調控CYP11A1啟動子於小鼠嗅覺上皮細胞表現之序列分析

Characterization of the DNA elements that regulate the expression of CYP11A1 promoter in mouse olfactory epithelium

梁凱豪(Kai-Hao Liang)

指導教授 ： 胡孟君

國立臺灣大學/醫學院/生理學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.02943

摘要

目前已證實許多類固醇荷爾蒙除了會在腎上腺、性腺與胎盤等處合成外，於中樞神經系統中也可以自行生成類固醇荷爾蒙，這些神經性類固醇荷爾蒙與許多神經生理功能相關，如神經細胞的凋亡、髓鞘的形成、影響記憶與學習的過程還有情緒的產生等。類固醇荷爾蒙以膽固醇 (cholesterone) 做為原料，經細胞色素P450膽固醇側鏈截切酶 (cytochrome P450 side chain cleavage，P450scc) 轉換成孕烯醇酮 (pregnenolone)，此步驟為整個類固醇生成步驟之第一步，同時也是速率決定步驟。因此，可表現P450scc的CYP11A1其調控機轉在類固醇荷爾蒙的生成以至於類固醇荷爾蒙所影響的生理功能中具有重大的意義。CYP11A1在一般類固醇荷爾蒙生成組織的調控已經被詳細探討，但在腦中卻因表現量低，其表現分布以及轉錄調控機制目前仍不清楚。本實驗室過去利用基因轉殖小鼠模式說明4.4 kb長之CYP11A1啟動子在腦中具有轉錄活性，更發現在3.3 kb左右之300 bp的序列決定了CYP11A1啟動子是否可以驅動報導基因在間腦、中腦以及嗅覺上皮細胞等腦區表現，因此我們判斷該段序列與CYP11A1啟動子的腦部調控有關。為了釐清這段啟動子範圍可能含有的調控序列，本論文將此300 bp分成數段，與小鼠嗅覺上皮細胞核蛋白質萃取物進行凝膠遷移 (electrophoresis mobility shift assay，EMSA) 反應，期望能找到可與核蛋白質反應的結合位。結果發現嗅覺上皮細胞之細胞核萃取液，至少有四種分子可辨識此段序列，具專一性之結合。顯示這些序列對CYP11A1啟動子於神經系統之轉錄調控可能具有相當的重要性。
以基因轉殖小鼠模式研究啟動子的調控耗時費力，因此我們想建立一套有效率的體外 (in vitro) 基因轉入系統，可驗證EMSA的結果。因此我建立了視網膜電穿孔轉染系統，測試啟動子在視網膜上的轉錄活性。當轉入CMV啟動子可成功驅動報導基因表現於視網膜組織，但轉入CYP11A1啟動子無法觀察到報導基因的表現，說明CYP11A1啟動子轉錄活性過低，無法利用體外視網膜培養系統偵測其活性。

關鍵字

CYP11A1啟動子 ； 嗅覺上皮細胞

並列摘要

Abstract
Steroid hormones are mainly synthesized in adrenal cortex, gonads and placenta. Recent studies indicate that they are also produced in CNS. These so-called neurosteroids do affect physiological functions including neural survival, myelination, neurogenesis, etc. CYP11A1 encodes P450scc (cytochrome P450 side chain cleavage), which catalyzes the first and the rate-limiting step during steroidogenesis that changes cholesterol into pregnenolone. Thus, CYP11A1 plays an important part in steroidogenesis. CYP11A1 can be expressed in steroidogenic tissue, but at low level in brain. This makes CYP11A1 difficult to be detected and studied in brain. We tried to investigate the regulation and distribution of CYP11A1 by transgenic mice and proved that 4.4 kb length of CYP11A1 promoter is able to promote Cre recombinase expression in brain. Furthermore, we have curtailed the range which may contain the potential DNA elements in CYP11A1 promoter. In this thesis, we spliced the potential regulatory sequence into short segments and incubated them with mouse olfactory epithelium nuclear extracts for electrophoretic mobility shift assay (EMSA). Our results indicate that the binding sequences of olfactory epithelium nuclear molecules do exist in CYP11A1 promoter and may play important roles in its neural regulation.
Since using transgenic mice for promoter research costs time and money, we are looking for some other in vitro transgenic system. Therefore, we constructed the retina electroporation system. We sent CYP11A1 promoter with Cre recombinase as the reporter gene in to retina tissue dissociated from mice cub. The results showed that although the retina electroporation system is able to transfect plasmids in to retina tissue, it still cannot help the weak promoter to trigger the reporter gene expression. In summary, this system is suitable for promoter studies except CYP11A1 promoter.

並列關鍵字

CYP11A1 promoter ； regulation ； olfactory epithelium

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