臺灣博碩士論文加值系統

近年研究指出，神經性類固醇影響腦內生理功能扮演相當程度的角色，其合成途徑的第一步是由CYP11A1基因產物，細胞色素P450膽固醇側鏈截切脢 (cytochrome P450 side chain cleavage, P450scc) 所催化，因CYP11A1在腦中含量很低，其表現分布與轉錄調控的了解有限。在本實驗室所建立的SCC-Cre/R26R動物模式，發現長度4.4 kb之人類CYP11A1啟動子可驅動轉殖基因Cre重組酶在小鼠腦區有專一性的表現。為了證明內生性P450scc在這些腦區的表現，本研究利用免疫組織染色分析，說明了在小鼠海馬迴結構，間腦內的上視丘疆核、視丘神經核、下視丘神經核，與新皮質等區域中皆可偵測到，且主要表現在神經細胞。為了了解CYP11A1基因的轉錄調控，我們利用SCC-Cre/R26R動物模式，建立了SCC-iCre暫時性基因轉殖小鼠，探討CYP11A1啟動子在腦內的表現活性。將長度為4.4 kb 與2.7 kb的人類CYP11A1啟動子接上Cre重組酶基因後，藉由顯微注射，送入ROSA26小鼠異合子型的原核胚，待胚胎發育第14.5天，即直接取胚胎，利用X-gal全胚胎染色，分析Cre重組酶於胚胎鼠腦的活性。結果發現，4.4 kb與2.7 kb之CYP11A1啟動子皆可使Cre重組酶表現於胚胎腦部，前者主要表現在中腦、間腦及嗅球，後者僅主要表現在中腦尾端，說明了CYP11A1在不同腦區表現所需的元素有所不同，可能有不同的調控機制。
肝受體同源體-1 (liver receptor homolog-1, LRH-1) 為核受器家族的成員之一，目前已知該蛋白大量表現於卵巢濾泡之顆粒細胞及黃體，但其生理功能不清楚。為了解LRH-1於小鼠卵巢之生理功能，我們利用人類CYP11A1啟動子驅動EGFP-mLRH11~240之融合蛋白，建立SCC-ELRHdn基因轉殖小鼠。mLRH-11~240片段具有顯性抑制效果，藉CYP11A1啟動子使其表現於卵巢，以降低內生性LRH-1。結果顯示，經反轉錄PCR反應，可測得轉殖基因之mRNA有表現，且兩個轉殖株之內生性Cyp11a1及Cyp19的表現量有下降趨勢。但對於卵巢的生理作用並無明顯的效應。

Recent studies indicated that neurosteroids play an important role in the physiological function of brain. The first and rate-limiting step of the steroid synthesis pathway is catalyzed by the cytochrome P450 side chain cleavage enzyme (P450scc) which is encoded by CYP11A1 gene. It is difficult to study because of the low expression levels. The expression pattern and transcriptional regulation of the CYP11A1 gene in nervous system is largely unknown. Our previous SCC-Cre/R26R transgenic model showed that the human CYP11A1 4.4 kb promoter can drive the transgene Cre recombinase expressed in specific regions of the mice brain. In order to verify the endogenous gene expression in these brain regions, we performed immunohistochemistry to detect the presence of P450scc in mice brain. Our result showed that P450scc expression was observed in hippocampus, habenular nucleus, neuron nucleus of thalamus and hypothalamus, and cerebral neocortex. The colocalization of P450scc and NeuN demonstrated that most of P450scc exist in neuron cell in these regions. In order to clarify the transcriptional regulation of CYP11A1 gene, established the transient SCC-iCre transgenic mice model by using R26R system to study the promoter activity of human CYP11A1 gene in the brain. The 4.4 kb or 2.7 kb of 5’-flanking region of the human CYP11A1 gene was fused to the Cre recombinase and the transgenes were injected to the ROSA26 heterozygote pronuclei by microinjection. The transgenic embryos were collected at embryonic day 14.5 and assayed for the activity of Cre recombinase by whole-mount X-gal staining. The results displayed that both 4.4 kb and 2.7 kb promoter can drive the Cre expression in the embryonic brain, but the distinct difference in expression pattern between the two promoters is observed. The activity of Cre recombinase is predominantly detected in the midbrain, diencephalon and olfactory bulb in the SCC4.4-iCre transgenic brain. However, it only can be observed in the caudal part of the midbrain in the SCC2.7-iCre brains. The results suggest that CYP11A1 gene expression could be differentially regulated in different regions of the brain.
Liver receptor homolog-1 (LRH-1) is one member of the nuclear receptor family. It has been show that LRH-1 is highly expressed in the granulose cells and luteal cells of the ovary, but its physiological function is not certain. In order to explore the potential role of LRH-1 in the ovary, we construct the EGFP-mLRH11~240 fusion protein which is driven by the human CYP11A1 promoter to generate the SCC-ELRHdn transgenic mice. The mLRH11~240 has the dominant-negative effect on the transactivity of mLRH-1. The transgene expression are detected by RT-PCR analysis in the transgenic mice. In addition, the potential target genes Cyp11a1 and Cyp19 are slightly down-regulated. However, the transgenic animals do not exhibit abnormalities in ovary development and fertility.

表次 I
圖次 I
中文摘要 II
英文摘要 III
第一章 引言 1
一、類固醇荷爾蒙生成途徑 1
二、神經性類固醇 (neurosteroids) 1
(一) 神經性類固醇的發現 1
(二) 神經性類固醇的生成 1
(三) 神經性類固醇的功能 2
三、CYP11A1基因在腦內的特性 3
(一) Cyp11a1於腦中的表現分布 3
(二) 人類CYP11A1啟動子於腦中的基因調控 4
四、Cre-loxP系統 5
(一) Cre-loxP的特性 5
(二) Cre-loxP系統於條件式基因剔除 (conditional knockout) 上的應用 6
(三) 檢測Cre重組酶之活性 6
五、Liver receptor homolog-1 (LRH-1) 的特性 7
(一) LRH-1的結構 7
(二) LRH-1的功能及基因調控角色 7
六、研究動機 8
第二章 材料與方法 10
一、轉殖基因的建構 10
二、基因轉殖小鼠的產生 10
三、小鼠基因型檢測 (Genotyping) 11
四、灌流 (perfusion) 12
五、冷凍組織切片 (frozen section) 12
六、免疫螢光組織染色 (immunohistochemistry) 13
七、X-gal 酵素活性染色 14
八、反轉錄-聚合酵素鏈鎖反應 (Reverse-transcription polymerase chain reaction; RT-PCR) 14
九、細胞培養 16
十、暫時轉染法 (Transient transfection) 16
第三章 結果 18
一、內生性Cyp11a1於小鼠腦中的表現與分布 18
(一) 海馬迴結構 (hippocampus formation) 18
(二) 疆核 (habenular nucleus) 19
(三) 視丘室旁核(pararventricular nucleus of thalamus, PV)19
(四) 下視丘室旁核 (paraventricular nucleus of hypothalamus, PaH) 20
(五) 腹內側下視丘核 (ventromedial hypothalamus nucleus, VMHDM) 20
(六) 大腦新皮質 (neocortex) 20
二、4.4 kb與2.7 kb CYP11A1啟動子在14.5天胚胎鼠腦活性表現之特異性 22
三、SCC-ELRHdn基因轉殖小鼠 24
(一) 轉殖基因SCC-ELRHdn的建構 24
(二) 轉殖基因於NCI-H295R細胞株的表現 24
(三) SCC-ELRHdn轉殖基因小鼠的產生 25
(四) 轉殖基因於SCC-ELRHdn小鼠之表現 25
第四章 討論 26
一、P450scc在小鼠腦中的表現 26
(一) 內生性P450scc於腦中的表現區域 26
(二) 與轉殖基因Cre重組酶表現分布之比較 27
(三) 具有P450scc表現的神經核區之功能探討 27
二、人類CYP11A1啟動子在腦的調控 29
三、SCC-ELRHdn基因轉殖小鼠的表現 30
參考文獻 32
表 41
圖 44

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