本文選題：丙烯酰胺 + 多巴胺轉(zhuǎn)運(yùn)體��； 參考：《華中科技大學(xué)》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(lái)的具有乙烯基的水溶性有機(jī)化合物，是國(guó)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(xué)原料。早在20世紀(jì)70年代就有職業(yè)性中毒的報(bào)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(cè)到單體丙烯酰胺的存在，由于其可以直接經(jīng)口進(jìn)入人體，從而引起了社會(huì)的高度關(guān)注。有關(guān)流行病學(xué)調(diào)查和實(shí)驗(yàn)研究發(fā)現(xiàn)，丙烯酰胺對(duì)人和實(shí)驗(yàn)動(dòng)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(jì)失調(diào)，而慢性中毒則以損害周?chē)窠?jīng)系統(tǒng)為主，關(guān)于其機(jī)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(gè)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(jìn)行合成、轉(zhuǎn)運(yùn)、釋放、降解，以達(dá)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(jìn)入突觸后膜發(fā)揮突觸后效應(yīng)，而多巴胺相關(guān)轉(zhuǎn)運(yùn)蛋白在整個(gè)過(guò)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(dāng)該平衡被打破后神經(jīng)系統(tǒng)就會(huì)出現(xiàn)不同程度的病癥，主要表現(xiàn)在感覺(jué)運(yùn)動(dòng)整合、軀體平衡運(yùn)動(dòng)及學(xué)習(xí)記憶等方面。以往對(duì)ACR的人群流行病學(xué)和實(shí)驗(yàn)研究時(shí)所出現(xiàn)的神經(jīng)癥狀和體征，似乎很大程度上預(yù)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(yùn)蛋白的研究報(bào)道較少，其具體機(jī)制仍未完全明確。因此，本研究擬通過(guò)體內(nèi)和體外實(shí)驗(yàn)的結(jié)合，檢測(cè)丙烯酰胺對(duì)多巴胺轉(zhuǎn)運(yùn)體、單胺囊泡轉(zhuǎn)運(yùn)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(jī)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(duì)大鼠神經(jīng)行為毒性的影響 目的：探討丙烯酰胺對(duì)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(xué)同濟(jì)醫(yī)學(xué)院實(shí)驗(yàn)動(dòng)物中心提供，合格證號(hào)：SCXK（鄂）2010-0007號(hào)。大鼠在10h：14h明暗光循環(huán)的動(dòng)物房?jī)?nèi)，自由飲水、攝食，檢疫觀察7天后用于實(shí)驗(yàn)。將大鼠按體重隨機(jī)分為4組，分別為對(duì)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(duì)應(yīng)的染毒劑量對(duì)各ACR劑量組進(jìn)行灌胃染毒，對(duì)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(chēng)量體重并進(jìn)行步態(tài)評(píng)分，于第0，19天測(cè)定大鼠的后肢支撐力和甩尾時(shí)間。末次給藥24小時(shí)后，斷頭處死大鼠，迅速取出腦組織，用預(yù)冷PBS緩沖液洗凈，稱(chēng)量大小腦重量，并于冰盤(pán)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(chēng)量重量并記錄。 結(jié)果：（1）在整個(gè)染毒期內(nèi)低劑量組大鼠雖然體重增長(zhǎng)始終低于對(duì)照組，但差異無(wú)顯著性；與對(duì)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(jì)學(xué)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(jì)學(xué)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(jì)學(xué)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(jì)學(xué)意義（P0.05或P0.01）。（3）染毒第6d、13d、19d，與對(duì)照組比較，低、中、高劑量組步態(tài)評(píng)分均明顯升高，差異有統(tǒng)計(jì)學(xué)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(shí)間—?jiǎng)┝俊?yīng)關(guān)系。另外，與低、中劑量組相比，高劑量組步態(tài)評(píng)分于第6d、13d、19d也明顯增加，差異有統(tǒng)計(jì)學(xué)意義(P0.05或P0.01)。（4）染毒第19d，與對(duì)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(jì)學(xué)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(jì)學(xué)意義(P0.01)（5）染毒第19d高劑量組的甩尾時(shí)間比對(duì)照組明顯縮短，差異有統(tǒng)計(jì)學(xué)意義(P0.01)。 結(jié)論：ACR亞急性染毒能導(dǎo)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(píng)分增高；后肢支撐力指數(shù)增加；甩尾時(shí)間縮短。具有明顯的神經(jīng)毒性，主要引起感覺(jué)運(yùn)動(dòng)功能異常。 第二部分丙烯酰胺對(duì)大鼠多巴胺相關(guān)轉(zhuǎn)運(yùn)蛋白mRNA和蛋白表達(dá)的影響 目的：探討丙烯酰胺對(duì)大鼠紋狀體多巴胺轉(zhuǎn)運(yùn)體(DAT)、單胺囊泡轉(zhuǎn)運(yùn)體(VMAT2)和酪氨酸羥化酶（TH）mRNA表達(dá)和蛋白水平的影響。 方法：動(dòng)物分組及處理同第一部分。實(shí)時(shí)熒光定量PCR技術(shù)檢測(cè)大腦皮層、小腦和紋狀體內(nèi)DAT、VMAT2、THmRNA表達(dá)水平，并檢測(cè)紋狀體中MAO-BmRNA表達(dá)水平；WesternBlot蛋白印跡技術(shù)檢測(cè)紋狀體內(nèi)DAT、VMAT2、TH的蛋白表達(dá)水平。 結(jié)果：（1）大腦皮層結(jié)果顯示：與對(duì)照組比較，各劑量組VMAT2mRNA表達(dá)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(jì)學(xué)意義(P0.05)，而高劑量組則降低了56%，差異具有統(tǒng)計(jì)學(xué)意義(P0.01)。DAT、TH各組間差異均無(wú)統(tǒng)計(jì)學(xué)意義。（2）小腦結(jié)果顯示：與對(duì)照組比較，低、中、高劑量組DAT、VMAT2、THmRNA表達(dá)均明顯降低，差異具有統(tǒng)計(jì)學(xué)意義(P0.01)。（3）紋狀體結(jié)果顯示：DATmRNA表達(dá)與對(duì)照組相比，低、中、高劑量組均明顯降低，差異具有統(tǒng)計(jì)學(xué)意義(P0.01)；Westernblot結(jié)果顯示，高劑量組的糖基化DAT蛋白表達(dá)明顯降低，僅為對(duì)照組的78%，差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。與對(duì)照組相比，高劑量組VMAT2mRNA表達(dá)顯著降低(P0.01)，VMAT2蛋白表達(dá)也顯著降低，差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。與對(duì)照組相比，高劑量組THmRNA和蛋白表達(dá)均明顯升高，差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。與對(duì)照組相比，高劑量組MAO-BmRNA表達(dá)明顯降低，差異具有統(tǒng)計(jì)學(xué)意義(P0.01)。 結(jié)論：ACR亞急性染毒不僅會(huì)使大鼠小腦中DAT、VMAT2、THmRNA表達(dá)降低，還會(huì)使紋狀體中DAT、VMAT2、MAO-BmRNA和蛋白表達(dá)降低，THmRNA和蛋白表達(dá)增加，提示ACR亞急性染毒會(huì)導(dǎo)致多巴胺神經(jīng)遞質(zhì)的合成和轉(zhuǎn)運(yùn)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(dǎo)致多巴胺神經(jīng)系統(tǒng)功能紊亂。 第三部分丙烯酰胺對(duì)PC12細(xì)胞多巴胺相關(guān)轉(zhuǎn)運(yùn)蛋白的蛋白表達(dá)的影響 目的：探討丙烯酰胺對(duì)PC12細(xì)胞中多巴胺轉(zhuǎn)運(yùn)體(DAT)、單胺囊泡轉(zhuǎn)運(yùn)體(VMAT2)和酪氨酸羥化酶（TH）蛋白表達(dá)的影響。 方法：取對(duì)數(shù)生長(zhǎng)期的PC12細(xì)胞，分為1個(gè)對(duì)照組和6個(gè)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(cè)PC12細(xì)胞中DAT、VMAT2、TH的蛋白表達(dá)水平。 結(jié)果：（1）DAT蛋白定量結(jié)果顯示：與對(duì)照組比較，0.6mmol/L和1.25mmol/L組PC12細(xì)胞中糖基化DAT明顯降低，差異具有統(tǒng)計(jì)學(xué)意義(P0.05)；與對(duì)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(jì)學(xué)意義(P0.05)。（2）VMAT2蛋白定量結(jié)果顯示：與對(duì)照組比較，0.6mmol/L-5mmol/L組PC12細(xì)胞中VMAT2明顯降低，差異具有統(tǒng)計(jì)學(xué)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(jì)學(xué)意義(P0.5或P0.01)。（3）TH蛋白定量結(jié)果顯示：與對(duì)照組比較，2.5mmol/L、5mmol/L組PC12細(xì)胞中TH含量明顯升高，差異具有統(tǒng)計(jì)學(xué)意義(P0.05，P0.01)。 結(jié)論：ACR可以引起PC12細(xì)胞中DAT、VMAT2蛋白含量降低，同時(shí)還可以引起TH蛋白含量升高，提示ACR可以引起PC12細(xì)胞中多巴胺神經(jīng)遞質(zhì)的合成和轉(zhuǎn)運(yùn)功能異常。 綜上所述，ACR亞急性染毒能導(dǎo)致大鼠體重下降，后肢支撐力指數(shù)增加，步態(tài)評(píng)分增高，甩尾時(shí)間縮短，說(shuō)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(huì)使大鼠小腦中DAT、VMAT2、THmRNA表達(dá)降低，還會(huì)使紋狀體中DAT、VMAT2、MAO-BmRNA和蛋白表達(dá)降低及THmRNA和蛋白表達(dá)增加。另外，ACR還能引起PC12細(xì)胞中DAT、VMAT2蛋白含量降低，同時(shí)還可以造成TH蛋白含量升高。因此，推測(cè)紋狀體DA系統(tǒng)功能紊亂可能與多巴胺相關(guān)轉(zhuǎn)運(yùn)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(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R114

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