本文選題：丙烯酰胺 + 多巴胺轉(zhuǎn)運體　； 參考：《華中科技大學》2012年碩士論文

【摘要】：丙烯酰胺(Acrylamide,ACR)是一種從水合丙烯腈中提取出來的具有乙烯基的水溶性有機化合物，是國內(nèi)外廣泛應(yīng)用于化工冶煉、污水處理、紡織加工及化妝品生產(chǎn)的一種化學原料。早在20世紀70年代就有職業(yè)性中毒的報道，主要表現(xiàn)為神經(jīng)系統(tǒng)癥狀，除職業(yè)接觸外，在飲用水、高溫油炸食品和食品的包裝材料中都可以檢測到單體丙烯酰胺的存在，由于其可以直接經(jīng)口進入人體，從而引起了社會的高度關(guān)注。有關(guān)流行病學調(diào)查和實驗研究發(fā)現(xiàn)，丙烯酰胺對人和實驗動物除了有生殖毒性、遺傳毒性及致癌性等作用以外，還有明顯的神經(jīng)毒性。丙烯酰胺急性、亞急性中毒主要以損傷中樞神經(jīng)系統(tǒng)為主，表現(xiàn)為神經(jīng)精神癥狀和小腦共濟失調(diào)，而慢性中毒則以損害周圍神經(jīng)系統(tǒng)為主，關(guān)于其機制目前尚不清楚。 紋狀體作為基底節(jié)最大的綜合處理元件，包含著大量的多巴胺能神經(jīng)元，在整個腦部中的多巴胺神經(jīng)遞質(zhì)含量最高。在多巴胺能系統(tǒng)神經(jīng)通路中多巴胺神經(jīng)遞質(zhì)首先在突觸前膜中進行合成、轉(zhuǎn)運、釋放、降解，以達到突出間隙中多巴胺神經(jīng)遞質(zhì)的平衡，突觸間隙中的多巴胺神經(jīng)遞質(zhì)進入突觸后膜發(fā)揮突觸后效應(yīng)，而多巴胺相關(guān)轉(zhuǎn)運蛋白在整個過程中發(fā)揮著重要作用，維持突觸間隙中多巴胺神經(jīng)遞質(zhì)的平衡，當該平衡被打破后神經(jīng)系統(tǒng)就會出現(xiàn)不同程度的病癥，主要表現(xiàn)在感覺運動整合、軀體平衡運動及學習記憶等方面。以往對ACR的人群流行病學和實驗研究時所出現(xiàn)的神經(jīng)癥狀和體征，似乎很大程度上預示著與紋狀體多巴胺神經(jīng)通路中多巴胺神經(jīng)遞質(zhì)平衡被打破有關(guān)。 但直至目前，有關(guān)丙烯酰胺影響紋狀體維持多巴胺神經(jīng)遞質(zhì)平衡的相關(guān)轉(zhuǎn)運蛋白的研究報道較少，其具體機制仍未完全明確。因此，本研究擬通過體內(nèi)和體外實驗的結(jié)合，檢測丙烯酰胺對多巴胺轉(zhuǎn)運體、單胺囊泡轉(zhuǎn)運體及多巴胺合成酶—酪氨酸酸羥化酶的影響，探討其引起神經(jīng)毒性的可能機制，為防治丙烯酰胺中毒提供理論依據(jù)。 第一部分丙烯酰胺對大鼠神經(jīng)行為毒性的影響 目的：探討丙烯酰胺對大鼠神經(jīng)行為毒性的影響。 方法：健康成年雄性SD大鼠40只，體重230±20g，由華中科技大學同濟醫(yī)學院實驗動物中心提供，合格證號：SCXK（鄂）2010-0007號。大鼠在10h：14h明暗光循環(huán)的動物房內(nèi)，自由飲水、攝食，檢疫觀察7天后用于實驗。將大鼠按體重隨機分為4組，分別為對照組、低劑量(20mg/kg)組、中劑量(30mg/kg)組、高劑量（40mg/kg）組，每組10只，均單籠飼養(yǎng)。按照對應(yīng)的染毒劑量對各ACR劑量組進行灌胃染毒，對照組則灌胃給予等容量的生理鹽水，每周連續(xù)染毒5天間隔2天，總共19天，并于第0，6，13，19天稱量體重并進行步態(tài)評分，于第0，19天測定大鼠的后肢支撐力和甩尾時間。末次給藥24小時后，斷頭處死大鼠，迅速取出腦組織，用預冷PBS緩沖液洗凈，稱量大小腦重量，并于冰盤上迅速分離紋狀體、大腦皮層及小腦，并用干凈濾紙吸干置于㧟80℃冰箱中保存?zhèn)溆�。取出大鼠心、肝、脾、肺、腎、睪丸等臟器，稱量重量并記錄。 結(jié)果：（1）在整個染毒期內(nèi)低劑量組大鼠雖然體重增長始終低于對照組，但差異無顯著性；與對照組比較，中劑量組大鼠平均體重于第19d明顯降低，差異有統(tǒng)計學意義(P0.01)；高劑量組大鼠平均體重于第6d、13d、19d也明顯下降，差異有統(tǒng)計學意義(P0.01)。另外，與低、中劑量組相比，高劑量組大鼠平均體重于第19d明顯降低，差異有統(tǒng)計學意義(P0.01)。（2）臟器系數(shù)顯示，高劑量組大小腦、心臟、肺臟、腎臟及睪丸臟器系數(shù)均有增加，且差異具有統(tǒng)計學意義（P0.05或P0.01）。（3）染毒第6d、13d、19d，與對照組比較，低、中、高劑量組步態(tài)評分均明顯升高，差異有統(tǒng)計學意義(P0.01)，且染毒結(jié)束后，其分值分別集中于1-2、2-3、3-4分之間，并呈現(xiàn)出時間—劑量—效應(yīng)關(guān)系。另外，與低、中劑量組相比，高劑量組步態(tài)評分于第6d、13d、19d也明顯增加，差異有統(tǒng)計學意義(P0.05或P0.01)。（4）染毒第19d，與對照組相比，低、中、高劑量組后肢支撐力指數(shù)均明顯增加，差異有統(tǒng)計學意義(P0.01)，且呈現(xiàn)出劑量—效應(yīng)關(guān)系。另外，與低、中劑量組相比，高劑量組后肢支撐力指數(shù)也明顯增加，差異有統(tǒng)計學意義(P0.01)（5）染毒第19d高劑量組的甩尾時間比對照組明顯縮短，差異有統(tǒng)計學意義(P0.01)。 結(jié)論：ACR亞急性染毒能導致大鼠體重下降；大小腦、心臟、肺臟、腎臟及睪丸臟器系數(shù)增加；步態(tài)評分增高；后肢支撐力指數(shù)增加；甩尾時間縮短。具有明顯的神經(jīng)毒性，主要引起感覺運動功能異常。 第二部分丙烯酰胺對大鼠多巴胺相關(guān)轉(zhuǎn)運蛋白mRNA和蛋白表達的影響 目的：探討丙烯酰胺對大鼠紋狀體多巴胺轉(zhuǎn)運體(DAT)、單胺囊泡轉(zhuǎn)運體(VMAT2)和酪氨酸羥化酶（TH）mRNA表達和蛋白水平的影響。 方法：動物分組及處理同第一部分。實時熒光定量PCR技術(shù)檢測大腦皮層、小腦和紋狀體內(nèi)DAT、VMAT2、THmRNA表達水平，并檢測紋狀體中MAO-BmRNA表達水平；WesternBlot蛋白印跡技術(shù)檢測紋狀體內(nèi)DAT、VMAT2、TH的蛋白表達水平。 結(jié)果：（1）大腦皮層結(jié)果顯示：與對照組比較，各劑量組VMAT2mRNA表達水平均明顯降低，其中低、中劑量組分別降低了30%、29%，差異具有統(tǒng)計學意義(P0.05)，而高劑量組則降低了56%，差異具有統(tǒng)計學意義(P0.01)。DAT、TH各組間差異均無統(tǒng)計學意義。（2）小腦結(jié)果顯示：與對照組比較，低、中、高劑量組DAT、VMAT2、THmRNA表達均明顯降低，差異具有統(tǒng)計學意義(P0.01)。（3）紋狀體結(jié)果顯示：DATmRNA表達與對照組相比，低、中、高劑量組均明顯降低，差異具有統(tǒng)計學意義(P0.01)；Westernblot結(jié)果顯示，高劑量組的糖基化DAT蛋白表達明顯降低，僅為對照組的78%，差異具有統(tǒng)計學意義(P0.05)。與對照組相比，高劑量組VMAT2mRNA表達顯著降低(P0.01)，VMAT2蛋白表達也顯著降低，差異具有統(tǒng)計學意義(P0.05)。與對照組相比，高劑量組THmRNA和蛋白表達均明顯升高，差異具有統(tǒng)計學意義(P0.05)。與對照組相比，高劑量組MAO-BmRNA表達明顯降低，差異具有統(tǒng)計學意義(P0.01)。 結(jié)論：ACR亞急性染毒不僅會使大鼠小腦中DAT、VMAT2、THmRNA表達降低，還會使紋狀體中DAT、VMAT2、MAO-BmRNA和蛋白表達降低，THmRNA和蛋白表達增加，提示ACR亞急性染毒會導致多巴胺神經(jīng)遞質(zhì)的合成和轉(zhuǎn)運功能失常，引起胞質(zhì)和突觸間隙中多巴胺含量改變，從而導致多巴胺神經(jīng)系統(tǒng)功能紊亂。 第三部分丙烯酰胺對PC12細胞多巴胺相關(guān)轉(zhuǎn)運蛋白的蛋白表達的影響 目的：探討丙烯酰胺對PC12細胞中多巴胺轉(zhuǎn)運體(DAT)、單胺囊泡轉(zhuǎn)運體(VMAT2)和酪氨酸羥化酶（TH）蛋白表達的影響。 方法：取對數(shù)生長期的PC12細胞，分為1個對照組和6個染毒組，各染毒組ACR終濃度分別為0.1mmol/L，0.3mmol/L，0.6mmol/L，1.25mmol/L，，2.5mmol/L，5mmol/L，染毒24h。用WesternBlot蛋白印跡技術(shù)檢測PC12細胞中DAT、VMAT2、TH的蛋白表達水平。 結(jié)果：（1）DAT蛋白定量結(jié)果顯示：與對照組比較，0.6mmol/L和1.25mmol/L組PC12細胞中糖基化DAT明顯降低，差異具有統(tǒng)計學意義(P0.05)；與對照組比較，0.1mmol/L、0.6mmol/L、1.25mmol/L組非糖基化DAT也明顯降低，差異具有統(tǒng)計學意義(P0.05)。（2）VMAT2蛋白定量結(jié)果顯示：與對照組比較，0.6mmol/L-5mmol/L組PC12細胞中VMAT2明顯降低，差異具有統(tǒng)計學意義(P0.01)；與0.1mmol/L和0.3mmol/L組比較，0.6mmol/L-5mmol/L組VMAT2也降低，差異具有統(tǒng)計學意義(P0.5或P0.01)。（3）TH蛋白定量結(jié)果顯示：與對照組比較，2.5mmol/L、5mmol/L組PC12細胞中TH含量明顯升高，差異具有統(tǒng)計學意義(P0.05，P0.01)。 結(jié)論：ACR可以引起PC12細胞中DAT、VMAT2蛋白含量降低，同時還可以引起TH蛋白含量升高，提示ACR可以引起PC12細胞中多巴胺神經(jīng)遞質(zhì)的合成和轉(zhuǎn)運功能異常。 綜上所述，ACR亞急性染毒能導致大鼠體重下降，后肢支撐力指數(shù)增加，步態(tài)評分增高，甩尾時間縮短，說明其神經(jīng)毒性作用比較明顯；其作用于小腦和紋狀體后，不僅會使大鼠小腦中DAT、VMAT2、THmRNA表達降低，還會使紋狀體中DAT、VMAT2、MAO-BmRNA和蛋白表達降低及THmRNA和蛋白表達增加。另外，ACR還能引起PC12細胞中DAT、VMAT2蛋白含量降低，同時還可以造成TH蛋白含量升高。因此，推測紋狀體DA系統(tǒng)功能紊亂可能與多巴胺相關(guān)轉(zhuǎn)運蛋白受到影響有關(guān)。
[Abstract]:acrylamide ( acrylamide , ACR ) is a kind of water - soluble organic compound which is extracted from hydrated acrylonitrile . It is a kind of chemical raw material widely used in chemical smelting , sewage treatment , textile processing and cosmetics .

The dopamine neurotransmitter in the synaptic cleft plays an important role in the process of synthesis , transport , release and degradation of the dopamine neurotransmitter in the synaptic cleft . The dopamine neurotransmitter in the synaptic cleft plays an important role in the process of synaptic cleft .

However , until now , there are few studies on the relative transport proteins involved in the maintenance of dopamine neurotransmitter balance by acrylamide , and the specific mechanism is still not completely clear . Therefore , this study intends to examine the effects of acrylamide on dopamine transporter , monoamine vesicle transporter and dopamine synthetase - tyrosine hydroxylase in vivo and in vitro experiments , and to explore the possible mechanism of causing neurotoxicity , and provide a theoretical basis for the prevention of acrylamide poisoning .

Effects of acrylamide on neurobehavioral toxicity in rats

Objective : To study the effect of acrylamide on neurobehavioral toxicity in rats .

Methods : 40 healthy adult male SD rats were randomly divided into four groups : control group , low - dose ( 20mg / kg ) group , middle - dose ( 30 mg / kg ) group and high - dose ( 40mg / kg ) group .

Results : ( 1 ) Although the weight gain of the low - dose group was lower than that of the control group during the whole exposure period , the difference was not significant .
Compared with the control group , the mean body weight of the rats in the middle dose group was significantly lower than that in the control group ( P0.01 ) .
The average body weight of rats in high dose group was significantly lower than that in control group ( P0.05 or P0.01 ) . ( 4 ) Compared with the control group , the support force index of the hindlimb of the high - dose group was significantly increased compared with the control group ( P0.01 ) , and the difference was statistically significant ( P0.01 ) ( P0.01 ) ( P0.01 ) .

Conclusion : ACR sub - acute toxicity can lead to a decrease in weight of rats .
The coefficients of brain , heart , lung , kidney and testis were increased .
and the gait score is increased ;
and the support force index of the hindlimb is increased ;
The spin - off time is shortened . It has obvious neurotoxicity , which mainly causes abnormal sensory motor function .

Effect of acrylamide on mRNA and protein expression of dopamine - related transporter in rats

Objective : To investigate the effects of acrylamide on the expression of dopamine transporter ( DAT ) , monoamine vesicle transporter ( VMAT2 ) and tyrosine hydroxylase ( TH ) mRNA and protein level in rats .

Methods : The levels of DAT , VMAT2 , THmRNA in cerebral cortex , cerebellum and striatum were detected by real - time fluorescence quantitative PCR , and the level of MAO - B mRNA in striatum was detected by real - time fluorescence quantitative PCR .
Western Blot was used to detect the protein expression level of DAT , VMAT2 , TH in striatum .

Results : ( 1 ) Compared with the control group , the expression level of VMAT2mRNA in each dose group was significantly lower than that in the control group ( P < 0.01 ) .
Compared with the control group , the expression of mRNA and protein in high - dose group significantly decreased ( P0.05 ) . Compared with the control group , the expression of THmRNA and protein in the high - dose group was significantly decreased ( P0.05 ) . Compared with the control group , the expression of THmRNA and protein in high - dose group was significantly decreased , and the difference was statistically significant ( P0.01 ) .

Conclusion : The expression of DAT , VMAT2 , THmRNA in the rat cerebellum decreased , and the expression of DAT , VMAT2 , MAO - B mRNA and protein in striatum decreased , THmRNA and protein expression increased , suggesting that ACR sub - acute intoxication could lead to the synthesis and transport of dopamine neurotransmitter , which caused the change of dopamine content in the cytoplasm and synaptic cleft .

Effect of acrylamide on protein expression of dopamine - associated transporter in PC12 cells

Objective : To investigate the effects of acrylamide on the expression of dopamine transporter ( DAT ) , monoamine vesicle transporter ( VMAT2 ) and tyrosine hydroxylase ( TH ) protein in PC12 cells .

Methods : PC12 cells with logarithmic growth were divided into 1 control group and 6 groups . The final concentrations of ACR were 0.1 mmol / L , 0.3 mmol / L , 0.6 mmol / L , 1.25 mmol / L , 2.5 mmol / L , 5 mmol / L and 24 h respectively . Western Blot was used to detect the protein expression level of DAT , VMAT2 , TH in PC12 cells .

Results : ( 1 ) The quantitative results of DAT protein showed that the levels of glycosylated DAT in PC12 cells were significantly lower than those in control group , 0.6mmol / L and 1.25 mmol / L PC12 cells ( P0.05 ) .
Compared with the control group , the non - glycosylated DAT of 0 . 1 mmol / L , 0.6 mmol / L and 1.25 mmol / L was significantly lower than that in the control group ( P0.05 ) .
Compared with the control group , the amount of TH in PC12 cells was significantly higher than that in the control group ( P0.05 , P0.01 ) .

Conclusion : ACR can induce the decrease of DAT and VMAT2 protein in PC12 cells , but also increase the content of TH protein , suggesting that ACR can cause abnormal synthesis and transport function of dopamine neurotransmitter in PC12 cells .

In conclusion , the acute toxicity of ACR could result in the decrease of weight of the rats , the increase of the support force index of the hind limbs , the increase of gait score and the shortening of the spin - tail time , which showed that the neurotoxicity was more obvious .
In addition , the amount of DAT , VMAT2 , MAO - BmRNA and protein in the striatum can be decreased , and the expression of THmRNA and protein can be increased . In addition , the ACR can also cause the decrease of DAT and VMAT2 protein in PC12 cells , and also increase the content of TH protein . Therefore , it is speculated that the dysfunction of the functional disorder of the striatum DA system may be related to the influence of dopamine - related transport protein .

【學位授予單位】：華中科技大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R114

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