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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 胡孟君(Meng-Chun Hu) | |
dc.contributor.author | Wen-Chun Lo | en |
dc.contributor.author | 羅文均 | zh_TW |
dc.date.accessioned | 2021-06-15T03:02:47Z | - |
dc.date.available | 2009-09-15 | |
dc.date.copyright | 2009-09-15 | |
dc.date.issued | 2009 | |
dc.date.submitted | 2009-07-30 | |
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Olds J, Milner P (1954) Positive reinforcement produced by electrical stimulation of septal area and other regions of rat brain. J Comp Physiol Psychol 47:419-427. Payne AH, Hales DB (2004) Overview of steroidogenic enzymes in the pathway from cholesterol to active steroid hormones. Endocr Rev 25:947-970. Richard S. Smith (Editor) SWMJE, Patsy M. Nishina (Editor), John P. Sundberg (Editor) (2001) Systematic Evaluation of the Mouse Eye: Anatomy, Pathology, and Biomethods. Risold PY, Swanson LW (1997) Connections of the rat lateral septal complex. Brain Res Brain Res Rev 24:115-195. Schumacher M, Akwa Y, Guennoun R, Robert F, Labombarda F, Desarnaud F, Robel P, De Nicola AF, Baulieu EE (2000) Steroid synthesis and metabolism in the nervous system: trophic and protective effects. J Neurocytol 29:307-326. Soriano P (1999) Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet 21:70-71. Swann JM, Wang J, Govek EK (2003) The MPN mag: introducing a critical area mediating pheromonal and hormonal regulation of male sexual behavior. Ann N Y Acad Sci 1007:199-210. Szymusiak R, Steininger T, Alam N, McGinty D (2001) Preoptic area sleep-regulating mechanisms. Arch Ital Biol 139:77-92. Tsutsui K (2006) Biosynthesis and organizing action of neurosteroids in the developing Purkinje cell. Cerebellum 5:89-96. Tsutsui K, Matsunaga M, Miyabara H, Ukena K (2006) Neurosteroid biosynthesis in the quail brain: a review. J Exp Zoolog A Comp Exp Biol 305:733-742. 33 Ukena K, Usui M, Kohchi C, Tsutsui K (1998) Cytochrome P450 side-chain cleavage enzyme in the cerebellar Purkinje neuron and its neonatal change in rats. Endocrinology 139:137-147. Wassle H (2004) Parallel processing in the mammalian retina. Nat Rev Neurosci 5:747-757. Wu HS, Lin HT, Wang CK, Chiang YF, Chu HP, Hu MC (2007) Human CYP11A1 promoter drives Cre recombinase expression in the brain in addition to adrenals and gonads. Genesis 45:59-65. Yawno T, Hirst JJ, Castillo-Melendez M, Walker DW (2009) Role of neurosteroids in regulating cell death and proliferation in the late gestation fetal brain. Neuroscience. Yu L, Romero DG, Gomez-Sanchez CE, Gomez-Sanchez EP (2002) Steroidogenic enzyme gene expression in the human brain. Mol Cell Endocrinol 190:9-17. 江衍楓(2007) The expression and reglation of CYP11A1 gene in the brain. 碩士論文. 吳曙序 (2006) Generation of SCC-Cre transgenic mice expressing Cre recombinase under control of human CYP11A1 promoter. 碩士論文. 林慧婷 (2006) Human CYP11A1 4.4 kb promoter drives Cre recombinase expression in mouse brain. 碩士論文. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/44522 | - |
dc.description.abstract | 近年研究指出神經性類固醇在神經系統中有多種功能,與情緒焦慮、記憶及認知有關,並可調控神經細胞之興奮性、存活與退化。類固醇生成第一個步驟是由位於粒腺體的細胞色素P450 側鏈截切酶(Cytochrome P450 side chain cleavage enzyme, P450scc) 所催化,可將膽固醇(cholesterol) 轉變為孕烯醇酮(pregnenolone)。雖然前人已發現腦部有P450scc 酵素之基因Cyp11a1 的表現,但其表現量很低,使得神經系統內生性的P450scc 不易被測得。本研究以P450scc 的一段胜肽產生之抗體與pre-immune serum 進行免疫組織染色分析內生性P450scc於成鼠腦中之表現與分佈,於小鼠海馬迴、杏仁核、側中隔、阿肯伯氏核及前視核大細胞中發現P450scc 之螢光表現,而pre-immune serum 則無法觀察到到螢光反應。本實驗室所建立的SCC-Cre 轉殖小鼠是以人類CYP11A1 啟動子驅動Cre 重組
酶之表現,於SCC-Cre 成鼠的視網膜可以看到明顯Cre 重組酶的活性,主要分佈於內細胞核層(INL) 及神經節細胞層(GCL) ,而外細胞核層(ONL) 及內網層(IPL) 則有較弱之呈色,說明CYP11A1 啟動子在視網膜具有轉錄活性。為了更進一步確認P450scc 表現的位置,以免疫組織染色偵測內生性P450scc 在視網膜的分布,發現在成鼠的視網膜中可於ONL、INL、IPL 及GCL 看到P450scc 的抗體反應。 已知神經性類固醇可影響神經細胞之存活與退化,為探討 Cyp11a1 對於小鼠視網膜發育可能之影響,以Cyp11a1+/+野生型及Cyp11a1-/-基因剔除鼠進行實驗。出生後五天之幼鼠視網膜發育尚未完全,主要以核層為主,以P450scc 抗體進行視網膜免疫組織染色,發現於Cyp11a1-/-與Cyp11a1+/+得到類似的螢光反應,無法說明P450scc 真正之表現。進一步以H & E 染色觀察出生後3 至5 天的Cyp11a1+/+與Cyp11a1-/-幼鼠視網膜之型態,則發現兩者沒有明顯差異。由於Cyp11a1-/-小鼠只能存活5 至6 天,因此無法觀察到Cyp11a1 基因功能喪失對後續發育可能造成的影響。 | zh_TW |
dc.description.abstract | Recent studies demonstrate that neurosteroids have many functions in the nervous
system, including involvement in anxiety, memory and cognition, and neuron survival and degeneration. The first step in de-novo synthesis of steroids is the conversion of cholesterol to pregnenolone by Cytochrome P450 side chain cleavage enzyme (P450scc) on the mitochondrial inner membrane. P450scc is encoded by the Cyp11a1 gene and expressed in brain. However, the presence of P450scc is very low, and therefore it is difficult to detect in vivo. We have produced an antibody against a 22-amino acid peptide of P450scc to analyze its expression and location using immunohistochemistry. Our results show that P450scc is expressed in the hippocampus, amygdala, lateral septal nucleus, nucleus accumbens, and magnocellular preoptic nucleus. Pre-immune serum has no fluorescence response. Additionally, We generated SCC-Cre transgenic mice, in which the human CYP11A1 promoter drives Cre recombination allowing for detection of Cre recombinase activity in retina, illustrate that Cre recombinase activity is most strongly expressed in inner nuclear layer (INL) and ganglion cell layer (GCL). The outer nuclear cell layer (ONL) and inner plexiform layer (IPL) show weak activity. Thus, the CYP11A1 promoter may have transcriptional activity in retina. To confirm the location of P450scc within the retina, we performed immunohistochemistry and observed immunoreactivity against P450scc in the ONL, INL, IPL and GCL. The above findings suggest that neurosteroid production via P450scc may affect survival and degeneration of neurons in many locations. To further investigate the potential effect of Cyp11a1 in retinal development, we generated Cyp11a1+/+ wild type and Cyp11a1-/- knock-out mice. Retina of postnatal five days pups have not developed completely, which mainly performed nuclear layer. Immunohistochemistry studies against P450scc, however, reveal that there are no significant differences in IX fluorescence responses between Cyp11a1-/- and Cyp11a1+/+mice. Finally, we observed the morphology of retinal cells in postnatal 3-5 days Cyp11a1-/- and Cyp11a1+/+ mice by Hematoxylin and Eosin-Y staining. Again, no significant morphological differences were noted between the wild type and knock-out mice. Cyp11a1 appears critical for mice development, as knock-outs do not survive past 5-6 days; as such, we couldn’t investigate the role of Cyp11a1 gene in late retinal development. We interpret the above findings as Cyp11a1 expresses in the nervous system, such as brain and retina, and it dose not obviously involved in early retinal development. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T03:02:47Z (GMT). No. of bitstreams: 1 ntu-98-R96441015-1.pdf: 2630784 bytes, checksum: 0602d01d94ae089071e987565d6dd32f (MD5) Previous issue date: 2009 | en |
dc.description.tableofcontents | 致謝.................................................. Ⅰ
目錄.................................................. Ⅲ 圖次.................................................. Ⅵ 中文摘要.............................................. Ⅶ 英文摘要.............................................. Ⅷ 第一章 導論......................................... 1 一、 類固醇荷爾蒙生成途徑 ................................................................... 1 二、 神經類固醇 (Neurosteroid) ............................................................. 1 1. 神經類固醇的發現 ........................................................................ 1 2. 神經類固醇之生成途徑.…..…………...................................... .... 2 3. 神經類固醇的功能…..………...……………........................ ........ 2 三、 Cyp11a1於腦內之特性 .................................................................... 4 四、 神經類固醇於視網膜之特性 ............................................................ 4 1. 視網膜結構………..………………….……………………... ...... 4 2. 神經類固醇於視網膜之表現………….……..……………........ . 5 3. 神經類固醇於視網膜之功能 ........................................................ 6 五、 Cre/loxP系統 ................................................................................... 7 六、 研究動機 .......................................................................................... 8 第二章 材料與方法 ................................................................................................. 9 一、 Cyp11a1-/-基因剔除鼠的產生 .......................................................... 9 二、 SCC-Cre/R26R 小鼠之產生 ............................................................ 9 1. SCC-Cre轉殖小鼠 ......................................................................... 9 2. SCC-Cre/R26R轉殖小鼠 .............................................................. 9 三、 小鼠基因型檢測 (genotyping) ........................................................ 10 四、 Anti-mouse P450scc抗體製備 ......................................................... 11 五、 灌流 (perfusion) ................................................................................ 11 六、 冷凍組織切片 (cryosection) ........................................................... 12 七、 免疫組織染色 (immunohistochemistry) .......................................... 12 1. 視網膜 ........................................................................................... 12 2. 腦組織 .......................................................................................... 13 八、 X-gal酵素活性染色 ......................................................................... 14 九、 石蠟組織切片 (paraffin section) ..................................................... 14 十、 蘇木紫-伊紅染色法 (Hematoxylin and Eosin-Y staining) ............. 14 十一、 Pregnenolone之測定 ..................................................................... 15 1. Pregnenolone萃取 ......................................................................... 15 2. Pregnenolone測量 ........................................................................ 15 第三章 結果 ............................................................................................................ 17 一、 內生性Cyp11a1於小鼠腦中的表現與分佈 ................................ 17 1. 杏仁核 (Central amygdaloid nuclei, CeA) ................................... 17 2. 側隔核 ( Lateral setpal neclei, LSD) ............................................ 17 3. 阿肯伯氏核 (nucleus accumbens, ACB) ...................................... 18 4. 大細胞視前核 (Magnocellular preoptic nucleus, MCPO) .......... 18 二、 Cre重組酶於SCC-Cre/R26R轉殖小鼠視網膜中的表現 .......... 18 三、 內生性Cyp11a1於小鼠視網膜中的表現與分佈 ......................... 19 四、 Cyp11a1基因剔除小鼠之產生 ..................................................... 19 五、 內生性Cyp11a1於幼鼠視網膜之表現 ....................................... 20 六、 視網膜pregnenolone之含量 ......................................................... 20 七、 Cyp11a1+/+與Cyp11a1-/-幼鼠視網膜發育型態之比較 ............... 21 第四章 討論 ............................................................................................................. 23 一、 Cyp11a1在小鼠腦中之表現 ......................................................... 23 (一) 內生性Cyp11a1於腦中的表現區域 ...................................... 23 (二) 具有P450scc表現神經核區功能之探討 .............................. 24 1. 杏仁核 ................................................................................ 24 2. 側隔核 ................................................................................ 24 3. 阿肯伯氏核 ........................................................................ 25 4. 大細胞視前核 ..................................................................... 25 二、 Cyp11a1於小鼠視網膜之表現 ....................................................... 25 (一) CYP11A1於成鼠視網膜中的表現分佈 .............................. 25 (二) 內生性Cyp11a1於幼鼠視網膜中的表現分佈 ..................... 26 三、 幼鼠視網膜pregnenolone之含量 ..................................................... 26 四、 Cyp11a1對幼鼠之視網膜發育之影響 ........................................... 27 參考文獻 ........................................................................................................ 28 圖 ................................................................................................................................ 34 | |
dc.language.iso | zh-TW | |
dc.title | Cyp11a1 於小鼠視網膜之表現與Cyp11a1 基因剔除小
鼠視網膜發育之探討 | zh_TW |
dc.title | Characterization of Cyp11a1 expression in mice retina
and retinal development in Cyp11a1 knockout mice | en |
dc.type | Thesis | |
dc.date.schoolyear | 97-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 李立安,應靜雯,盧主欽 | |
dc.subject.keyword | 神經性類固醇,細胞色素P450 側鏈截切酶,腦,視網膜, | zh_TW |
dc.subject.keyword | neurosteroid,P450scc,brain,retina, | en |
dc.relation.page | 45 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2009-07-30 | |
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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