圍生期雙酚A暴露對(duì)子代小鼠海馬突觸結(jié)構(gòu)發(fā)育的影響
發(fā)布時(shí)間：2018-09-18 11:02
【摘要】：環(huán)境雌激素是環(huán)境中干擾體內(nèi)激素生成、釋放、輸送、代謝、結(jié)合或消除過(guò)程的外源性化合物,表現(xiàn)出擬雌激素或抗雌激素作用。雙酚A(Bisphenol A, BPA)是一種典型的環(huán)境雌激素,廣泛用于、食品和飲料包裝材料、金屬罐頭樹(shù)脂內(nèi)膜、牙齒固封劑、添加劑等。BPA能夠通過(guò)食品和飲料進(jìn)入人體,也可以從垃圾滲析到周圍生態(tài)系統(tǒng)污染環(huán)境而危害健康。BPA具類雌激素活性,通過(guò)與雌激素受體結(jié)合干擾內(nèi)源性雌激素活性。 近來(lái)有研究發(fā)現(xiàn),低于風(fēng)險(xiǎn)評(píng)估值的BPA對(duì)嚙齒類動(dòng)物中樞神經(jīng)系統(tǒng)發(fā)育存在低劑量效應(yīng)。圍生期母體BPA暴露可影響子代大鼠大腦皮層結(jié)構(gòu)以及皮層和海馬神經(jīng)元的形態(tài)發(fā)育,改變其多種神經(jīng)行為及其性別分化,說(shuō)明發(fā)育中的腦是對(duì)BPA敏感的靶器官。本實(shí)驗(yàn)室前期研究發(fā)現(xiàn),圍生期母體BPA (0.05～50mg/kg/d)暴露后,仔鼠的活動(dòng)性、探究、焦慮和抑郁、學(xué)習(xí)記憶等多種非生殖行為發(fā)生改變；體外培養(yǎng)的海馬神經(jīng)元暴露于BPA后快速促進(jìn)樹(shù)突絲狀的運(yùn)動(dòng)性和密度。鑒于突觸是腦可塑性變化和信息傳遞的關(guān)鍵部位,與學(xué)習(xí)記憶等行為密切相關(guān),而突觸結(jié)構(gòu)修飾包括突觸界面結(jié)構(gòu)、突觸數(shù)密度、突觸傳遞等容易受環(huán)境和藥物的影響,因此,本研究通過(guò)圍生期母體BPA暴露,觀察BPA對(duì)雄性仔鼠生后發(fā)育過(guò)程中海馬突觸結(jié)構(gòu)的影響,并通過(guò)檢測(cè)相關(guān)腦區(qū)突觸前標(biāo)志蛋白synapsinI和突觸后標(biāo)志蛋白PSD-95,以及與學(xué)習(xí)記憶密切相關(guān)的突觸后谷氨酸NMDA和AMPA受體的表達(dá)水平進(jìn)一步探討B(tài)PA影響突觸結(jié)構(gòu)的分子機(jī)制。 方法：取清潔級(jí)ICR雄鼠(30-35g)和雌鼠(25-30g)適應(yīng)1周后合籠,母鼠從妊娠第7天至斷乳期(產(chǎn)后21天)進(jìn)行雙酚A (Bisphenol A, BPA,0.04、0.4、4mg/kg/day)灌胃染毒,同時(shí)設(shè)對(duì)照組給予溶媒花生油。子代雄鼠每組10只,分別在生后14天、21天、56天處死,按要求取海馬組織樣品,分別制備電鏡超薄切片觀察海馬CA1區(qū)錐體細(xì)胞的突觸數(shù)密度和突觸界面參數(shù),應(yīng)用western-blot和免疫組織化學(xué)技術(shù)分別檢測(cè)海馬synapsin I、NMDA受體亞基NR1和AMPA受體亞基GluR1蛋白表達(dá)。 用Quantity one軟件測(cè)量目的蛋白條帶光密度值,用Image-Pro Plus6.0分析測(cè)定突觸形態(tài)和免疫組織化學(xué)圖片。實(shí)驗(yàn)數(shù)據(jù)采用SPSS15.0統(tǒng)計(jì)軟件中的One-way ANOVA法分析,測(cè)定值用平均值±標(biāo)準(zhǔn)誤差(M±SE)表示,進(jìn)行t檢驗(yàn),P0.05作為具有統(tǒng)計(jì)學(xué)差異顯著性。 結(jié)果：1、圍生期母體BPA暴露后,與對(duì)照組相比,BPA(0.04和4mg/kg/d)顯著減少PND14和PND56的突觸數(shù)密度(P0.01或P0.001),但對(duì)PND21沒(méi)有顯著差異。說(shuō)明圍生期BPA暴露抑制子代小鼠突觸形成。 2、圍生期母體BPA暴露改變突觸界面結(jié)構(gòu)。BPA所有劑量組均極顯著增大不同發(fā)育階段的突觸間隙寬度(P0.01)；所有劑量組使PSD厚度極顯著減小(P0.01)；縮短不同發(fā)育階段的突觸活性帶長(zhǎng)度,其中,生后21天影響最顯著(P0.05或P0.01)；但對(duì)突觸界面曲率影響較小,主要是發(fā)育早期(PND14和21)有顯著上升(P0.05)。說(shuō)明突觸界面參數(shù)對(duì)BPA的敏感程度不一樣,其中突觸間隙和PSD厚度最為敏感。 3、Western blot分析結(jié)果發(fā)現(xiàn),圍生期BPA暴露,所有劑量組顯著降低生后21天和56天synapsin I蛋白表達(dá)(P0.05或P0.01)；顯著下調(diào)PSD-95蛋白表達(dá),其中生后56天下降最顯著(P0.05,P0.01或P0.001)；BPA(0.4和4mg/kg/d)顯著降低PND21的NR1表達(dá),所有劑量組使56天表達(dá)極顯著下降(P0.05,P0.01或P0.001),但對(duì)PND14無(wú)顯著影響；GluRl蛋白表達(dá)被抑制,低劑量組和高劑量組顯著或極顯著降低發(fā)育階段的表達(dá)量(P0.05或P0.01)。 4、免疫組化技術(shù)分析結(jié)果與WB結(jié)果相似。 結(jié)論：低于日允許攝入劑量(tolerable daily intake,0.05mg/kg/d)的BPA也可影響腦的突觸發(fā)育和突觸可塑性；圍生期母體BPA暴露影響仔鼠發(fā)育期海馬突觸結(jié)構(gòu),并降低興奮性氨基酸NMDA和AMPA受體、突觸蛋白PSD-95和synapsin I蛋白的表達(dá)。推測(cè)這可能是圍生期母體BPA暴露影響仔鼠發(fā)育期和成年后多種行為的原因之一。
[Abstract]:Bisphenol A (BPA) is a typical environmental estrogen widely used in food and beverage packaging materials, canned metal resin lining, dental sealants, etc. BPA can enter the human body through food and beverage, and can also be leached from landfill to the surrounding ecosystem to pollute the environment and endanger health. BPA has estrogenic activity and interferes with endogenous estrogenic activity by binding with estrogen receptors.
Recent studies have found that BPA below risk assessment has a low-dose effect on the development of the central nervous system in rodents. Perinatal exposure to BPA can affect the cortical structure and morphological development of cortical and hippocampal neurons in offspring rats, alter a variety of neurobehavioral and sexual differentiation, suggesting that the developing brain is BPA. Sensitive target organs. Previous studies in our laboratory found that after exposure to maternal BPA (0.05-50mg/kg/d) during perinatal period, the activity, exploration, anxiety and depression, learning and memory and other non-reproductive behaviors of offspring were altered; in vitro cultured hippocampal neurons exposed to BPA rapidly promoted dendritic filamentous mobility and density. The changes of plasticity and the key parts of information transmission are closely related to learning and memory. Synaptic modifications include the structure of synaptic interface, number density of synapses, synaptic transmission and so on, which are susceptible to the influence of environment and drugs. Therefore, this study observed the effects of BPA on the hippocampal synapses of male offspring during postnatal development through exposure of maternal BPA. The effects of BPA on synaptic structure were further explored by detecting synapsin I and PSD-95, as well as the expression levels of NMDA and AMPA receptors, which are closely related to learning and memory.
METHODS: Clean grade ICR male rats (30-35g) and female rats (25-30g) were caged after one week. Bisphenol A (BPA, 0.04, 0.4, 4mg/kg/day) was administered to female rats from the 7th day of pregnancy to the 21st day of postpartum. The control group was given solvent peanut oil. Ten male offspring in each group were sacrificed on the 14th, 21st and 56th days after birth. The synaptic density and synaptic interface parameters of hippocampal CA1 pyramidal cells were observed by ultrathin sections. The expression of synapsin I, NMDA receptor subunit NR1 and AMPA receptor subunit GluR1 in hippocampal CA1 pyramidal cells was detected by Western blot and immunohistochemistry.
Quantity one software was used to measure the optical density of the target protein bands, and image-Pro Plus 6.0 was used to analyze the synaptic morphology and immunohistochemical images. Sex.
Results: 1. Compared with the control group, BPA (0.04 and 4 mg/kg/d) significantly decreased the synaptic number density of PND14 and PND56 (P 0.01 or P 0.001), but there was no significant difference in PND21 between the two groups.
2. Perinatal maternal exposure to BPA altered the structure of synaptic interface. All BPA doses significantly increased the width of synaptic gap at different developmental stages (P 0.01); all doses significantly decreased the thickness of PSD (P 0.01); shortened the length of synaptic active bands at different developmental stages, of which 21 days after birth had the most significant effect (P 0.05 or P 0.01); but on the process. Contact curvature had little effect on BPA, mainly due to a significant increase in early development (PND14 and 21) (P 0.05).
3. Western blot analysis showed that BPA exposure significantly decreased the expression of synapsin I protein at 21 and 56 days postnatal (P 0.05 or P 0.01); significantly decreased the expression of PSD-95 protein at 56 days postnatal (P 0.05, P 0.01 or P 0.001); BPA (0.4 and 4 mg/kg/d) significantly decreased the expression of NR1 at 56 days postnatal. The expression of GluRl protein was inhibited, and the expression of GluRl protein in low dose group and high dose group was significantly or extremely significantly decreased at the development stage (P 0.05 or P 0.01).
4, the results of immunohistochemical technique were similar to those of WB.
CONCLUSION: BPA at a dose below tolerable daily intake (0.05 mg/kg/d) can also affect synaptic development and synaptic plasticity in the brain, and maternal BPA exposure during perinatal period affects synaptic structure in the hippocampus of offspring rats and decreases the expression of excitatory amino acid NMDA and AMPA receptors, synaptic protein PSD-95 and synapsin I. It is one of the reasons that perinatal maternal BPA exposure affects the development of offspring and adult behavior.
【學(xué)位授予單位】：浙江師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R114
本文編號(hào)：2247707
[Abstract]:Bisphenol A (BPA) is a typical environmental estrogen widely used in food and beverage packaging materials, canned metal resin lining, dental sealants, etc. BPA can enter the human body through food and beverage, and can also be leached from landfill to the surrounding ecosystem to pollute the environment and endanger health. BPA has estrogenic activity and interferes with endogenous estrogenic activity by binding with estrogen receptors.
Recent studies have found that BPA below risk assessment has a low-dose effect on the development of the central nervous system in rodents. Perinatal exposure to BPA can affect the cortical structure and morphological development of cortical and hippocampal neurons in offspring rats, alter a variety of neurobehavioral and sexual differentiation, suggesting that the developing brain is BPA. Sensitive target organs. Previous studies in our laboratory found that after exposure to maternal BPA (0.05-50mg/kg/d) during perinatal period, the activity, exploration, anxiety and depression, learning and memory and other non-reproductive behaviors of offspring were altered; in vitro cultured hippocampal neurons exposed to BPA rapidly promoted dendritic filamentous mobility and density. The changes of plasticity and the key parts of information transmission are closely related to learning and memory. Synaptic modifications include the structure of synaptic interface, number density of synapses, synaptic transmission and so on, which are susceptible to the influence of environment and drugs. Therefore, this study observed the effects of BPA on the hippocampal synapses of male offspring during postnatal development through exposure of maternal BPA. The effects of BPA on synaptic structure were further explored by detecting synapsin I and PSD-95, as well as the expression levels of NMDA and AMPA receptors, which are closely related to learning and memory.
METHODS: Clean grade ICR male rats (30-35g) and female rats (25-30g) were caged after one week. Bisphenol A (BPA, 0.04, 0.4, 4mg/kg/day) was administered to female rats from the 7th day of pregnancy to the 21st day of postpartum. The control group was given solvent peanut oil. Ten male offspring in each group were sacrificed on the 14th, 21st and 56th days after birth. The synaptic density and synaptic interface parameters of hippocampal CA1 pyramidal cells were observed by ultrathin sections. The expression of synapsin I, NMDA receptor subunit NR1 and AMPA receptor subunit GluR1 in hippocampal CA1 pyramidal cells was detected by Western blot and immunohistochemistry.
Quantity one software was used to measure the optical density of the target protein bands, and image-Pro Plus 6.0 was used to analyze the synaptic morphology and immunohistochemical images. Sex.
Results: 1. Compared with the control group, BPA (0.04 and 4 mg/kg/d) significantly decreased the synaptic number density of PND14 and PND56 (P 0.01 or P 0.001), but there was no significant difference in PND21 between the two groups.
2. Perinatal maternal exposure to BPA altered the structure of synaptic interface. All BPA doses significantly increased the width of synaptic gap at different developmental stages (P 0.01); all doses significantly decreased the thickness of PSD (P 0.01); shortened the length of synaptic active bands at different developmental stages, of which 21 days after birth had the most significant effect (P 0.05 or P 0.01); but on the process. Contact curvature had little effect on BPA, mainly due to a significant increase in early development (PND14 and 21) (P 0.05).
3. Western blot analysis showed that BPA exposure significantly decreased the expression of synapsin I protein at 21 and 56 days postnatal (P 0.05 or P 0.01); significantly decreased the expression of PSD-95 protein at 56 days postnatal (P 0.05, P 0.01 or P 0.001); BPA (0.4 and 4 mg/kg/d) significantly decreased the expression of NR1 at 56 days postnatal. The expression of GluRl protein was inhibited, and the expression of GluRl protein in low dose group and high dose group was significantly or extremely significantly decreased at the development stage (P 0.05 or P 0.01).
4, the results of immunohistochemical technique were similar to those of WB.
CONCLUSION: BPA at a dose below tolerable daily intake (0.05 mg/kg/d) can also affect synaptic development and synaptic plasticity in the brain, and maternal BPA exposure during perinatal period affects synaptic structure in the hippocampus of offspring rats and decreases the expression of excitatory amino acid NMDA and AMPA receptors, synaptic protein PSD-95 and synapsin I. It is one of the reasons that perinatal maternal BPA exposure affects the development of offspring and adult behavior.
【學(xué)位授予單位】：浙江師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R114
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