臺灣博碩士論文加值系統

γ-胺基丁酸（GABA）和甘胺酸（glycine）為中樞神經主要的抑制性傳導物質。GABA主要與γ-胺基丁酸A型受體結合，導致過極化而抑制神經元的活性，使神經元不易被興奮。γ-胺基丁酸A型受體(GABAA receptors)經純化與選殖證明為由配體(ligand)控制的氯離子通道﹔受體由五個亞型單位組成，分別由六種不同的基因(α、β、γ、δ、ε和ρ)製造，每種基因又有多種的同功異構型(α1-6、β1-3、γ1-3、δ1、ε1和ρ1-3)。GABAA受體上含有GABA和調節物質的結合位點，包括benzodiazepines、β-carbolines（例如β-CCM）和picrotoxin（離子通道阻斷劑）。離體的表達系統實驗證明不同基因組成的受體對GABA、benzodiazepines或picrotoxin等不同的藥物具有不同的敏感性。不同的腦區受體蛋白的組成及ｍRNA的表達皆不同﹔但在文獻上並沒有動物種類或品系之間的差異的報告。GABA拮抗劑或痙攣藥物可在實驗動物如大白鼠或小白鼠引起痙攣﹔若它們對這些藥物的敏感性具有差異性，則可能表示腦中GABAA受體的組成不一樣。我們在痙攣劑閾值的實驗發現兩種不同品系的小白鼠(NMRI和Mf1)對痙攣藥物picrotoxin具有相同的敏感性，但對β-CCM(一種benzodiazepine的逆向致效劑)的敏感性則相差2~10倍以上，Mf1小白鼠較不敏感。在投予bicuculline(一種GABA結合位點的拮抗劑)則發現NMRI小白鼠較不敏感。此暗示兩種不同品系小白鼠的GABAA受體，其蛋白質亞型單位的組成可能不一樣。在蛋白質分析實驗結果發現Mf1小白鼠，其α1亞型單位在大腦皮質和除去大腦皮質與小腦之外腦區的表達程度多於NMRI小白鼠26%和17%，這可能是造成Mf1小白鼠對bicuculline較敏感之原因；γ3亞型單位在大腦皮質的蛋白質密度較NMRI高出49%。而γ2亞型單位在兩種品系小白鼠的大腦皮質、小腦及其他腦區的表達結果皆沒有差異。另外，投予strychnine (glycine受體拮抗劑)所出現的痙攣閾質發現Mf1小白鼠是較NMRI小白鼠低，表示NMRI小白鼠較不敏感。在蛋白質分析結果也證明glycine受體在Mf1的表達多於NMRI。此初步結果驗證體外表達系統的結論，證明不同的受體組成可決定動物對致痙攣劑的敏感度。

γ-Aminobutyric acid (GABA) and glycine are the major inhibitory neurotransmitters in the CNS. GABA binds to GABAA receptor resulting in hyperpolarization and inhibition of neuronal firing. GABAA receptor, a pentameric Cl- channel, consists of 5 homologus subunits derived from 6 separate gene families with multiple variants (α1-6, β1-3, γ1-3, δ, ε, ρ1-3), GABAA receptors contains recognition sites for GABA and several allosteric modulators, including benzodiazepines, β-carbolines (such as β-CCM), and picrotoxin (a channel blocker). Subunit mRNA expression studies demonstrated that receptors expressed with different subunit combinations showed differential sensitivities toward GABA, benzodiazepines, or picrotoxin. This observation implys that if different species or strains of animals exhibit differential sensitivities toward convulsants, the composition of GABAA receptors in animals may be different. In convulsants threshold experiments, we found that in both NMRI and Mf1 mice, the convulsive thresholds for picrotoxin were similar (tonic hindlimb extension, mg/kg, mean±S.E.M., n=8, 11.49±2.08 for Mf1, 11.44±2.30 for NMRI) but differing by more than 2 to 10 folds for β-CCM , with Mf1 mice showing less sensitivity (forelimb clonus, mg/kg, mean ± S.E.M., n=7, >30 for Mf1, 3.82±0.76 for NMRI; n=39, 4.71±0.93 for Mf1, 2.49±0.33 for NMRI). NMRI mice had less sensitivity(tonic hindlimb extension, mg/kg, mean±S.E.M., n=8, 1.82±0.12 for Mf1, 2.42±0.19 for NMRI) than Mf1 mice for bicuculline (a competitive antagonist at the GABA binding site). The subunit composition of GABAA receptor on these two strains mice may be different. Protein immunoblotting demonstrated that Mf1 mice had higher level of α1 subunit protein than that of NMRI mice. The result may be associated with Mf1 mice showing greater sensitivity toward bicuculline. The γ3 subunit protein level in cortex of Mf1 mice was higher than that of NMRI mice. The γ2 subunit protein level in cortex and cerebellum were similar for both strains. In addition, the convulsive threshold for strychnine (glycine receptor antagonist) for Mf1 mice was lower than that for NMRI mice. The glycine receptor level in Mf1 mice was higher than that of NMRI mice. These results indicate that different receptor subunit compositions may determine differential sensitivities of NMRI and Mf1 mice toward convulsants.

中文摘要....................................1 英文摘要....................................2 壹、緒論....................................3 （一）GABA的合成、釋放、回收和代謝..........4 （二）GABA受體的分類........................5 （三）GABAA受體結構、藥理特性及生理功能.....7 （1）受體亞型單位的分子結構.................7 （2）受體的藥理及生理功能...................9 （四）GABAA受體與神經性疾病................11 （五）GABAA受體的實驗動物模式..............11 （六）Glycine合成與代謝....................12 （七）Glycine受體的藥理、生理和病理作用....13 貳、研究目的...............................14 參、材料與方法.............................16 肆、實驗結果...............................21 伍、討論...................................42 陸、附圖...................................49 柒、參考文獻...............................52

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