持續(xù)氟鋁聯(lián)合暴露對二代仔鼠空間學(xué)習(xí)記憶能力及cAMP-PKA-CREB信號通路的影響
發(fā)布時間：2018-08-24 16:34
【摘要】：目的:探討妊娠期和哺乳期持續(xù)染毒對二代仔鼠空間學(xué)習(xí)記憶能力和海馬組織內(nèi)環(huán)磷酸腺苷(cAMP)-蛋白激酶A(PKA)-環(huán)磷酸腺苷反應(yīng)元件結(jié)合蛋白(CREB)信號通路的影響。方法:將36只健康成年清潔級SD孕鼠隨機分為9組,每組4只。各組飲水中氟化鈉、三氯化鋁濃度分別為(0,0)、(100,0)、(200,0)、(0,500)、(0,1 000)、(100,500)、(100,1 000)、(200,500)、(200,1 000)mg/L。采用自由飲水方式進行染毒,母鼠從妊娠第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)每組隨機選取6只(雌∶雄=2∶1)并進行合籠,從其PND22(斷乳后)至二代仔鼠PND21繼續(xù)染毒;每組隨機選取二代仔鼠8只(雌雄各半),自PND22(斷乳后)~PND60(成年)繼續(xù)染毒。隔1周稱1次體重。仔鼠處死前,觀察仔鼠生理發(fā)育及神經(jīng)反射行為指標,Morris水迷宮測試空間學(xué)習(xí)記憶能力,收集12h尿液。仔鼠處死后,留取大腦組織。采用氟離子選擇電極法測定尿氟、腦氟濃度,用電感耦合等離子體發(fā)射光譜法測定尿鋁、腦鋁濃度,用酶聯(lián)免疫吸附法檢測海馬組織中cAMP蛋白的含量,用實時熒光定量聚合酶鏈式反應(yīng)(qRT-PCR)方法檢測海馬組織中PKA和CREB mRNA表達水平,用免疫組織化學(xué)方法檢測腦組織中PKA和p-CREB蛋白的表達水平。結(jié)果:1、尿、腦的氟、鋁水平:與對照組比較,除單獨鋁染毒組外,其余各染毒組仔鼠尿氟、腦氟水平升高,差異有統(tǒng)計學(xué)意義(P0.05);除單獨氟染毒組外,其余各染毒組仔鼠尿鋁、腦鋁水平升高,差異有統(tǒng)計學(xué)意義(P0.05);且高氟+低鋁聯(lián)合染毒組氟、鋁濃度較其他染毒組高。2、二代仔鼠生長發(fā)育及神經(jīng)反射情況:與對照組相比,第0周,除低氟、高鋁單獨染毒組及低氟+低鋁聯(lián)合染毒組二代仔鼠體重差異無統(tǒng)計學(xué)意義外,其余各染毒組的體重均較低;第2、4周,高鋁單獨染毒組的體重較高,而低氟+高鋁和高氟+低鋁聯(lián)合染毒組的體重較低;第6周,除氟單獨染毒組及高鋁單獨染毒組,其余各染毒組的體重均較低;第8周,低氟單獨染毒組的體重較高,其余各染毒組的體重均較低,差異有統(tǒng)計學(xué)意義(P0.05)。各染毒組腦重系數(shù)、開眼和出牙,與對照組比較差異無統(tǒng)計學(xué)意義;與對照組比較,高氟組、低氟+低鋁和高氟+低鋁組平面翻正反射時間延長;除低氟組、低鋁組及低氟+低鋁,其余染毒組懸崖回避反射時間延長,差異有統(tǒng)計學(xué)意義(P0.05)。3、空間學(xué)習(xí)記憶能力:與對照組比較,第2天,除低氟組、低鋁組及低氟+低鋁組,第3、4天,除低氟+低鋁組外,其余各染毒組仔鼠的逃避潛伏期均延長,差異有統(tǒng)計學(xué)意義(P0.05)。與對照組比較,除低氟組、低鋁組和低氟+低鋁組外,其余各染毒組仔鼠的首次達平臺時間均延長及穿越平臺次數(shù)均減少,差異均有統(tǒng)計學(xué)意義(P0.05)。4、海馬組織中c AMP-PKA-CREB信號通路相關(guān)蛋白和基因的表達情況:與對照組比較,各濃度單獨氟染毒組、低鋁組及低氟+高鋁組cAMP蛋白的含量降低,各濃度單獨氟染毒組及各濃度氟鋁聯(lián)合染毒組PKA蛋白的表達水平均降低,鋁單獨染毒組PKA蛋白的表達水平升高,各濃度氟、鋁單獨及聯(lián)合染毒組p-CREB蛋白的表達水平均降低,低鋁組及低氟+高鋁組PKA mRNA的表達水平較高,低鋁組及低氟+高鋁組及高氟+各濃度鋁組CREB mRNA的表達水平較高,差異有統(tǒng)計學(xué)意義(P0.05)。5、氟鋁聯(lián)合對各指標的聯(lián)合作用:氟鋁聯(lián)合染毒對二代仔鼠空間學(xué)習(xí)記憶能力及海馬組織中PKA和CREB mRNA表達水平和cAMP蛋白含量及腦組織中PKA、p-CREB蛋白的表達水平均存在拮抗型交互作用,但對后期體重表現(xiàn)為協(xié)同型交互作用。結(jié)論:在本實驗條件下,1、持續(xù)氟鋁聯(lián)合暴露可抑制二代仔鼠的生長發(fā)育;2、持續(xù)氟鋁聯(lián)合暴露可損害二代仔鼠的空間學(xué)習(xí)記憶能力;3、氟鋁聯(lián)合暴露可能通過抑制海馬cAMP-PKA-CREB信號通路導(dǎo)致二代仔鼠學(xué)習(xí)記憶能力受損;4、氟鋁聯(lián)合作用形式主要表現(xiàn)為拮抗型交互作用。
[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.
【學(xué)位授予單位】：貴州醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R114
本文編號：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.
【學(xué)位授予單位】：貴州醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R114
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