Characterization of Cyp11a1 expression in mice retinand retinal development in Cyp11a1 knockout mice

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amp;#37238;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 nervousystem, including involvement in anxiety, memory and cognition, and neuron survivalnd degeneration. The first step in de-novo synthesis of steroids is the conversion ofholesterol to pregnenolone by Cytochrome P450 side chain cleavage enzyme (P450scc)n the mitochondrial inner membrane. P450scc is encoded by the Cyp11a1 gene andxpressed in brain. However, the presence of P450scc is very low, and therefore it isifficult to detect in vivo. We have produced an antibody against a 22-amino acideptide of P450scc to analyze its expression and location using immunohistochemistry.ur results show that P450scc is expressed in the hippocampus, amygdala, lateral septalucleus, nucleus accumbens, and magnocellular preoptic nucleus. Pre-immune serumas no fluorescence response. Additionally, We generated SCC-Cre transgenic mice, inhich the human CYP11A1 promoter drives Cre recombination allowing for detectionf Cre recombinase activity in retina, illustrate that Cre recombinase activity is mosttrongly expressed in inner nuclear layer (INL) and ganglion cell layer (GCL). Theuter nuclear cell layer (ONL) and inner plexiform layer (IPL) show weak activity. Thus,he CYP11A1 promoter may have transcriptional activity in retina. To confirm theocation of P450scc within the retina, we performed immunohistochemistry andbserved immunoreactivity against P450scc in the ONL, INL, IPL and GCL.he above findings suggest that neurosteroid production via P450scc may affecturvival and degeneration of neurons in many locations. To further investigate theotential effect of Cyp11a1 in retinal development, we generated Cyp11a1+/+ wild typend Cyp11a1-/- knock-out mice. Retina of postnatal five days pups have not developedompletely, which mainly performed nuclear layer. Immunohistochemistry studiesgainst P450scc, however, reveal that there are no significant differences inXluorescence responses between Cyp11a1-/- and Cyp11a1+/+mice. Finally, we observedhe morphology of retinal cells in postnatal 3-5 days Cyp11a1-/- and Cyp11a1+/+ mice byematoxylin and Eosin-Y staining. Again, no significant morphological differencesere noted between the wild type and knock-out mice. Cyp11a1 appears critical forice development, as knock-outs do not survive past 5-6 days; as such, we couldn’tnvestigate the role of Cyp11a1 gene in late retinal development. We interpret the aboveindings as Cyp11a1 expresses in the nervous system, such as brain and retina, and itose not obviously involved in early retinal development.致謝.................................................. Ⅰ錄.................................................. Ⅲ次.................................................. Ⅵ文摘要.............................................. Ⅶ文摘要.............................................. Ⅷ一章 導論......................................... 1、 類固醇荷爾蒙生成途徑 ................................................................... 1 二、 神經類固醇 (Neurosteroid) ............................................................. 1. 神經類固醇的發現 ........................................................................ 1. 神經類固醇之生成途徑.…..…………...................................... .... 2. 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. 視網膜結構………..………………….……………………... ...... 4. 神經類固醇於視網膜之表現………….……..……………........ . 5. 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 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. SCC-Cre轉殖小鼠 ......................................................................... 9. 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. Pregnenolone萃取 ......................................................................... 15. Pregnenolone測量 ........................................................................ 15三章 結果 ............................................................................................................ 17、 內生性Cyp11a1於小鼠腦中的表現與分佈 ................................ 17. 杏仁核 (Central amygdaloid nuclei, CeA) ................................... 17. 側隔核 ( Lateral setpal neclei, LSD) ............................................ 17. 阿肯伯氏核 (nucleus accumbens, ACB) ...................................... 18. 大細胞視前核 (Magnocellular preoptic nucleus, MCPO) .......... 18、 Cre重組酶於SCC-Cre/R26R轉殖小鼠視網膜中的表現 .......... 18、 內生性Cyp11a1於小鼠視網膜中的表現與分佈 ......................... 19、 Cyp11a1基因剔除小鼠之產生 ..................................................... 19、 內生性Cyp11a1於幼鼠視網膜之表現 ....................................... 20、 視網膜pregnenolone之含量 ......................................................... 20、 Cyp11a1+/+與Cyp11a1-/-幼鼠視網膜發育型態之比較 ............... 21四章 討論 ............................................................................................................. 23、 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. 杏仁核 ................................................................................ 24. 側隔核 ................................................................................ 24. 阿肯伯氏核 ........................................................................ 25. 大細胞視前核 ..................................................................... 25、 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、 幼鼠視網膜pregnenolone之含量 ..................................................... 26、 Cyp11a1對幼鼠之視網膜發育之影響 ........................................... 27考文獻 ........................................................................................................ 28 ................................................................................................................................ 3