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  1. NTU Theses and Dissertations Repository
  2. 醫學院
  3. 生理學科所
請用此 Handle URI 來引用此文件: http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/44522
標題: 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
Characterization of Cyp11a1 expression in mice retina
and retinal development in Cyp11a1 knockout mice
作者: Wen-Chun Lo
羅文均
指導教授: 胡孟君(Meng-Chun Hu)
關鍵字: 神經性類固醇,細胞色素P450 側鏈截切&#37238,腦,視網膜,
neurosteroid,P450scc,brain,retina,
出版年 : 2009
學位: 碩士
摘要: 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。
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.
URI: http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/44522
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