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題名 以典型瑞特氏症模式小鼠研究運動障礙之療癒
In search of interventions to ameliorate motor deficits in mouse models of typical Rett syndrome
作者 黃弈博
Huang, Yi Bo
貢獻者 廖文霖
Liao, Wen Lin
黃弈博
Huang, Yi Bo
關鍵詞 瑞特氏症
第二型甲基CpG結合蛋白
紋狀體
γ-胺基丁酸
厚朴生藥
和厚朴酚合成純化物
Rett syndrome
Mecp2
Striatum
GABA
cortex Magnoliae
MH101
日期 2014
上傳時間 1-Apr-2015 10:02:46 (UTC+8)
摘要 瑞特氏症 (Rett syndrome, RTT)是由於第二型甲基CpG結合蛋白 (methyl - CpG binding protein 2, MECP2) 基因發生突變所造成的一種神經發育疾病。當小鼠的MeCP2缺失時，會產生許多類似瑞特氏症患者的運動障礙，其中包含運動活力低下、運動協調與運動學習能力缺損，此運動障礙可能由於γ-胺基丁酸(GABA)與多巴胺神經訊號傳遞功能失調所造成。先前研究發現Mecp2基因剔除小鼠的運動障礙伴隨大腦皮質與紋狀體中GABA合成的減少，我們因此嘗試利用藥理的方式增加GABA傳訊，測試其是否可改善Mecp2基因剔除小鼠的運動障礙。我們發現Mecp2基因剔除以及條件缺失公鼠，經由管餵方式給予100 mg/kg厚朴生藥 (cortex Magnoliae)連續七天後，對於平衡桿行走測試以及加速滾輪測試有改善的趨勢，但是對於Mecp2T158A點突變公鼠以及條件缺失母鼠卻沒有效果。我們進而檢測和厚朴酚合成純化物 (以下簡稱MH101)對運動障礙的改善效果。在行為測試前九十分鐘腹腔給予 1 mg/kg的MH101並觀察待測鼠在加速滾輪上的掉落延宕時間， 發現Mecp2條件缺失母鼠在投藥後，與控制組母鼠在同樣藥物處理下相近，顯示MH101有改善運動障礙的效果。以免疫染色法觀察c-Fos蛋白表現量檢測神經細胞的活性，發現在投予MH101後，控制組母鼠的c-Fos蛋白表現量在紋狀體之內背側區有顯著的增加，且Mecp2條件缺失母鼠相對應的腦區也有增加的趨勢，而其他皮質腦區卻皆無明顯改變，其結果顯示內背側紋狀體的活性增加可能與Mecp2條件缺失母鼠的運動協調能力增加有所關聯。另一方面，先前研究指出瑞特氏症模式小鼠的前端紋狀體中，有μ型類鴉片受體表現量減少以及第二型多巴胺受體的過度表現，所以我們嘗試在野生型小鼠的前端紋狀體活化其μ型類鴉片受體或是減少第二型多巴胺受體的神經傳導，檢測是否能夠增加其運動能力。實驗結果顯示，不論是給予μ型類鴉片受體促效劑或是第二型多巴胺受體拮抗劑皆可有效防止手術後之運動活力表現的缺損，因此，於前端紋狀體活化μ型類鴉片受體或阻斷第二型多巴胺受體可能改善小鼠之運動障礙。綜上所述，本研究藉由改善神經傳導的缺失緩解瑞特氏症模式小鼠部份的運動障礙，提供典型瑞特氏症可能有效的治療方式。
Rett Syndrome (RTT) is a neurodevelopmental disorder caused by mutations of the methyl-CpG-binding protein 2 (MECP2). Mice with deficient MeCP2 recapitulate many RTT-like motor symptoms, including hypoactivity, deficits in motor coordination, and motor learning, those are associated with hypofunction of GABAergic and dopaminergic neurotransmission. Previous study found that decreased GABA synthesis in the striatum is accompanied by motor deficits in Mecp2-null mice, here we attempt to examine whether pharmacological interventions by increasing GABA transmission could ameliorate motor deficits in Mecp2 mutant mice. We found that Mecp2-null mice and conditional knockout (cKO) male mice, but not mice carrying Mecp2T158A point mutation and cKO old female mice, administered with the cortex Magnoliae for 7 days via oral gavage at the dose of 100 mg/kg, showed a trend of improvement on open field test, beam walking task or rotarod task. We next tested the therapeutic effects of MH101 (a synthetic analog of honokiol),which is a GABAA receptor agonist derived from the bark of the plant Magnolia officinalis. We found that female cKO mice showed improvement on the rotarod performance after administration of 1 mg/kg MH101 90 mins prior to behavior tests. By examining expression of c-Fos protein, an indicator of neuronal activity, in different cortical and striatal regions with immunohistochemistry, we found that the MH101 treatment increased c-Fos expression in dorsomedial part of the rostral striatum in both control and cKO mice, but without significant alteration in the cortical neuron activity, suggesting that neuronal activity in the dorsomedial part of the rostral striatum may be related to increased motor learning in Mecp2 cKO female mice. On the other hand, the reduced expression of mu opioid receptor 1 and increased dopamine D2 receptor in the rostral striatum of RTT-like mice had been demonstrated in our previous study, we thus try to test whether activation of MOR1 or blockade of DRD2 in the rostral striatum could enhance motor function in wild-type mice. The results indicated that both treatments prevented post-surgery hypoactivity, suggesting that opioid and dopaminergic drugs could be alternative choices for improving motor deficits in RTT-like animal models. Taken together, our findings provide a proof-of-principle for novel pharmacological therapeutics to ameliorate motor deficits in mouse models of typical Rett syndrome.
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描述 碩士
國立政治大學
神經科學研究所
101754007
103
資料來源 http://thesis.lib.nccu.edu.tw/record/#G0101754007
資料類型 thesis
dc.contributor.advisor 廖文霖zh_TW
dc.contributor.advisor Liao, Wen Linen_US
dc.contributor.author (Authors) 黃弈博zh_TW
dc.contributor.author (Authors) Huang, Yi Boen_US
dc.creator (作者) 黃弈博zh_TW
dc.creator (作者) Huang, Yi Boen_US
dc.date (日期) 2014en_US
dc.date.accessioned 1-Apr-2015 10:02:46 (UTC+8)-
dc.date.available 1-Apr-2015 10:02:46 (UTC+8)-
dc.date.issued (上傳時間) 1-Apr-2015 10:02:46 (UTC+8)-
dc.identifier (Other Identifiers) G0101754007en_US
dc.identifier.uri (URI) http://nccur.lib.nccu.edu.tw/handle/140.119/74246-
dc.description (描述) 碩士zh_TW
dc.description (描述) 國立政治大學zh_TW
dc.description (描述) 神經科學研究所zh_TW
dc.description (描述) 101754007zh_TW
dc.description (描述) 103zh_TW
dc.description.abstract (摘要) 瑞特氏症 (Rett syndrome, RTT)是由於第二型甲基CpG結合蛋白 (methyl - CpG binding protein 2, MECP2) 基因發生突變所造成的一種神經發育疾病。當小鼠的MeCP2缺失時，會產生許多類似瑞特氏症患者的運動障礙，其中包含運動活力低下、運動協調與運動學習能力缺損，此運動障礙可能由於γ-胺基丁酸(GABA)與多巴胺神經訊號傳遞功能失調所造成。先前研究發現Mecp2基因剔除小鼠的運動障礙伴隨大腦皮質與紋狀體中GABA合成的減少，我們因此嘗試利用藥理的方式增加GABA傳訊，測試其是否可改善Mecp2基因剔除小鼠的運動障礙。我們發現Mecp2基因剔除以及條件缺失公鼠，經由管餵方式給予100 mg/kg厚朴生藥 (cortex Magnoliae)連續七天後，對於平衡桿行走測試以及加速滾輪測試有改善的趨勢，但是對於Mecp2T158A點突變公鼠以及條件缺失母鼠卻沒有效果。我們進而檢測和厚朴酚合成純化物 (以下簡稱MH101)對運動障礙的改善效果。在行為測試前九十分鐘腹腔給予 1 mg/kg的MH101並觀察待測鼠在加速滾輪上的掉落延宕時間， 發現Mecp2條件缺失母鼠在投藥後，與控制組母鼠在同樣藥物處理下相近，顯示MH101有改善運動障礙的效果。以免疫染色法觀察c-Fos蛋白表現量檢測神經細胞的活性，發現在投予MH101後，控制組母鼠的c-Fos蛋白表現量在紋狀體之內背側區有顯著的增加，且Mecp2條件缺失母鼠相對應的腦區也有增加的趨勢，而其他皮質腦區卻皆無明顯改變，其結果顯示內背側紋狀體的活性增加可能與Mecp2條件缺失母鼠的運動協調能力增加有所關聯。另一方面，先前研究指出瑞特氏症模式小鼠的前端紋狀體中，有μ型類鴉片受體表現量減少以及第二型多巴胺受體的過度表現，所以我們嘗試在野生型小鼠的前端紋狀體活化其μ型類鴉片受體或是減少第二型多巴胺受體的神經傳導，檢測是否能夠增加其運動能力。實驗結果顯示，不論是給予μ型類鴉片受體促效劑或是第二型多巴胺受體拮抗劑皆可有效防止手術後之運動活力表現的缺損，因此，於前端紋狀體活化μ型類鴉片受體或阻斷第二型多巴胺受體可能改善小鼠之運動障礙。綜上所述，本研究藉由改善神經傳導的缺失緩解瑞特氏症模式小鼠部份的運動障礙，提供典型瑞特氏症可能有效的治療方式。zh_TW
dc.description.abstract (摘要) Rett Syndrome (RTT) is a neurodevelopmental disorder caused by mutations of the methyl-CpG-binding protein 2 (MECP2). Mice with deficient MeCP2 recapitulate many RTT-like motor symptoms, including hypoactivity, deficits in motor coordination, and motor learning, those are associated with hypofunction of GABAergic and dopaminergic neurotransmission. Previous study found that decreased GABA synthesis in the striatum is accompanied by motor deficits in Mecp2-null mice, here we attempt to examine whether pharmacological interventions by increasing GABA transmission could ameliorate motor deficits in Mecp2 mutant mice. We found that Mecp2-null mice and conditional knockout (cKO) male mice, but not mice carrying Mecp2T158A point mutation and cKO old female mice, administered with the cortex Magnoliae for 7 days via oral gavage at the dose of 100 mg/kg, showed a trend of improvement on open field test, beam walking task or rotarod task. We next tested the therapeutic effects of MH101 (a synthetic analog of honokiol),which is a GABAA receptor agonist derived from the bark of the plant Magnolia officinalis. We found that female cKO mice showed improvement on the rotarod performance after administration of 1 mg/kg MH101 90 mins prior to behavior tests. By examining expression of c-Fos protein, an indicator of neuronal activity, in different cortical and striatal regions with immunohistochemistry, we found that the MH101 treatment increased c-Fos expression in dorsomedial part of the rostral striatum in both control and cKO mice, but without significant alteration in the cortical neuron activity, suggesting that neuronal activity in the dorsomedial part of the rostral striatum may be related to increased motor learning in Mecp2 cKO female mice. On the other hand, the reduced expression of mu opioid receptor 1 and increased dopamine D2 receptor in the rostral striatum of RTT-like mice had been demonstrated in our previous study, we thus try to test whether activation of MOR1 or blockade of DRD2 in the rostral striatum could enhance motor function in wild-type mice. The results indicated that both treatments prevented post-surgery hypoactivity, suggesting that opioid and dopaminergic drugs could be alternative choices for improving motor deficits in RTT-like animal models. Taken together, our findings provide a proof-of-principle for novel pharmacological therapeutics to ameliorate motor deficits in mouse models of typical Rett syndrome.en_US
dc.description.tableofcontents 中文摘要 I
英文摘要 II
目錄 III
表次 V
圖次 VI
縮寫對照表 VII
第一章 緒論 01
第一節 瑞特氏症 (Rett syndrome, RTT) 01
一、症狀 01
二、病源學 01
三、MECP2基因 03
第二節 瑞特氏症模式小鼠 03
一、Mecp2基因剔除小鼠 04
二、Mecp2 點突變小鼠 04
三、Mecp2條件缺失小鼠 04
第三節 瑞特氏症模式小鼠的療癒 05
一、基因治療 05
二、藥物治療 06
第四節 實驗目的與策略 07
第二章 材料與方法 09
第一節 實驗藥物……...………………………………………………………….……..09
一、厚朴生藥與和厚朴酚合成純化物 09
二、DAMGO與Raclopride 09
第二節 實驗動物 …….………………………………………………………………..09
第三節 基因轉殖鼠之基因型鑑定 .……………………………………………..10
一、基因組去氧核醣核酸 (Genomic DNA)之萃取 10
二、聚合酶鏈鎖反應 (polymerase chain reaction, PCR) 10
第四節 小鼠行為測試 ……..……………………………………………………… 12
一、敞箱測試 (open field test, OFT) 12
二、平衡桿行走測試 (beam walking test, BWT) 12
三、加速滾輪測試 (accelerating rotarod, RTR) 12
四、行為實驗之統計分析 13
第五節 腦部藥物注射之立體定位手術 ……..……………………………………… 13
第六節免疫組織染色 ……..………..………………………………………………… 13
一、腦組織的灌流固定與切片 13
二、免疫組織染色 14
三、cFos細胞計數定量分析 14
第三章 結果 16
第一節 厚朴生藥對Mecp2基因剔除公鼠運動障礙之影響 ……..………………..16
第二節 厚朴生藥對Mecp2點突變公鼠運動障礙之影響……..……………………..20
第三節 厚朴生藥對Mecp2條件缺失公鼠運動障礙之影響 ……………………….24
第四節 厚朴生藥對Mecp2條件缺失母鼠運動障礙之影響 .…………………..28
第五節 和厚朴酚合成純化物對Mecp2點突變公鼠運動障礙之影響 .………..32
第六節 和厚朴酚合成純化物對Mecp2條件缺失母鼠運動障礙之影響 .……..35
第七節 和厚朴酚合成純化物對Mecp2條件缺失母鼠腦中神經活性的影響 ...38
第八節 μ型類鴉片受體促效劑在前端紋狀體對於運動表現的影響 .....…..41
第九節 第二型多巴胺受體拮抗劑在前端紋狀體對於運動表現的影響 ..…...44
第四章 討論 47
第一節 瑞特氏症模式小鼠表現運動活力低下與運動協調能力缺失 ………...…..47
第二節 厚朴生藥對不同瑞特氏症模式小鼠產生不同影響.…………………………..47
第三節 和厚朴酚合成純化物對於不同腦區的神經細胞活性的影響..……………….49
第四節 紋狀體前端活化μ型類鴉片受體促效劑對於運動表現的影響.………..…....50
第五節 紋狀體前端抑制第二型多巴胺受體拮抗劑對於運動表現的影響..........…....51
第六節 瑞特氏症模式小鼠運動障礙之療癒.………………………....………………..52
第七節 本篇研究重要性………..………………………………………………….……52
第五章 結論 54
參考文獻 56
附錄 VIII		zh_TW
dc.format.extent 3462851 bytes-
dc.format.mimetype application/pdf-
dc.source.uri (資料來源) http://thesis.lib.nccu.edu.tw/record/#G0101754007en_US
dc.subject (關鍵詞) 瑞特氏症zh_TW
dc.subject (關鍵詞) 第二型甲基CpG結合蛋白zh_TW
dc.subject (關鍵詞) 紋狀體zh_TW
dc.subject (關鍵詞) γ-胺基丁酸zh_TW
dc.subject (關鍵詞) 厚朴生藥zh_TW
dc.subject (關鍵詞) 和厚朴酚合成純化物zh_TW
dc.subject (關鍵詞) Rett syndromeen_US
dc.subject (關鍵詞) Mecp2en_US
dc.subject (關鍵詞) Striatumen_US
dc.subject (關鍵詞) GABAen_US
dc.subject (關鍵詞) cortex Magnoliaeen_US
dc.subject (關鍵詞) MH101en_US
dc.title (題名) 以典型瑞特氏症模式小鼠研究運動障礙之療癒zh_TW
dc.title (題名) In search of interventions to ameliorate motor deficits in mouse models of typical Rett syndromeen_US
dc.type (資料類型) thesisen
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