丙烯酰胺对纹状体神经末梢多巴胺相关转运蛋白的影响

发布时间：2018-04-19 04:14

本文选题：丙烯酰胺 + 多巴胺转运体　；参考：《华中科技大学》2012年硕士论文

【摘要】：丙烯酰胺(Acrylamide,ACR)是一种从水合丙烯腈中提取出来的具有乙烯基的水溶性有机化合物，是国内外广泛应用于化工冶炼、污水处理、纺织加工及化妆品生产的一种化学原料。早在20世纪70年代就有职业性中毒的报道，主要表现为神经系统症状，除职业接触外，在饮用水、高温油炸食品和食品的包装材料中都可以检测到单体丙烯酰胺的存在，由于其可以直接经口进入人体，从而引起了社会的高度关注。有关流行病学调查和实验研究发现，丙烯酰胺对人和实验动物除了有生殖毒性、遗传毒性及致癌性等作用以外，还有明显的神经毒性。丙烯酰胺急性、亚急性中毒主要以损伤中枢神经系统为主，表现为神经精神症状和小脑共济失调，而慢性中毒则以损害周围神经系统为主，关于其机制目前尚不清楚。纹状体作为基底节最大的综合处理元件，包含着大量的多巴胺能神经元，在整个脑部中的多巴胺神经递质含量最高。在多巴胺能系统神经通路中多巴胺神经递质首先在突触前膜中进行合成、转运、释放、降解，以达到突出间隙中多巴胺神经递质的平衡，突触间隙中的多巴胺神经递质进入突触后膜发挥突触后效应，而多巴胺相关转运蛋白在整个过程中发挥着重要作用，维持突触间隙中多巴胺神经递质的平衡，当该平衡被打破后神经系统就会出现不同程度的病症，主要表现在感觉运动整合、躯体平衡运动及学习记忆等方面。以往对ACR的人群流行病学和实验研究时所出现的神经症状和体征，似乎很大程度上预示着与纹状体多巴胺神经通路中多巴胺神经递质平衡被打破有关。但直至目前，有关丙烯酰胺影响纹状体维持多巴胺神经递质平衡的相关转运蛋白的研究报道较少，其具体机制仍未完全明确。因此，本研究拟通过体内和体外实验的结合，检测丙烯酰胺对多巴胺转运体、单胺囊泡转运体及多巴胺合成酶—酪氨酸酸羟化酶的影响，探讨其引起神经毒性的可能机制，为防治丙烯酰胺中毒提供理论依据。第一部分丙烯酰胺对大鼠神经行为毒性的影响目的：探讨丙烯酰胺对大鼠神经行为毒性的影响。方法：健康成年雄性SD大鼠40只，体重230±20g，由华中科技大学同济医学院实验动物中心提供，合格证号：SCXK（鄂）2010-0007号。大鼠在10h：14h明暗光循环的动物房内，自由饮水、摄食，检疫观察7天后用于实验。将大鼠按体重随机分为4组，分别为对照组、低剂量(20mg/kg)组、中剂量(30mg/kg)组、高剂量（40mg/kg）组，每组10只，均单笼饲养。按照对应的染毒剂量对各ACR剂量组进行灌胃染毒，对照组则灌胃给予等容量的生理盐水，每周连续染毒5天间隔2天，总共19天，并于第0，6，13，19天称量体重并进行步态评分，于第0，19天测定大鼠的后肢支撑力和甩尾时间。末次给药24小时后，断头处死大鼠，迅速取出脑组织，用预冷PBS缓冲液洗净，称量大小脑重量，并于冰盘上迅速分离纹状体、大脑皮层及小脑，并用干净滤纸吸干置于㧟80℃冰箱中保存备用。取出大鼠心、肝、脾、肺、肾、睾丸等脏器，称量重量并记录。结果：（1）在整个染毒期内低剂量组大鼠虽然体重增长始终低于对照组，但差异无显著性；与对照组比较，中剂量组大鼠平均体重于第19d明显降低，差异有统计学意义(P0.01)；高剂量组大鼠平均体重于第6d、13d、19d也明显下降，差异有统计学意义(P0.01)。另外，与低、中剂量组相比，高剂量组大鼠平均体重于第19d明显降低，差异有统计学意义(P0.01)。（2）脏器系数显示，高剂量组大小脑、心脏、肺脏、肾脏及睾丸脏器系数均有增加，且差异具有统计学意义（P0.05或P0.01）。（3）染毒第6d、13d、19d，与对照组比较，低、中、高剂量组步态评分均明显升高，差异有统计学意义(P0.01)，且染毒结束后，其分值分别集中于1-2、2-3、3-4分之间，并呈现出时间—剂量—效应关系。另外，与低、中剂量组相比，高剂量组步态评分于第6d、13d、19d也明显增加，差异有统计学意义(P0.05或P0.01)。（4）染毒第19d，与对照组相比，低、中、高剂量组后肢支撑力指数均明显增加，差异有统计学意义(P0.01)，且呈现出剂量—效应关系。另外，与低、中剂量组相比，高剂量组后肢支撑力指数也明显增加，差异有统计学意义(P0.01)（5）染毒第19d高剂量组的甩尾时间比对照组明显缩短，差异有统计学意义(P0.01)。结论：ACR亚急性染毒能导致大鼠体重下降；大小脑、心脏、肺脏、肾脏及睾丸脏器系数增加；步态评分增高；后肢支撑力指数增加；甩尾时间缩短。具有明显的神经毒性，主要引起感觉运动功能异常。第二部分丙烯酰胺对大鼠多巴胺相关转运蛋白mRNA和蛋白表达的影响目的：探讨丙烯酰胺对大鼠纹状体多巴胺转运体(DAT)、单胺囊泡转运体(VMAT2)和酪氨酸羟化酶（TH）mRNA表达和蛋白水平的影响。方法：动物分组及处理同第一部分。实时荧光定量PCR技术检测大脑皮层、小脑和纹状体内DAT、VMAT2、THmRNA表达水平，并检测纹状体中MAO-BmRNA表达水平；WesternBlot蛋白印迹技术检测纹状体内DAT、VMAT2、TH的蛋白表达水平。结果：（1）大脑皮层结果显示：与对照组比较，各剂量组VMAT2mRNA表达水平均明显降低，其中低、中剂量组分别降低了30%、29%，差异具有统计学意义(P0.05)，而高剂量组则降低了56%，差异具有统计学意义(P0.01)。DAT、TH各组间差异均无统计学意义。（2）小脑结果显示：与对照组比较，低、中、高剂量组DAT、VMAT2、THmRNA表达均明显降低，差异具有统计学意义(P0.01)。（3）纹状体结果显示：DATmRNA表达与对照组相比，低、中、高剂量组均明显降低，差异具有统计学意义(P0.01)；Westernblot结果显示，高剂量组的糖基化DAT蛋白表达明显降低，仅为对照组的78%，差异具有统计学意义(P0.05)。与对照组相比，高剂量组VMAT2mRNA表达显著降低(P0.01)，VMAT2蛋白表达也显著降低，差异具有统计学意义(P0.05)。与对照组相比，高剂量组THmRNA和蛋白表达均明显升高，差异具有统计学意义(P0.05)。与对照组相比，高剂量组MAO-BmRNA表达明显降低，差异具有统计学意义(P0.01)。结论：ACR亚急性染毒不仅会使大鼠小脑中DAT、VMAT2、THmRNA表达降低，还会使纹状体中DAT、VMAT2、MAO-BmRNA和蛋白表达降低，THmRNA和蛋白表达增加，提示ACR亚急性染毒会导致多巴胺神经递质的合成和转运功能失常，引起胞质和突触间隙中多巴胺含量改变，从而导致多巴胺神经系统功能紊乱。第三部分丙烯酰胺对PC12细胞多巴胺相关转运蛋白的蛋白表达的影响目的：探讨丙烯酰胺对PC12细胞中多巴胺转运体(DAT)、单胺囊泡转运体(VMAT2)和酪氨酸羟化酶（TH）蛋白表达的影响。方法：取对数生长期的PC12细胞，分为1个对照组和6个染毒组，各染毒组ACR终浓度分别为0.1mmol/L，0.3mmol/L，0.6mmol/L，1.25mmol/L，，2.5mmol/L，5mmol/L，染毒24h。用WesternBlot蛋白印迹技术检测PC12细胞中DAT、VMAT2、TH的蛋白表达水平。结果：（1）DAT蛋白定量结果显示：与对照组比较，0.6mmol/L和1.25mmol/L组PC12细胞中糖基化DAT明显降低，差异具有统计学意义(P0.05)；与对照组比较，0.1mmol/L、0.6mmol/L、1.25mmol/L组非糖基化DAT也明显降低，差异具有统计学意义(P0.05)。（2）VMAT2蛋白定量结果显示：与对照组比较，0.6mmol/L-5mmol/L组PC12细胞中VMAT2明显降低，差异具有统计学意义(P0.01)；与0.1mmol/L和0.3mmol/L组比较，0.6mmol/L-5mmol/L组VMAT2也降低，差异具有统计学意义(P0.5或P0.01)。（3）TH蛋白定量结果显示：与对照组比较，2.5mmol/L、5mmol/L组PC12细胞中TH含量明显升高，差异具有统计学意义(P0.05，P0.01)。结论：ACR可以引起PC12细胞中DAT、VMAT2蛋白含量降低，同时还可以引起TH蛋白含量升高，提示ACR可以引起PC12细胞中多巴胺神经递质的合成和转运功能异常。综上所述，ACR亚急性染毒能导致大鼠体重下降，后肢支撑力指数增加，步态评分增高，甩尾时间缩短，说明其神经毒性作用比较明显；其作用于小脑和纹状体后，不仅会使大鼠小脑中DAT、VMAT2、THmRNA表达降低，还会使纹状体中DAT、VMAT2、MAO-BmRNA和蛋白表达降低及THmRNA和蛋白表达增加。另外，ACR还能引起PC12细胞中DAT、VMAT2蛋白含量降低，同时还可以造成TH蛋白含量升高。因此，推测纹状体DA系统功能紊乱可能与多巴胺相关转运蛋白受到影响有关。
[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 .

【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R114

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