位於真核細胞之細胞質及細胞核中的蛋白質大多數經由泛肽素-蛋白質分解體 (ubiquitin-proteasome) 途徑進行代謝作用。泛肽素主要的作用是將所結合之蛋白質帶入蛋白質分解體進行代謝作用。泛肽素羧基端水解酶-L1 (ubiquitin carboxyl-terminal hydrolase L1; UCH-L1) 是中樞及周邊神經元特有的酵素，負責將小片段胜肽自泛肽素鏈移除，並協助泛肽素之基因產物轉譯過程，以產生游離性之泛肽素單體。在惡劣環境下，細胞可向上調節熱休克蛋白之表現，以利細胞修復及保護機轉。熱休克蛋白70為熱休克蛋白家族中保護細胞對抗死亡最主要的誘導性蛋白。有機磷具有膽鹼酯酶抑制劑之藥理性質，可使乙醯膽鹼於周邊及中樞神經突觸部位累積及過度刺激突觸傳導而導致有機磷中毒症狀，包括腺體分泌增加、肌肉緊張、木僵、心肺功能衰退、呼吸衰竭、昏迷或死亡。對於中毒時可能保護機轉的研究有助於吾人進一步了解有機磷中毒。因此，關於有機磷中毒時細胞及分子機轉方面的訊息值得探討。
本實驗室先前研究發現P=O形式之有機磷農藥--美文松，作用於腦幹之決定性部位是延腦鼻端腹外側區 (rostral ventrolateral medulla; RVLM)，此處為前運動交感性神經元之起源，負責維持交感神經性血管運動張力。由於急性美文松中毒所導致之心臟血管作用分期變化與來自RVLM之 “生-死” 訊號的波動有相關聯性。本論文依據有機磷中毒之動物模式所提供之RVLM連續性的細胞分子變化機轉，探討在美文松中毒時RVLM中UCH-L1及熱休克蛋白70兩類蛋白質表現量的變化與死亡之關聯性，並評估此兩類蛋白質於RVLM對抗有機磷中毒造成死亡之神經保護作用。
本實驗首先評估於美文松中毒所引發之心臟血管毒性作用時，UCH-L1在延腦交感神經性血管運動張力作用之起源-- RVLM中，是否具有神經保護角色。使用propofol麻醉下的Sprague-Dawley品系大白鼠，以靜脈注射美文松 (960

In eukaryotic cells, most proteins in the cytosol and nucleus are degraded via the ubiquitin-proteasome pathway. Ubiquitin is best known for its role in targeting proteins for degradation by the proteasome. Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is found specifically in central and peripheral neurons, and is responsible for the removal of small peptide fragments from the ubiquitin chain and for co-translational processing of ubiquitin gene products to generate free monomeric ubiquitin. In response to extreme conditions, cells exhibit an up-regulation of heat shock protein (HSP) expression, which contributes to repair and protective mechanisms. Within the HSP family, HSP70 is the major inducible member that protects against cell death. Based on the pharmacologic property of organophosphates as an inhibitor of cholinesterase, it is generally contended that manifestations of organophosphate poisoning, including secretion and muscle fasciculation, stupor, cardiopulmonary collapse, respiratory failure, coma or death, result from accumulation of, and over-stimulation by acetylcholine at peripheral of central synapses. One approach in furthering our understanding on organophosphate poisoning is delineation of its potential protective mechanisms. In this regard, the information on the cellular and molecular mechanisms that underlie organophosphate poisoning has received attention.
Our laboratory demonstrated previously that a crucial brain site via which mevinphos (Mev), an organophosphate insecticide of the P=O type, acts is the rostral ventrolateral medulla (RVLM), the medullary origin of premotor sympathetic neurons that are responsible for the maintenance of vasomotor tone. The phasic changes in cardiovascular events over the course of acute Mev intoxication also parallel fluctuations of the “life-and-death” signals that emanate form the RVLM. Based on a rat model of organophosphate poisoning that provides continuous information on cellular and molecular mechanisms in the RVLM, the present study was undertaken to evaluate whether changes in protein level of UCH-L1 or HSP70 are associated with death arising from Mev intoxication. We also evaluated the efficacy of both of them in the neuroprotection against fatality during Mev intoxication.
The first part of this study investigated whether UCH-L1 plays a neuroprotective role at the RVLM, where Mev acts to elicit cardiovascular toxicity. In Sprague-Dawley rats maintained under propofol anesthesia, Mev (960 µg/kg, i.v.) induced a parallel and progressive augmentation in UCH-L1 or ubiquitin expression at the ventrolateral medulla during the course of Mev intoxication. The increase in UCH-L1 level was significantly blunted on pretreatment with microinjection bilaterally into the RVLM of a transcription inhibitor, actinomycin D (5 nmol) or a translation inhibitor, cycloheximide (20 nmol). Compared to artificial cerebrospinal fluid (aCSF) or sense uch-L1 oligonucleotide (100 pmol) pretreatment, microinjection of an antisense uch-L1 oligonucleotide (100 pmol) bilaterally into the RVLM significantly increased mortality, reduced the duration of the phase I (“pro- life” phase), blunted the increase in ubiquitin expression in ventrolateral medulla, and augmented the induced hypotension in rats that received Mev.
The second part of this study investigated whether HSP70 plays a neuroprotective role at the RVLM. Intravenous administration of Mev (960

摘要 …………………………………………………………………………… i
Abstract ……………………………………………………………………… iv
Table of Contents …………………………………………………………… vii
List of Tables ………………………………………………………………… ix
List of Figures ………………………………………………………………… x
Abbreviations ……………………………………………………………… xiii
Chapter 1. Introduction and Literature Review ……………………………… 1
1-1. Organophosphates ………………………………………………… 1
1-2. Rostral Ventrolateral Medulla …………………………………… 7
1-3. Mev and RVLM …………………………………………………… 8
1-4. Ubiquitin-Proteasome Pathway …………………………………… 9
1-5. Heat Shock Proteins ……………………………………………… 13
1-6. Cardiovascular Parameters ……………………………………… 14
Chapter 2. Motive and Research Objective ………………………………… 17
2-1. Motive …………………………………………………………… 17
2-2. Research Objective ……………………………………………… 18
Chapter 3. Materials and Methods …………………………………………… 19
3-1. General Preparation of Animals ………………………………… 19
3-2. Measurement of Cardiovascular Parameters …………………… 20
3-3. Mev Intoxication ………………………………………………… 21
3-4. Pretreatments …………………………………………………… 21
3-5. Protein Extraction and Western Blot Analysis …………………… 23
3-6. Histology ………………………………………………………… 24
3-7. Statistical Analysis ……………………………………………… 35
Chapter 4. Neuroprotective Role of Ubiquitin Carboxyl-Terminal Hydrolase L1 in the Rostral Ventrolateral Medulla During Acute Mevinphos Intoxication in the Rat …………………………………………… 36
4-1. Introduction ……………………………………………………… 36
4-2. Materials and Methods …………………………………………… 38
4-3. Results …………………………………………………………… 39
4-4. Discussion ……………………………………………………… 59
Chapter 5. Neuroprotective Role of Heat Shock Protein 70 in the Rostral Ventrolateral Medulla During Acute Mevinphos Intoxication in the Rat ……………………………………………………………… 64
5-1. Introduction ……………………………………………………… 64
5-2. Materials and Methods …………………………………………… 66
5-3. Results …………………………………………………………… 66
5-4. Discussion ……………………………………………………… 86
Chapter 6. Conclusion and Future Work …………………………………… 90
6-1. Conclusion ……………………………………………………… 90
6-2. Future Work ……………………………………………………… 90
References …………………………………………………………………… 92
Appendices ………………………………………………………………… 109

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