测海马组织中PKA和CREB mRNA表达水平,用免疫组织化学方法检测脑组织中PKA和p-CREB蛋白的表达水平。结果:1、尿、脑的氟、铝水平:与对照组比较,除单独铝染毒组外,其余各染毒组仔鼠尿氟、脑氟水平升高,差异有统计学意义(P0.05);除单独氟染毒组外,其余各染毒组仔鼠尿铝、脑铝水平升高,差异有统计学意义(P0.05);且高氟+低铝联合染毒组氟、铝浓度较其他染毒组高。2、二代仔鼠生长发育及神经反射情况:与对照组相比,第0周,除低氟、高铝单独染毒组及低氟+低铝联合染毒组二代仔鼠体重差异无统计学意义外,其余各染毒组的体重均较低;第2、4周,高铝单独染毒组的体重较高,而低氟+高铝和高氟+低铝联合染毒组的体重较低;第6周,除氟单独染毒组及高铝单独染毒组,其余各染毒组的体重均较低;第8周,低氟单独染毒组的体重较高,其余各染毒组的体重均较低,差异有统计学意义(P0.05)。各染毒组脑重系数、开眼和出牙,与对照组比较差异无统计学意义;与对照组比较,高氟组、低氟+低铝和高氟+低铝组平面翻正反射时间延长;除低氟组、低铝组及低氟+低铝,其余染毒组悬崖回避反射时间延长,差异有统计学意义(P0.05)。3、空间学习记忆能力:与对照组比较,第2天,除低氟组、低铝组及低氟+低铝组,第3、4天,除低氟+低铝组外,其余各染毒组仔鼠的逃避潜伏期均延长,差异有统计学意义(P0.05)。与对照组比较,除低氟组、低铝组和低氟+低铝组外,其余各染毒组仔鼠的首次达平台时间均延长及穿越平台次数均减少,差异均有统计学意义(P0.05)。4、海马组织中c AMP-PKA-CREB信号通路相关蛋白和基因的表达情况:与对照组比较,各浓度单独氟染毒组、低铝组及低氟+高铝组cAMP蛋白的含量降低,各浓度单独氟染毒组及各浓度氟铝联合染毒组PKA蛋白的表达水平均降低,铝单独染毒组PKA蛋白的表达水平升高,各浓度氟、铝单独及联合染毒组p-CREB蛋白的表达水平均降低,低铝组及低氟+高铝组PKA mRNA的表达水平较高,低铝组及低氟+高铝组及高氟+各浓度铝组CREB mRNA的表达水平较高,差异有统计学意义(P0.05)。5、氟铝联合对各指标的联合作用:氟铝联合染毒对二代仔鼠空间学习记忆能力及海马组织中PKA和CREB mRNA表达水平和cAMP蛋白含量及脑组织中PKA、p-CREB蛋白的表达水平均存在拮抗型交互作用,但对后期体重表现为协同型交互作用。结论:在本实验条件下,1、持续氟铝联合暴露可抑制二代仔鼠的生长发育;2、持续氟铝联合暴露可损害二代仔鼠的空间学习记忆能力;3、氟铝联合暴露可能通过抑制海马cAMP-PKA-CREB信号通路导致二代仔鼠学习记忆能力受损;4、氟铝联合作用形式主要表现为拮抗型交互作用。
[Abstract]:AIM: To investigate the effects of continuous exposure during pregnancy and lactation on spatial learning and memory ability and cyclic adenosine monophosphate (cAMP) - protein kinase A (PKA) - cyclic adenosine monophosphate response element binding protein (CREB) signaling pathway in hippocampus of offspring rats. The concentrations of sodium fluoride and aluminum trichloride were (0,0), (100,0), (200,0), (0,500), (0,1,000), (100,500), (100,1,000), (200,500), (200,1,000) mg/L. Free drinking water was used to infect the mother rats from the tenth day of pregnancy to the 21st day of birth (PND21) of the first generation offspring, and six (PND90) mice in each group were randomly selected. Female: male = 2:1) and caged, from its PND22 (after weaning) to the second generation of offspring PND21 continued to be exposed; each group randomly selected eight offspring (half male and half female) from PND22 (after weaning) to PND60 (adult) continued to be exposed. Every other week weighed. Before the offspring were executed, the physiological development and neuroreflex behavior of offspring were observed, and Morris water maze test space was used. The urine samples were collected for 12 hours. The brain tissues were taken after the offspring were sacrificed. The urinary fluoride and brain fluoride concentrations were determined by fluoride ion selective electrode method, urinary aluminum and brain aluminum concentrations were determined by ICP-AES, the content of cAMP protein in hippocampus was detected by enzyme-linked immunosorbent assay, and real-time fluorescence quantitative polymerase chain reaction was used. The expression levels of PKA and CREB mRNA in hippocampus were detected by qRT-PCR, and the expression levels of PKA and p-CREB protein in brain were detected by immunohistochemistry. Results: 1. Fluoride and aluminum levels in urine and brain were significantly higher than those in control group (P Except for the fluoride-exposed group, the urinary and brain aluminum levels of the other groups increased significantly (P 0.05); and the fluoride and aluminum concentrations of the high-fluoride + low-aluminium group were higher than those of the other groups. 2. The growth and development of the second-generation offspring and nerve reflexes of the two groups: Compared with the control group, in the first week, except the low-fluoride, high-aluminium group and the low-fluoride + low group, respectively. There was no significant difference in the body weight of the second generation offspring between the two groups except that of the other groups; the body weight of the high aluminum group was higher than that of the low fluorine + high aluminum group and the high fluorine + low aluminum group at the 2nd and 4th week; the body weight of the other groups was lower than that of the high aluminum group and the fluorine + high aluminum group at the 6th week. In the 8th week, the body weight of the low fluoride group was higher than that of the other groups, and the difference was statistically significant (P 0.05). Compared with the control group, the escape latencies of the rats in the low fluoride group, low aluminium group and low fluoride + low aluminium group were prolonged on the second day, except the low fluoride group, low aluminium group and low fluoride + low aluminium group, the escape latencies of the other groups were prolonged and worse. Compared with the control group, except the low fluoride group, low aluminum group and low fluoride + low aluminum group, the first plateau arrival time and the number of plateau crossing were prolonged and decreased in the other groups. The differences were statistically significant (P 0.05). 4. The expression of C AMP-PKA-CREB signaling pathway related proteins and genes in hippocampus tissue were compared with the control group. Compared with the control group, the content of cAMP protein in the fluoride-exposed group, the low-aluminum group and the low-fluoride + high-aluminum group decreased, the expression level of PKA protein in the fluoride-exposed group and the combined fluoride-aluminum group decreased, the expression level of PKA protein in the aluminum-exposed group increased, and the expression of p-CREB protein in the fluoride-exposed group and the aluminum-exposed group increased. The expression level of PKA mRNA was higher in low aluminum group and low fluoride + high aluminum group, and higher in low aluminum group and low fluoride + high aluminum group and high fluoride + high aluminum group. The difference was statistically significant (P 0.05). 5. The combined effects of fluoride and aluminum on the spatial learning and memory ability and the sea of the second generation offspring rats: the combined effects of fluoride and aluminum on the spatial learning and memory ability of the second generation rats. The expression levels of PKA and CREB mRNA, cAMP protein and PKA, p-CREB protein in horse tissues were antagonistic to each other, but they had synergistic interaction with body weight at later stage. Dew can impair the spatial learning and memory abilities of the second-generation offspring. 3. The combined exposure of fluoride and aluminum may lead to the impairment of learning and memory abilities of the second-generation offspring by inhibiting the cAMP-PKA-CREB signaling pathway in the hippocampus. 4. The combined action of fluoride and aluminum is mainly antagonistic interaction.
【学位授予单位】：贵州医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R114
本文编号：2201387
[Abstract]:AIM: To investigate the effects of continuous exposure during pregnancy and lactation on spatial learning and memory ability and cyclic adenosine monophosphate (cAMP) - protein kinase A (PKA) - cyclic adenosine monophosphate response element binding protein (CREB) signaling pathway in hippocampus of offspring rats. The concentrations of sodium fluoride and aluminum trichloride were (0,0), (100,0), (200,0), (0,500), (0,1,000), (100,500), (100,1,000), (200,500), (200,1,000) mg/L. Free drinking water was used to infect the mother rats from the tenth day of pregnancy to the 21st day of birth (PND21) of the first generation offspring, and six (PND90) mice in each group were randomly selected. Female: male = 2:1) and caged, from its PND22 (after weaning) to the second generation of offspring PND21 continued to be exposed; each group randomly selected eight offspring (half male and half female) from PND22 (after weaning) to PND60 (adult) continued to be exposed. Every other week weighed. Before the offspring were executed, the physiological development and neuroreflex behavior of offspring were observed, and Morris water maze test space was used. The urine samples were collected for 12 hours. The brain tissues were taken after the offspring were sacrificed. The urinary fluoride and brain fluoride concentrations were determined by fluoride ion selective electrode method, urinary aluminum and brain aluminum concentrations were determined by ICP-AES, the content of cAMP protein in hippocampus was detected by enzyme-linked immunosorbent assay, and real-time fluorescence quantitative polymerase chain reaction was used. The expression levels of PKA and CREB mRNA in hippocampus were detected by qRT-PCR, and the expression levels of PKA and p-CREB protein in brain were detected by immunohistochemistry. Results: 1. Fluoride and aluminum levels in urine and brain were significantly higher than those in control group (P Except for the fluoride-exposed group, the urinary and brain aluminum levels of the other groups increased significantly (P 0.05); and the fluoride and aluminum concentrations of the high-fluoride + low-aluminium group were higher than those of the other groups. 2. The growth and development of the second-generation offspring and nerve reflexes of the two groups: Compared with the control group, in the first week, except the low-fluoride, high-aluminium group and the low-fluoride + low group, respectively. There was no significant difference in the body weight of the second generation offspring between the two groups except that of the other groups; the body weight of the high aluminum group was higher than that of the low fluorine + high aluminum group and the high fluorine + low aluminum group at the 2nd and 4th week; the body weight of the other groups was lower than that of the high aluminum group and the fluorine + high aluminum group at the 6th week. In the 8th week, the body weight of the low fluoride group was higher than that of the other groups, and the difference was statistically significant (P 0.05). Compared with the control group, the escape latencies of the rats in the low fluoride group, low aluminium group and low fluoride + low aluminium group were prolonged on the second day, except the low fluoride group, low aluminium group and low fluoride + low aluminium group, the escape latencies of the other groups were prolonged and worse. Compared with the control group, except the low fluoride group, low aluminum group and low fluoride + low aluminum group, the first plateau arrival time and the number of plateau crossing were prolonged and decreased in the other groups. The differences were statistically significant (P 0.05). 4. The expression of C AMP-PKA-CREB signaling pathway related proteins and genes in hippocampus tissue were compared with the control group. Compared with the control group, the content of cAMP protein in the fluoride-exposed group, the low-aluminum group and the low-fluoride + high-aluminum group decreased, the expression level of PKA protein in the fluoride-exposed group and the combined fluoride-aluminum group decreased, the expression level of PKA protein in the aluminum-exposed group increased, and the expression of p-CREB protein in the fluoride-exposed group and the aluminum-exposed group increased. The expression level of PKA mRNA was higher in low aluminum group and low fluoride + high aluminum group, and higher in low aluminum group and low fluoride + high aluminum group and high fluoride + high aluminum group. The difference was statistically significant (P 0.05). 5. The combined effects of fluoride and aluminum on the spatial learning and memory ability and the sea of the second generation offspring rats: the combined effects of fluoride and aluminum on the spatial learning and memory ability of the second generation rats. The expression levels of PKA and CREB mRNA, cAMP protein and PKA, p-CREB protein in horse tissues were antagonistic to each other, but they had synergistic interaction with body weight at later stage. Dew can impair the spatial learning and memory abilities of the second-generation offspring. 3. The combined exposure of fluoride and aluminum may lead to the impairment of learning and memory abilities of the second-generation offspring by inhibiting the cAMP-PKA-CREB signaling pathway in the hippocampus. 4. The combined action of fluoride and aluminum is mainly antagonistic interaction.
【学位授予单位】：贵州医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R114
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