自噬在TOCP诱发成年母鸡迟发性神经毒性中的作用探讨

发布时间：2018-04-19 13:41

  本文选题：TOCP + OPIDN　； 参考：《南华大学》2013年硕士论文

【摘要】：目的： 利用三邻甲苯基磷酸酯（Tri-ortho-cresyl phosphate, TOCP）诱导建立成年母鸡迟发性神经病（OPIDN）的模型，观察细胞自噬在TOCP诱发的迟发性神经毒性发生、发展过程中的变化，揭示自噬与TOCP诱发OPIDN的联系，探索OPIDN发生的可能机制。为进一步了解和揭示神经退行性病变的机理提供理论依据。 方法： 将实验母鸡随机分为对照组和实验组，实验组35只，对照组7只。实验组喂以装有TOCP（剂量为750mg/kg）的医用胶囊，进行一次染毒,对照组则喂以装有等量植物油的胶囊，观察实验鸡的迟发性神经毒性的发生情况，分别在不同时间点（染毒1d、3d、5d、7d、10d、14d、21d）处死动物，分离脊髓与坐骨神经，Western blot法测定自噬相关蛋白LC3的表达，取部分新鲜脊髓与坐骨神经组织固定供作制备石蜡切片和电镜切片，光镜下观察常规病理学改变，免疫组织化学法检测溶酶体相关膜糖蛋白LAMP-1的表达，透射电镜技术观察组织的超微结构。 结果： 1.一般情况：染毒后实验鸡活动减少，食量减退，于染毒第三天后活动和食量恢复到染毒前水平,第7天开始出现OPIDN症状，实验组第14天、第21天母鸡体重增长率与对照组比较下降（P0.05）。 2. OPIDN临床症状：实验组鸡在TOCP染毒后第7天左右出现肢体活动减少、下肢行走轻微不协调；第10天时出现共济失调，好蹲坐，双下肢无力，行走不稳，症状随时间延长呈进行性加重，第15天，行走极不协调，经常跌倒，并逐渐加重，第18天，症状严重的动物出现瘫痪，不能站立，直至第21天实验结束，期间动物正常进食及饮水。对照组动物未观察到任何OPIDN症状。 3. HE染色结果：脊髓神经元细胞较对照组明显肿胀，部分细胞胞浆淡染，胞核模糊不清；神经元细胞出现核固缩和细胞胞浆空泡化；坐骨神经纤维排列稀疏、肿胀，着色变淡和结构紊乱。 4.电镜结果：正常的神经组织中小胶质细胞和神经元形态正常，髓鞘紧密，板层清晰。坐骨神经的髓鞘葱管样改变明显且模糊不清晰，，多处板层松散、破裂甚至崩解。 5.随着染毒时间的延长，实验鸡的OPIDN症状加重，分别用Western blot、免疫组化法检测鸡脊髓、坐骨神经自噬相关蛋白LC3-Ⅱ的表达，随着染毒时间的延长，蛋白表达增多，说明TOCP诱发的OPIDN可能跟自噬有关。 6.免疫组化法检测实验鸡脊髓、坐骨神经LAMP-1的表达，随着染毒时间的延长，蛋白表达增多，存在一定的时效关系。 结论： 1. TOCP（750mg/kg）一次性胶囊喂饲法染毒成年母鸡21天，实验母鸡出现迟发性神经毒性，受试动物脊髓、坐骨神经出现中毒表现，染毒时间越长，中毒表现越严重。 2.TOCP对实验母鸡脊髓、坐骨神经中的自噬相关蛋白LC3和溶酶体相关膜糖蛋白（LAMP-1）的表达有影响，随着染毒时间的延长，蛋白表达均增多；TOCP诱发的成年母鸡OPIDN可能与自噬的发生有关。
[Abstract]:Objective:The model of delayed neuropathy (OPIDN) in adult hens induced by tri-ortho-cresyl phosphate (Top) was established. The late neurotoxicity induced by autophagy in TOCP was observed and the changes in the course of development were observed. The relationship between autophagy and OPIDN induced by TOCP was revealed.To explore the possible mechanism of OPIDN.To provide theoretical basis for further understanding and revealing the mechanism of neurodegenerative diseases.Methods:The hens were randomly divided into control group (n = 35) and experimental group (n = 7).The experimental group was fed with medical capsules containing TOCP (750mg / kg) and the control group was given capsules containing the same amount of vegetable oil to observe the occurrence of delayed neurotoxicity in experimental chickens.The animals were killed at different time points (1 day, 3 days, 5 days, 7 days, 10 days, 14 days and 21 days). The expression of autophagy associated protein LC3 was determined by Western blot method in isolated spinal cord and sciatic nerve. Some fresh spinal cord and sciatic nerve tissue were fixed for paraffin section and electron microscope section.The expression of lysosomal membrane glycoprotein (LAMP-1) was detected by immunohistochemical method and the ultrastructure of the tissue was observed by transmission electron microscopy (TEM).Results:1.General conditions: after exposure, the activity of experimental chickens decreased, the amount of food decreased, the activity and the amount of food returned to the pre-exposure level on the third day of exposure, the symptoms of OPIDN began to appear on the 7th day, and the experimental group showed symptoms on the 14th day.On the 21st day, the weight growth rate of hens was lower than that of the control group (P 0.05).2.The clinical symptoms of OPIDN were as follows: the experimental group had decreased limb activity on the 7th day after TOCP exposure, slight disharmony in walking of lower limbs, ataxia, squatting, weakness of both legs and unstable walking on the 10th day.The symptoms increased progressively over time. On the 15th day, the walking was extremely uncoordinated, often fell down, and gradually aggravated. On the 18th day, the severely symptomatic animals became paralyzed and could not stand up until the end of the 21st day of the experiment.During the period of normal eating and drinking animals.No OPIDN symptoms were observed in the control group.3.The results of HE staining showed that the spinal cord neurons were obviously swollen compared with the control group, some of them were slightly stained in cytoplasm, the nuclei were blurred, the neurons had nuclear shrinkage and cytoplasmic vacuolation, the fibers of sciatic nerve were sparse and swollen,Coloring and disordered structure.4.The results of electron microscope showed that the morphology of glial cells and neurons in normal nerve tissue was normal, the myelin sheath was close and the lamellar layer was clear.The medullary scallion tube of sciatic nerve was obviously changed and unclear, and many lamellar layers were loose, ruptured and even disintegrated.5.With the prolongation of exposure time, the symptoms of OPIDN in experimental chickens were aggravated. The expression of LC3- 鈪

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