本文選題：TOCP + OPIDN��； 參考：《南華大學(xué)》2013年碩士論文

【摘要】：目的： 利用三鄰甲苯基磷酸酯（Tri-ortho-cresyl phosphate, TOCP）誘導(dǎo)建立成年母雞遲發(fā)性神經(jīng)�。∣PIDN）的模型，觀察細(xì)胞自噬在TOCP誘發(fā)的遲發(fā)性神經(jīng)毒性發(fā)生、發(fā)展過(guò)程中的變化，揭示自噬與TOCP誘發(fā)OPIDN的聯(lián)系，探索OPIDN發(fā)生的可能機(jī)制。為進(jìn)一步了解和揭示神經(jīng)退行性病變的機(jī)理提供理論依據(jù)。 方法： 將實(shí)驗(yàn)?zāi)鸽u隨機(jī)分為對(duì)照組和實(shí)驗(yàn)組，實(shí)驗(yàn)組35只，對(duì)照組7只。實(shí)驗(yàn)組喂以裝有TOCP（劑量為750mg/kg）的醫(yī)用膠囊，進(jìn)行一次染毒,對(duì)照組則喂以裝有等量植物油的膠囊，觀察實(shí)驗(yàn)雞的遲發(fā)性神經(jīng)毒性的發(fā)生情況，分別在不同時(shí)間點(diǎn)（染毒1d、3d、5d、7d、10d、14d、21d）處死動(dòng)物，分離脊髓與坐骨神經(jīng)，Western blot法測(cè)定自噬相關(guān)蛋白LC3的表達(dá)，取部分新鮮脊髓與坐骨神經(jīng)組織固定供作制備石蠟切片和電鏡切片，光鏡下觀察常規(guī)病理學(xué)改變，免疫組織化學(xué)法檢測(cè)溶酶體相關(guān)膜糖蛋白LAMP-1的表達(dá)，透射電鏡技術(shù)觀察組織的超微結(jié)構(gòu)。 結(jié)果： 1.一般情況：染毒后實(shí)驗(yàn)雞活動(dòng)減少，食量減退，于染毒第三天后活動(dòng)和食量恢復(fù)到染毒前水平,第7天開(kāi)始出現(xiàn)OPIDN癥狀，實(shí)驗(yàn)組第14天、第21天母雞體重增長(zhǎng)率與對(duì)照組比較下降（P0.05）。 2. OPIDN臨床癥狀：實(shí)驗(yàn)組雞在TOCP染毒后第7天左右出現(xiàn)肢體活動(dòng)減少、下肢行走輕微不協(xié)調(diào)；第10天時(shí)出現(xiàn)共濟(jì)失調(diào)，好蹲坐，雙下肢無(wú)力，行走不穩(wěn)，癥狀隨時(shí)間延長(zhǎng)呈進(jìn)行性加重，第15天，行走極不協(xié)調(diào)，經(jīng)常跌倒，并逐漸加重，第18天，癥狀嚴(yán)重的動(dòng)物出現(xiàn)癱瘓，不能站立，直至第21天實(shí)驗(yàn)結(jié)束，期間動(dòng)物正常進(jìn)食及飲水。對(duì)照組動(dòng)物未觀察到任何OPIDN癥狀。 3. HE染色結(jié)果：脊髓神經(jīng)元細(xì)胞較對(duì)照組明顯腫脹，部分細(xì)胞胞漿淡染，胞核模糊不清；神經(jīng)元細(xì)胞出現(xiàn)核固縮和細(xì)胞胞漿空泡化；坐骨神經(jīng)纖維排列稀疏、腫脹，著色變淡和結(jié)構(gòu)紊亂。 4.電鏡結(jié)果：正常的神經(jīng)組織中小膠質(zhì)細(xì)胞和神經(jīng)元形態(tài)正常，髓鞘緊密，板層清晰。坐骨神經(jīng)的髓鞘蔥管樣改變明顯且模糊不清晰，，多處板層松散、破裂甚至崩解。 5.隨著染毒時(shí)間的延長(zhǎng)，實(shí)驗(yàn)雞的OPIDN癥狀加重，分別用Western blot、免疫組化法檢測(cè)雞脊髓、坐骨神經(jīng)自噬相關(guān)蛋白LC3-Ⅱ的表達(dá)，隨著染毒時(shí)間的延長(zhǎng)，蛋白表達(dá)增多，說(shuō)明TOCP誘發(fā)的OPIDN可能跟自噬有關(guān)。 6.免疫組化法檢測(cè)實(shí)驗(yàn)雞脊髓、坐骨神經(jīng)LAMP-1的表達(dá)，隨著染毒時(shí)間的延長(zhǎng)，蛋白表達(dá)增多，存在一定的時(shí)效關(guān)系。 結(jié)論： 1. TOCP（750mg/kg）一次性膠囊喂飼法染毒成年母雞21天，實(shí)驗(yàn)?zāi)鸽u出現(xiàn)遲發(fā)性神經(jīng)毒性，受試動(dòng)物脊髓、坐骨神經(jīng)出現(xiàn)中毒表現(xiàn)，染毒時(shí)間越長(zhǎng)，中毒表現(xiàn)越嚴(yán)重。 2.TOCP對(duì)實(shí)驗(yàn)?zāi)鸽u脊髓、坐骨神經(jīng)中的自噬相關(guān)蛋白LC3和溶酶體相關(guān)膜糖蛋白（LAMP-1）的表達(dá)有影響，隨著染毒時(shí)間的延長(zhǎng)，蛋白表達(dá)均增多；TOCP誘發(fā)的成年母雞OPIDN可能與自噬的發(fā)生有關(guān)。
[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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