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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 胡孟君 | |
dc.contributor.author | Tzu-Hsuan W | en |
dc.contributor.author | 吳姿萱 | zh_TW |
dc.date.accessioned | 2021-06-13T17:29:46Z | - |
dc.date.available | 2016-10-05 | |
dc.date.copyright | 2011-10-05 | |
dc.date.issued | 2011 | |
dc.date.submitted | 2011-07-12 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/39488 | - |
dc.description.abstract | 神經類固醇於神經系統的作用很廣,主要藉由神經傳導物質受器,如γ-aminobutyric acidA (GABAA) 和 N-methyl-D-aspartate (NMDA) receptor 來調控腦部的神經發育以及生理上功能。pregnenolone (PREG), dehydroepiandrosterone (DHEA), progesterone (PROG) 以及 allopregnanolone (Allo) 等多種類固醇皆扮演保護神經細胞存活之角色,此外,也會參與神經纖維的生長發育。由於 Cyp11a1 基因是類固醇生成之關鍵酵素,本篇研究利用 Cyp11a1 基因剔除小鼠探討類固醇缺乏對發育中小鼠腦部之影響。
Cyp11a1 基因剔除 (KO) 鼠於在出生後約六天死亡,因此取五天大幼鼠進行實驗。利用 TUNEL assay 觀察 Cyp11a1 KO 與 WT 幼鼠大腦皮質 (cortex)、海馬迴 (hippocampus)、胼胝體與扣帶迴 (corpus callosum and cingulum bundle)、以及下視丘 (hypothalamus) 此四個腦區細胞凋亡之情形,結果顯示 KO 幼鼠此四個腦區細胞凋亡程度高於同胎之 WT 幼鼠,表示類固醇缺乏會導致腦部細胞凋亡增加。為了探討造成此現象之分子機制,利用 western blotting分析參與細胞凋亡相關分子之蛋白質表現量,結果顯示 WT 與 KO 幼鼠海馬迴以及下視丘之 Procaspase-3、Bcl-2 以及Bax 的蛋白質表現量皆沒有顯著差異。接著使用 caspase-3 colorimetric assay分析 caspase-3 的活性,其結果與 western blotting 類似,也沒有看到兩者活性上的差異。進一步利用 Golgi-Cox 染色法評估海馬迴 CA1 以及 CA3錐狀細胞樹突形態,結果顯示樹突分支總長度、分岔點以及分節數量皆沒有顯著差異,但在樹突分支情形以及複雜度,KO 與 WT 幼鼠相比有較簡單之趨勢。以上實驗結果顯示,類固醇缺乏會導致發育中幼鼠腦部細胞凋亡增加以及樹突形態受到影響。 | zh_TW |
dc.description.abstract | Steroids can be synthesized de novo in the nervous system that influence the brain development and many neurophysiological functions. Neurosteroids have the ability to change neuronal excitability by modulating neurotransmitter receptors such as γ-aminobutyric acidA (GABAA) and N-methyl-D-aspartate (NMDA) receptors. Some neurosteroids, including pregnenolone (PREG), dehydroepiandrosterone (DHEA), progesterone (PROG) and allopregnanolone (Allo), have been shown to prevent neuronal cell death elicited by different stimuli. Besides, these steroids also affect neurite growth during development. In this study, we utilize knockout mice disrupted with Cyp11a1, the key gene controlling steroidogenesis, to elucidate the role of endogenous steroids in the neuronal survival and neurite outgrowth of brain.
Cyp11a1 null mice lack the synthesis of steroids and die about postnatal 6 days. We used TUNEL assay to investigate the apoptosis in various brain areas of wild-type (WT) and knockout (KO) mice on postnatal day 5. Our data showed that when compared with the wild-type littermate, the number of TUNEL-positive cells in cerebral cortex, hippocampus, corpus callosum, cingulum bundle and hypothalamus were higher in the Cyp11a1 KO mice. It indicated that steroid deficiency may enhance the apoptosis in the developing brain. To understand the molecular pathway, we analyzed the expression of procaspase-3、Bcl-2 and Bax. Besides we examined the activation of caspase-3 by colorimetric assay in hippocampus and hypothalamus. However, there is no significant difference in the levels of these proteins and the activity of caspase-3 between WT and KO mice. On the other hand, we used Golgi-Cox stain to study the dendritic morphology of hippocampal pyramidal cells. Total dendritic length, the number of nodes and segments between WT and KO mice were not significantly different; however, the pattern of dendritic branching and the dendritic arbor distribution in KO mice were simpler than WT mice. These studies suggested that steroids may play a role in regulating cell survival and the hippocampal dendritic outgrowth in developing brain. | en |
dc.description.provenance | Made available in DSpace on 2021-06-13T17:29:46Z (GMT). No. of bitstreams: 1 ntu-100-R98441016-1.pdf: 2070697 bytes, checksum: 0ebda73e125e5163da116e49780b931c (MD5) Previous issue date: 2011 | en |
dc.description.tableofcontents | 目錄
致謝 I 目錄 III 表次 V 圖次 V 摘要 VI Abstract VII 第一章 導論 1 一、類固醇荷爾蒙 (steroid hormone) 之生成 1 二、神經類固醇 (neurosteroid) 之發現與生成 1 三、神經類固醇在腦部作用方式 2 四、類固醇於神經系統之功能 3 1.神經細胞之保護作用 (neuroprotection) 3 2.神經纖維的生長 (neurite growth) 4 3.神經新生 (neurogenesis) 5 五、研究目的 6 第二章 材料與方法 7 一、Cyp11a1 基因剔除鼠之產生 7 二、小鼠基因型鑑定 (Genotyping) 7 三、灌流 (Perfusion) 8 四、石蠟組織切片 (Paraffin Section) 8 五、細胞凋亡 ( apoptosis) 之分析 9 六、西方墨點法 (Western blotting) 9 七、Caspase-3 活性測量 11 八、Golgi-Cox 染色法 11 九、海馬迴錐狀細胞樹突形態 (Dendritic morphology) 之分析 12 十、統計分析 13 第三章 結果 14 一、Cyp11a1 基因剔除鼠之產生與基因型鑑定 14 二、Cyp11a1 KO 鼠之腦部細胞凋亡數目高於 WT 幼鼠 14 三、細胞凋亡相關分子之分析 15 四、類固醇缺乏對幼鼠海馬迴錐狀細胞樹突形態之影響 16 第四章 討論 19 一、KO 與 WT 幼鼠腦部細胞凋亡之情形 19 二、導致 KO 與 WT 幼鼠腦部細胞凋亡差異之分子機制 20 三、類固醇缺乏對幼鼠海馬迴之錐狀細胞樹突形態之影響 21 參考文獻 Reference 24 表次 表一、五天大 Cyp11a1 雌鼠 KO 中相對於 WT 細胞凋亡之比例 33 表二、五天大 Cyp11a1 雄鼠 KO 中相對於 WT 細胞凋亡之比例 34 圖次 圖一、小鼠之Cyp11a1 基因型鑑定 35 圖二、大腦細胞凋亡分析 36 圖三、Cyp11a1 KO 雌鼠腦部細胞之凋亡數多於 WT 幼鼠 37 圖四、Cyp11a1 KO 雄鼠腦部細胞之凋亡數多於 WT 幼鼠 38 圖五、Procaspase-3 與 Bcl-2 在五天大 WT 和 KO 雌鼠海馬迴之表現 39 圖六、Procaspase-3、Bcl-2 與 Bax 在五天大 WT 和 KO 雌鼠下視丘之表現 40 圖七、Procaspase-3、Bcl-2 與 Bax 在五天大 WT 和 KO 雄鼠海馬迴之表現 41 圖八、Procaspase-3、Bcl-2 與 Bax 在五天大 WT 和 KO 雄鼠下視丘之表現 42 圖九、五天大 WT 與 KO 雌鼠海馬迴與下視丘之 caspase-3 的活性分析 43 圖十、幼鼠海馬迴錐狀細胞樹突形態示意圖 44 圖十一、五天大 WT 與 KO 幼鼠海馬迴 CA1 樹突分支情形之分析 45 圖十二、五天大 WT 與 KO 幼鼠海馬迴 CA3 樹突分支情形之分析 46 圖十三、五天大 WT 與 KO 幼鼠海馬迴的樹突分支層級與分節的數量關係圖 47 圖十四、五天大 WT 與 KO 幼鼠海馬迴的樹突之 Sholl concentric sphere analysis 48 | |
dc.language.iso | zh-TW | |
dc.title | 分析 Cyp11a1 基因剔除對發育中小鼠腦部之影響 | zh_TW |
dc.title | Analysis of Cyp11a1 null in developing mice brain | en |
dc.type | Thesis | |
dc.date.schoolyear | 99-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 蘇慧敏,王致恬,李立仁 | |
dc.subject.keyword | 神經類固醇,細胞色素 P450 側鏈截切酶,基因剔除鼠,細胞凋亡,神經纖維生長, | zh_TW |
dc.subject.keyword | neurosteroid,Cyp11a1,P450scc,apoptosis,TUNEL,caspase-3,Bcl-2,neurite outgrowth, | en |
dc.relation.page | 48 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2011-07-12 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 生理學研究所 | zh_TW |
顯示於系所單位: | 生理學科所 |
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