【作者】 黄璐；

【导师】 于素芳；

【作者基本信息】 山东大学 ， 劳动卫生与环境卫生学， 2010， 硕士

【摘要】 目的丙烯酰胺(ACR)是一种用途广泛的工业毒物。它是一种神经毒性物质和人类可能致癌物,它的神经毒性主要表现为中枢-周围性远端轴索病,临床表现为共济失调、骨骼肌无力,其病理特征是远端轴突肿胀、变性、神经丝(neurofilament,NF)聚集：我们前面的研究发现,ACR中毒大鼠坐骨神经组织中NFs(包括NF-L、NF-M和NF-H)的含量均是下降的。但其神经毒性的作用机制仍未完全阐明。基于ACR中毒NFs及其超微结构的改变,为进一步探讨ACR中毒性外周神经病变发生的分子机制,我们建立了ACR亚慢性中毒大鼠模型,测定了坐骨神经中与NFs磷酸化有关的三种蛋白激酶：蛋白激酶A(PKA)、蛋白激酶C(PKC)和细胞周期依赖性蛋白激酶5 (CDK5),及其相关蛋白包括钙调蛋白(CaM)、环磷腺苷(cAMP)、CDK5的两个激活因子P35和P25的变化。此外还对血清PKA和PKC的含量进行了分析,以期为ACR神经损伤生物标志物的探索提供依据。方法1.成年雄性Wistar大鼠随机分为对照组、低剂量组和高剂量组。低、高剂量组分别以20 mg/kg体重和40 mg/kg体重剂量腹腔注射ACR染毒,每周3次,连续8周；对照组腹腔注射等体积双蒸水。2.神经行为的改变和步态评分密切观察各组大鼠的一般状况和神经行为的改变,测量大鼠体重,给出步态评分。3.分离坐骨神经组织,匀浆后超速离心,取上清-80℃保存备用。用SDS-PAGE和Western Blotting方法测定PKA、PKC、CDK、P35和P25蛋白含量；用ELISA试剂盒测定CaM和cAMP的含量。4.取大鼠坐骨神经组织匀浆上清,利用放射性同位素技术研究ACR染毒对大鼠神经组织中PKA和PKC活性的影响。5.断头取血,常规分离血清,利用SDS-PAGE和Western Blotting方法测定血清中PKA和PKC的含量。结果1.体重结果与中毒表现：与对照组相比,ACR染毒组大鼠体重减轻,高剂量组第5-8周体重下降(P<0.05)。高剂量组大鼠在染毒第8周后肢完全瘫痪,走路时后肢后拖,足背着地,无法支撑身体,步态得分增高；染毒终止时,低、高剂量组分别呈轻度和重度受损步态。与对照组比较,ACR染毒组大鼠步态评分增高,低剂量组第8周出现增高(P<0.01),高剂量组第4-8周出现增高(P<0.01)。2.蛋白测定结果：与对照组相比,低剂量组和高剂量组坐骨神经中的蛋白激酶及其相关蛋白都发生了不同程度的变化。(1)坐骨神经中CaM含量的变化：与对照组相比,低剂量组无明显变化,高剂量组增高了50%(P<0.01)；与低剂量组相比,高剂量组升高了50%(P<0.01)。(2)坐骨神经中cAMP含量的变化：cAMP蛋白含量在ACR亚慢性中毒大鼠坐骨神经中明显降低,与对照相比,cAMP在低剂量组降低了11%,在高剂量组降低了95%(P<0.01)；与低剂量组相比,cAMP在高剂量组降低了94%(P<0.01)。(3)坐骨神经中PKA和PKC的含量及活性的变化：与对照组相比,PKA和PKC的含量在低剂量组分别升高了18%和21%,在高剂量组分别增高了73%(P<0.01)和170%(P<0.01)；与低剂量组相比,这两者的含量在高剂量组分别升高了46%(P<0.01)和125%(P<0.01)。PKA活性与对照组相比,低剂量组增高了60%(P<0.01),高剂量组降低了50%(P<0.01)；与低剂量组相比,高剂量组降低了220%(P<0.01)。PKC活性与对照组相比,低剂量组增高了50%,高剂量组增高了6倍(P<0.01)；与低剂量组相比,高剂量组增高了3倍(P<0.01)。(4)坐骨神经中CDK5、P25和P35含量的变化：与对照组相比,CDK5在低剂量组降低了51%(P<0.01),高剂量组降低了22%(P<0.05)；与低剂量组相比,高剂量组升高了59%(P<0.01)。P25和P35含量与对照组相比,低剂量组分别降低了10%和21%(P<0.01),高剂量组分别降低了77%(P<0.01)和82%(P<0.01)；与低剂量组相比,高剂量组分别降低了74%(P<0.01)和77%(P<0.01)。(5)血清中PKC和PKA的含量与对照组相比,低剂量组分别升高了6%和6%,高剂量组分别升高了45%(P<0.01)和55%(P<0.01)；与低剂量组相比,高剂量组分别升高了37%(P<0.01)和47%(P<0.01)。结论1.ACR亚慢性染毒可以引起神经系统损伤,表现为大鼠步态评分异常。2.ACR染毒可明显升高坐骨神经组织中CaM含量,说明ACR可明显增加细胞内Ca2+浓度。3.坐骨神经中蛋白激酶的变化可能与ACR的周围神经毒性有关,表现为ACR亚慢染毒可明显改变坐骨神经中cAMP、PKA、PKC、CDK5、P35和P25蛋白的含量以及PKA和PKC的活性,从而导致NFs磷酸化状态变化。4.血清中PKC和PKA有可能作为ACR神经病变的生物效应标志物,表现为ACR亚慢性染毒可引起大鼠血清中PKC和PKA相对含量改变。5.ACR的周围神经毒性作用机制可能与上述蛋白的变化相关。更多还原

【Abstract】 ObjectiveAcrylamide (ACR) is a kind of widely-used industrial material. It is regarded as a neurotoxicant and human potential carcinogen through the studies of more than thirty years. ACR is known to produce central-peripheral distal axonopathy, which is characterized by ataxia and distal skeletal muscle weakness. The major pathological hallmarks are distal swellings and secondary degeneration both in experimental animals and human companioning excessive accumulation of neurofilaments (NFs) in the distal swollen axon. However, the exact mechanism of action of its neurotoxicity has not been completely clear until now.Based on the pathological alterations, we built ACR-intoxicated rats models and investigated the relative levels of proteins in the tissue of sciatic nerves (SN) to determine the molecular mechanisms. We detected the changes of three protein kinases and four relative proteins, include calmodulin (CAM), protein kinase C (PKC), protein kinase A (PKA), Cyclic Adenosine monophosphate (cAMP), cyclin dependent kinase 5 (CDK5) and CDK5-related factors (P35 and P25) in SN of ACR-treated rats. Moreover, the contents of PKA and PKC in the serum of rats were also tested, which will offer the cues and evidences for exploring the mechanism of ACR neurotoxicity and searching biomarkers.Methods 1. Male Wistar rats weighing 180-220g were divided into three groups, each 9 animals. The rats in group 1 served as control, and received double distilled water (DDW). The animals in group 2 and 3 were given ACR dissolved in DDW(20、40 mg/kg i.p.3 days/week) for 8 weeks.2. Neurobehavioral index were determined per week. The changes of general status, body weight of rats and neurobehavioral function were observed. In addition, we observed abnormal gait and got the gait score.3. The excised nervous tissues of SN were homogenized in ice-cold homogenizing buffer and then centrifuged at a high speed. The supernatant was saved in-80℃.The relative levels of PKA, PKC, CDK5, P35 and P25 in the supernatant of SN were determined by SDS-PAGE and Western Blotting. The contents of CaM and cAMP in SN were determined by ELISA kits.4. The activities of PKA and PKC were determined by using corresponding radioactivated 32P assay kits in corresponding cytosolic fractions of SN of control and experimental group rats.5. The decapitated bloods were sampled, and the serum was separated routinely. The contents of PKA and PKC in the rat serum were investigated by SDS-PAGE and immunoblotting.Results1. The body weight and gait scoreCompared with the control, the body weight of rats lost in the ACR-induced groups, i.e. the body weight of the 5th to 8th week decreased significantly (P<0.05) of high ACR group. Compared with the control, gait score increased remarkably at 8th week (P<0.01) of low ACR group and increased remarkably at 4th to 8th week (P<0.01) of high ACR group. 2. The changes of proteins(1) The change of CaM contents in sciatic nerves:Compared with control, no significant changes observed in the low dose group and high dose group had increased 50%(P<0.01). Compared with the low dose group, CaM content increased by 50%(P<0.01) in the high dose group.(2) The change of cAMP contents in sciatic nerves:The exposure to ACR resulted in a significant decrease in cAMP contents. Compared with the control, the level of cAMP decreased by 11% in the low dose group and by 95%(P<0.01) in the high dose group. Compared with the low dose group, cAMP content decreased by 94%(P<0.01) in the high dose group.(3) The contents and activities of PKA and PKC in sciatic nerves:In comparison with the control rats, the levels of PKA and PKC increased respectively by 18%and 21% in low ACR group, by73% (P<0.01) and 170%(P<0.01) in high ACR group. Compared with the control group, PKA activity had increased 60% (P＜0.01) in low dose group but decreased 50%(P<0.01) in high dose group. While compared with the low dose group, it decreased 220%(P<0.01) in high dose group. Compared with the control, PKC activity was significantly increased in 20 (by 50%, P< 0.01) and 40 (6 folds, P< 0.01) mg/kg ACR group rats, respectively. Compared with the 20 mg/kg ACR group, PKC activity increased 3 folds (P<0.01).(4) The exposure to ACR resulted in a significant decrease in CDK5, P25 and P35 contents in sciatic nerve. In comparison with the control rats, CDK5 in the low dose group decreased 51%(P<0.01), in the high dose group decreased 22%(P<0.05). Compared with the low dose group, CDK5 in the high dose group increased by 59%(P<0.01). In comparison with the control rats, the levels of P25 and P35 in the high dose group decreased respectively by 77%(P<0.01) and 82%(P<0.01). (5) The levels of PKA and PKC in serum significantly increased by 6% and 6%in 20mg/kg ACR rats,by 45%(P<0.01) and 55%(P<0.01)in 40 mg/kg ACR rats, respectively.Conclusions1. Subchronic intoxation of ACR can induce the injury of nerve system. It was showed that the gait abnormal.2. ACR exposure could increase the content of CaM in sciatic nerve tissue, which indicates ACR significantly increased the intracellular Ca2+ concentration.3. The changes of protein kinases in sciatic nerve may be related to ACR induced peripheral nerve toxicity. ACR exposure could significantly change the contents of cAMP, PKA, PKC, CDK5, P35 and P25, and, the activities of PKA and PKC in sciatic nerve. As a result, the phosphorylation state of NFs changed.4. The contents of PKA and PKC were significantly increased in serum of ACR-treated rats, which suggested they might be served as some of the biomarkers of ACR-induced neuropathy for earlier diagnosis.5. The peripheral nerve toxicity mechanism of ACR may be associated with the changes of the above-mentioned proteins.更多还原