氟化鈉對(duì)中華大蟾蜍胚胎發(fā)育的影響
發(fā)布時(shí)間：2018-07-23 19:37
【摘要】：氟是存在于地球上最豐富的化學(xué)元素之一,由于氟化學(xué)性質(zhì)活潑,在自然界中幾乎不以氟單體的形式存在,而是與其它元素結(jié)合形成無(wú)機(jī)和有機(jī)氟化物。氟是動(dòng)物及人體發(fā)育過(guò)程中必需的微量元素之一。適量攝入氟有益于齲齒的預(yù)防和骨骼發(fā)育,然而過(guò)量的氟攝取會(huì)對(duì)動(dòng)物及人體產(chǎn)生毒性效應(yīng),造成氟斑牙、氟骨癥外,還會(huì)引起機(jī)體神經(jīng)系統(tǒng)、內(nèi)分泌腺等非骨相組織器官的損傷。近年來(lái),隨著現(xiàn)代工業(yè)的發(fā)展,人類生產(chǎn)活動(dòng)造成大量氟化物的產(chǎn)生,如金屬冶煉、有機(jī)肥料的加工、含氟“三廢”的排放,導(dǎo)致水環(huán)境中氟含量持續(xù)升高。中華大蟾蜍作為我國(guó)兩棲類最具代表性的物種,其胚胎發(fā)育和幼體發(fā)育均在水域中進(jìn)行,水域氟污染會(huì)直接影響其發(fā)育。在本研究中,首先研究了氟化鈉對(duì)中華大蟾蜍胚胎的急性毒性效應(yīng),以及亞致死濃度下氟化鈉對(duì)中華大蟾蜍胚胎生長(zhǎng)發(fā)育的影響,其次對(duì)胚胎的體重和體長(zhǎng)進(jìn)行了測(cè)量和分期,和電鏡觀察,最后采用RT-PCR和Real-time PCR方法,對(duì)不同分期中華大蟾蜍胚胎樣本中的Dio2, Dio3, TRα和TRβ mRNA水平進(jìn)行定量研究。本文的主要研究結(jié)果如下：1.采用半靜態(tài)生物毒性試驗(yàn)方法對(duì)中華大蟾蜍G11期-G22期(本研究依照Gosner (1960)對(duì)蛙類胚胎的分期方法來(lái)確定蛙胚的不同發(fā)育時(shí)期)的胚胎進(jìn)行氟化鈉的高濃度(200-500 mg/L)毒性試驗(yàn),統(tǒng)計(jì)了氟化鈉對(duì)中華大蟾蜍胚胎的致畸個(gè)數(shù)和致死個(gè)數(shù),并計(jì)算了半數(shù)致畸濃度(EC50)和半數(shù)致死濃度(LC50)。結(jié)果顯示中華大蟾蜍胚胎暴露在氟化鈉中216小時(shí)的ECso和LC50分別為416.37mg/L和561.64 m/L,并且暴露在500 mg/L的畸形率和致死率分別是66.25%和47.50%。2.氟化鈉可以造成中華大蟾蜍胚胎畸形,主要包括卵黃囊水腫、體軸彎曲和組織畸形增生。掃描電鏡進(jìn)一步了解氟化鈉對(duì)胚胎的致畸效應(yīng),結(jié)果顯示氟化鈉會(huì)導(dǎo)致胚胎尾部表皮細(xì)胞畸形增生和變性。3.采用半靜態(tài)生物毒性試驗(yàn)方法對(duì)中華大蟾蜍G7期-G22期的胚胎進(jìn)行氟化鈉的亞致死濃度(1-150 mg/L)毒性試驗(yàn),通過(guò)測(cè)定體重和體長(zhǎng)等相關(guān)形態(tài)學(xué)數(shù)據(jù),推測(cè)氟化鈉對(duì)中華大蟾蜍胚胎生長(zhǎng)發(fā)育的影響。結(jié)果顯示,氟暴露對(duì)體重和體長(zhǎng)均呈現(xiàn)出了劑量相關(guān)性的影響。1 mg/L氟暴露對(duì)胚胎的體重和體長(zhǎng)沒(méi)有顯著性影響(p0.05),10-150 mg/L氟暴露會(huì)造成胚胎的體重和體長(zhǎng)的顯著降低(p0.05)。4.在亞致死濃度毒性試驗(yàn)中,記錄暴露在不同濃度氟化鈉,中華大蟾蜍胚胎發(fā)育到G22期的累計(jì)發(fā)育時(shí)間；記錄6d-9d,胚胎發(fā)育的平均時(shí)期。采用兩種不同的統(tǒng)計(jì)方法,分析氟化鈉對(duì)中華大蟾蜍胚胎生長(zhǎng)發(fā)育的影響。累計(jì)發(fā)育時(shí)間結(jié)果的統(tǒng)計(jì)分析顯示,在氟化鈉處理組,胚胎的發(fā)育延遲顯著(p0.05)；胚胎發(fā)育分期的結(jié)果統(tǒng)計(jì)分析表明,1 mg/L氟暴露對(duì)胚胎的發(fā)育進(jìn)程無(wú)顯著性影響(p0.05),10-150 mg/L氟暴露會(huì)顯著延遲胚胎發(fā)育(p0.05)5.采用實(shí)時(shí)定量Real-time PCR方法,檢測(cè)氟化鈉在中華大蟾蜍胚胎發(fā)育過(guò)程中對(duì)脫碘酶基因Dio2,Dio3以及甲狀腺激素受體基因TRa和TRβ表達(dá)水平的影響。結(jié)果顯示：在G11期(原腸胚期),氟化鈉處理組中Dio3和TRβ基因的表達(dá)量顯著性增加p0.05),而Dio2和TRa基因的表達(dá)水平與對(duì)照組相比,無(wú)顯著性差異(p0.05)；在G16期(神經(jīng)胚期),經(jīng)氟化鈉暴露的實(shí)驗(yàn)組,其Dio2,Dio3,TRα和TRβ基因的表達(dá)水平與對(duì)照組相比均有顯著性升高(p0.05)；在G18期(肌肉效應(yīng)期),經(jīng)100 mg/L氟化鈉處理的實(shí)驗(yàn)組中,Dio2和TRa基因的表達(dá)量顯著性降低(p0.05),而Dio3和TRβ基因的表達(dá)量在100和150 mg/L氟化鈉暴露組中顯著性增加(p0.05)；在G22期(尾鰭循環(huán)期),經(jīng)氟化鈉暴露的實(shí)驗(yàn)組,其Dio2,Dio3,TRα和TRβ基因的表達(dá)水平與對(duì)照組相比均有顯著性降低(p0.05)。推測(cè)氟化鈉可能通過(guò)干擾脫碘酶基因Dio2和Dio3,以及甲狀腺激素受體基因TRa和TRβ的正常表達(dá),從而影響中華大蟾蜍胚胎的發(fā)育。本研究首次從傳統(tǒng)毒理學(xué)和現(xiàn)代分子生物學(xué)水平較系統(tǒng)的研究了氟化鈉對(duì)中華大蟾蜍胚胎發(fā)育的影響,取得了較完整的研究數(shù)據(jù),揭示了氟化鈉對(duì)中華大蟾蜍胚胎的中毒機(jī)理,為兩棲類毒理學(xué)研究及其棲息地的保護(hù)研究提供了參考。
[Abstract]:Fluorine is one of the most abundant chemical elements in the earth. Because of the active chemical properties of fluorine, it is almost not in the form of fluorine monomers in nature, but is combined with other elements to form inorganic and organic fluorides. Fluorine is one of the essential trace elements in the development of animals and human beings. Bone development, however, excessive fluoride intake can produce toxic effects on animals and human body, causing dental fluorosis, fluorosis, and the injury of non osteogenic organs such as the nervous system and endocrine gland. In recent years, with the development of modern industry, human production activities caused a large number of fluoride production, such as metal smelting, organic fertilizer. As the most representative species of amphibians in China, Bufo bufo gargarizans, as the most representative species of amphibians in China, are the most representative species in the water environment. The development of the fluorine pollution in the waters will directly affect its development. In this study, the first study of sodium fluoride to Bufo Bufo bufo gargarizans embryos The effects of acute toxicity and sublethal concentration of sodium fluoride on the growth and development of Bufo bufo gargarizans were observed. Secondly, the weight and body length of the embryos were measured and staging and observed by electron microscopy. Finally, the RT-PCR and Real-time PCR methods were used to improve the levels of Dio2, Dio3, TR a and TR beta mRNA in different stages of the embryo samples of Bufo bufo gargarizans. The main results of this study are as follows: 1. the high concentration (200-500 mg/L) toxicity test of sodium fluoride (200-500 mg/L) in the G11 phase -G22 phase of Bufo bufo gargarizans (this study was determined by Gosner (1960) stage method for frog embryo at different developmental stages) was carried out, and the fluorination was statistically analyzed. The number of teratogenicity and lethal number of sodium to Bufo gargarizans embryos were calculated, and half of the teratogenic concentration (EC50) and half lethal concentration (LC50) were calculated. The results showed that the 216 hours of ECso and LC50 were 416.37mg/L and 561.64 m/L respectively in the embryo exposure of Bufo bufo gargarizans, respectively, and the malformation rate and mortality rate of 500 mg/L were 66.25% and 47, respectively. .50%.2. sodium fluoride can cause fetal malformation of Bufo bufo gargarizans, mainly including ovoid sac edema, body axis bending and tissue malformation. The scanning electron microscope is used to further understand the teratogenic effect of sodium fluoride on embryos. The results show that sodium fluoride can lead to abnormal proliferation and denaturation of embryonic tail epidermal cells in.3. by semi static biotoxicity test method. The sublethal concentration (1-150 mg/L) of sodium fluoride (1-150 mg/L) toxicity test of sodium fluoride in the -G22 phase of Bufo bufo gargarizans was conducted. The effects of sodium fluoride on the growth and development of Bufo bufo gargarizans were estimated by measuring the morphological data of body weight and body length. The results showed that fluorine exposure had a dose-dependent effect on the.1 mg/L fluorine storm. Exposure had no significant influence on the weight and body length of the embryo (P0.05). 10-150 mg/L fluorine exposure could cause a significant decrease in the body weight and body length of the embryo (P0.05).4. in the sublethal concentration toxicity test, to record the cumulative time of exposure to different concentrations of sodium fluoride, the embryo development of Bufo bufo gargarizans to the G22 period, and record 6d-9d, the flat development of embryo. Two different statistical methods were used to analyze the effects of sodium fluoride on the growth and development of Bufo bufo gargarizans. The statistical analysis of the cumulative development time results showed that the development delay of embryos was significant (P0.05) in the sodium fluoride treatment group, and the results of embryo development staging showed that 1 mg/L fluorine exposure had no effect on the development of embryos. Significant effect (P0.05), 10-150 mg/L fluorine exposure significantly delayed embryo development (P0.05) 5. using real-time quantitative Real-time PCR method to detect the effect of sodium fluoride on the expression level of deiodonase gene Dio2, Dio3, and thyroid hormone receptor gene TRa and TR beta during the development of Bufo bufo gargarizans. The results showed that in the G11 phase (the primary gut embryo) The expression of Dio3 and TR beta gene in the sodium fluoride treatment group was significantly increased by P0.05), while the expression level of Dio2 and TRa genes had no significant difference compared with the control group (P0.05). In the G16 phase (neural embryo stage), the expression level of Dio2, Dio3, TR A and TR beta gene in the experimental group with sodium fluoride exposure was significantly higher than that of the control group (P). 0.05); in the G18 phase (muscle effect period), the expression of Dio2 and TRa genes decreased significantly in the experimental group treated with 100 mg/L sodium fluoride (P0.05), while the expression of Dio3 and TR beta genes increased significantly in the 100 and 150 mg/L sodium fluoride exposure group (P0.05); in the G22 phase (caudal fin cycle), the experimental group of sodium fluoride exposure was Dio2, Dio3, and alpha. The expression level of the TR beta gene was significantly lower than that in the control group (P0.05). It is speculated that the sodium fluoride may affect the normal expression of the deiodinase gene Dio2 and Dio3, as well as the normal expression of the thyroid hormone receptor gene TRa and TR beta, thus affecting the development of the embryo of Bufo bufo gargarizans. The effect of sodium fluoride on the embryo development of Bufo bufo gargarizans was studied in a relatively systematic way. The complete research data were obtained, and the poisoning mechanism of sodium fluoride on Bufo bufo gargarizans embryos was revealed, which provided a reference for the study of amphibian toxicology and the protection of its habitat.
【學(xué)位授予單位】：陜西師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：X171.5;Q954.4
[Abstract]:Fluorine is one of the most abundant chemical elements in the earth. Because of the active chemical properties of fluorine, it is almost not in the form of fluorine monomers in nature, but is combined with other elements to form inorganic and organic fluorides. Fluorine is one of the essential trace elements in the development of animals and human beings. Bone development, however, excessive fluoride intake can produce toxic effects on animals and human body, causing dental fluorosis, fluorosis, and the injury of non osteogenic organs such as the nervous system and endocrine gland. In recent years, with the development of modern industry, human production activities caused a large number of fluoride production, such as metal smelting, organic fertilizer. As the most representative species of amphibians in China, Bufo bufo gargarizans, as the most representative species of amphibians in China, are the most representative species in the water environment. The development of the fluorine pollution in the waters will directly affect its development. In this study, the first study of sodium fluoride to Bufo Bufo bufo gargarizans embryos The effects of acute toxicity and sublethal concentration of sodium fluoride on the growth and development of Bufo bufo gargarizans were observed. Secondly, the weight and body length of the embryos were measured and staging and observed by electron microscopy. Finally, the RT-PCR and Real-time PCR methods were used to improve the levels of Dio2, Dio3, TR a and TR beta mRNA in different stages of the embryo samples of Bufo bufo gargarizans. The main results of this study are as follows: 1. the high concentration (200-500 mg/L) toxicity test of sodium fluoride (200-500 mg/L) in the G11 phase -G22 phase of Bufo bufo gargarizans (this study was determined by Gosner (1960) stage method for frog embryo at different developmental stages) was carried out, and the fluorination was statistically analyzed. The number of teratogenicity and lethal number of sodium to Bufo gargarizans embryos were calculated, and half of the teratogenic concentration (EC50) and half lethal concentration (LC50) were calculated. The results showed that the 216 hours of ECso and LC50 were 416.37mg/L and 561.64 m/L respectively in the embryo exposure of Bufo bufo gargarizans, respectively, and the malformation rate and mortality rate of 500 mg/L were 66.25% and 47, respectively. .50%.2. sodium fluoride can cause fetal malformation of Bufo bufo gargarizans, mainly including ovoid sac edema, body axis bending and tissue malformation. The scanning electron microscope is used to further understand the teratogenic effect of sodium fluoride on embryos. The results show that sodium fluoride can lead to abnormal proliferation and denaturation of embryonic tail epidermal cells in.3. by semi static biotoxicity test method. The sublethal concentration (1-150 mg/L) of sodium fluoride (1-150 mg/L) toxicity test of sodium fluoride in the -G22 phase of Bufo bufo gargarizans was conducted. The effects of sodium fluoride on the growth and development of Bufo bufo gargarizans were estimated by measuring the morphological data of body weight and body length. The results showed that fluorine exposure had a dose-dependent effect on the.1 mg/L fluorine storm. Exposure had no significant influence on the weight and body length of the embryo (P0.05). 10-150 mg/L fluorine exposure could cause a significant decrease in the body weight and body length of the embryo (P0.05).4. in the sublethal concentration toxicity test, to record the cumulative time of exposure to different concentrations of sodium fluoride, the embryo development of Bufo bufo gargarizans to the G22 period, and record 6d-9d, the flat development of embryo. Two different statistical methods were used to analyze the effects of sodium fluoride on the growth and development of Bufo bufo gargarizans. The statistical analysis of the cumulative development time results showed that the development delay of embryos was significant (P0.05) in the sodium fluoride treatment group, and the results of embryo development staging showed that 1 mg/L fluorine exposure had no effect on the development of embryos. Significant effect (P0.05), 10-150 mg/L fluorine exposure significantly delayed embryo development (P0.05) 5. using real-time quantitative Real-time PCR method to detect the effect of sodium fluoride on the expression level of deiodonase gene Dio2, Dio3, and thyroid hormone receptor gene TRa and TR beta during the development of Bufo bufo gargarizans. The results showed that in the G11 phase (the primary gut embryo) The expression of Dio3 and TR beta gene in the sodium fluoride treatment group was significantly increased by P0.05), while the expression level of Dio2 and TRa genes had no significant difference compared with the control group (P0.05). In the G16 phase (neural embryo stage), the expression level of Dio2, Dio3, TR A and TR beta gene in the experimental group with sodium fluoride exposure was significantly higher than that of the control group (P). 0.05); in the G18 phase (muscle effect period), the expression of Dio2 and TRa genes decreased significantly in the experimental group treated with 100 mg/L sodium fluoride (P0.05), while the expression of Dio3 and TR beta genes increased significantly in the 100 and 150 mg/L sodium fluoride exposure group (P0.05); in the G22 phase (caudal fin cycle), the experimental group of sodium fluoride exposure was Dio2, Dio3, and alpha. The expression level of the TR beta gene was significantly lower than that in the control group (P0.05). It is speculated that the sodium fluoride may affect the normal expression of the deiodinase gene Dio2 and Dio3, as well as the normal expression of the thyroid hormone receptor gene TRa and TR beta, thus affecting the development of the embryo of Bufo bufo gargarizans. The effect of sodium fluoride on the embryo development of Bufo bufo gargarizans was studied in a relatively systematic way. The complete research data were obtained, and the poisoning mechanism of sodium fluoride on Bufo bufo gargarizans embryos was revealed, which provided a reference for the study of amphibian toxicology and the protection of its habitat.
【學(xué)位授予單位】：陜西師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：X171.5;Q954.4
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