臺灣博碩士論文加值系統

中文摘要
鮫鯊烯是天然存在以碳氫鍵結的三萜類化合物，也是內生性膽固醇合成過程中的重要中間物質。鮫鯊烯有益於健康的報導日漸增多，主要是針對其對膽固醇代謝的影響。最近，有研究人員提出另一項假說，指出鮫鯊烯可能是橄欖油降低癌症風險中的關鍵。流行病學的文獻報告指出鮫鯊烯有抗癌功效，一些動物實驗也顯示鮫鯊烯可抑制癌化。儘管如此，截至目前尚未有人體試驗研究報導。歸究其原因，可能是機制尚未明朗的緣故。鮫鯊烯的合成反應由鮫鯊烯合成酶所催化。鮫鯊烯合成酶也是固醇類合成途徑的首要酵素。研究報告指出鮫鯊烯合成酶的調節，對固醇及非固醇三萜類化合物合成的平衡扮演著樞紐角色。基於我們對鮫鯊烯預防疾病的效力及其可能機制的興趣，我們企盼找尋其催化酵素--鮫鯊烯合成酶的可靠來源。因此，本研究的總目標為選殖並構築人類鮫鯊烯合成酶的cDNA，以供後續評估此選殖基因的功能及對鮫鯊烯甚至膽固醇生合成調節之探討。本實驗從人類肝癌細胞(human HepG2 cell line)中抽取出total RNA後再利用自行設計的primers進行逆轉錄聚合酶連鎖反應(RT-PCR)增幅放大出人類鮫鯊烯合成酶的基因片段，長度大約1.2kb左右，之後再將基因連接入載體(pQE9)中，並轉形至勝任細胞(JM 109)，運用PCR檢測方法確認轉形成功後進行單一菌落的培養，再純化plasmid DNA定序，並將所得的序列進行分析和已發表相關基因序列比對。此外為分析酵素活性，我們將重組表達質體轉形至宿主細胞(JM 15)，再利用IPTG誘導進而大量表現外源基因所製造的蛋白質。再以Ni-NTA純化人類鮫鯊烯合成酶之後以放射線[3H]標定的FPP為受質分析酵素活性。人類鮫鯊烯合成酶為合成膽固醇的前驅酵素，若影響此酵素的活性，或許可調節膽固醇的生合成，進而提供生技保健或醫藥的基礎。因此我們挑選文獻報導有降血脂功效的山楂、鞣花酸及醫療用藥品Lovastatin來測試其對人類鮫鯊烯合成酶活性的影響。結果顯示山楂及鞣花酸皆隨劑量的增加而抑制酵素活性，相對的儘管Lovastatin抑制HMG-CoA reductase的活性其對人類鮫鯊烯合成酶卻有活化作用。



關鍵詞：鮫鯊烯、膽固醇、鮫鯊烯合成酶

ABSTRACT
Squalene, a naturally occurring hydrocarbon triterpene type containing six isoprene units, is an important intermediate in the endogenous biosynthesis of cholesterol. The beneficial effects of squalene on health are accumulating and primarily attributed to its impact on the cholesterol metabolism. Recently, one working hypothesis has been proposed that squalene may be a key factor in the cancer-risk reducing effect of olive oil. Although epidemiological and animal studies suggest anti-cancer properties of squalene, to date no human trials have been conducted. It could be due to the lacking of clear mechanisms. The formation of squalene is catalyzed by squalene synthase (SQS), the first enzyme committed exclusively to sterol synthesis. Studies with mammalian cells have shown that regulation of SQS is crucial for balancing the sterol and nonsterol isoprene synthesis in response to changing cellular requirements. In connection with our interest in the disease prevention efficacy of squalene and its possible mechanisms, we sought a reliable source of the catalyzed enzyme, squalene synthase. Therefore, the overall objective of this study was designed to clone and construct a cDNA for human squalene syntase (hSQS) for future examination on functions and modulations of squalene and even cholesterol biosynthesis. In this study, total RNA was extracted from human hepatic carcinoma cell (HepG2), followed by RT-PCR to amplify the targeted DNA, which is about 1.2 kb. The desired gene was then subcloned to pQE9 and transformed to E coli (JM109) and the purified DNA was sequenced and compared with the published data. For enzyme activity analysis. In the recombinant expression plasmid was transformed to JM15 and the squalene synthase protein was amplified under IPTG induction. The enzyme activity was determined after Ni-NTA purification and using [3H] FPP as substrate. Since squalene synthase is the first enzyme in the cholesterol biosynthesis pathway, modulation of this enzyme may further regulate cholesterol formation and this may serve as the basis for nutraceutical and pharmaceutical design. Accordingly, we examined the effect of the hawthorn, ellagic acid and lovastatin, all have been demonstrated having the potential to reduce cholesterol, on squalene synthase activity. The results showed hawthorn and ellagic acid inhibited the squalene synthase activity with dose-dependent and on the other hand, lovastatin activated the squalene synthase even it inhibited HMG-CoA reductase.

Keyword：squalene、cholesterol、squalene synthase

中文摘要……………………………………………………………………………I
英文摘要……………………………………………………………………………II
壹、緒論……………………………………………………………………………1
一、研究背景………………………………………………………………1
貳、文獻探討………………………………………………………………………3
一、動脈粥狀硬化與疾病……………………………………………………3
二、疾病與鮫鯊烯……………………………………………………………5
三、鮫鯊烯簡介………………………………………………………………7
參、研究大綱………………………………………………………………………11
肆、實驗材料………………………………………………………………………12
一、質體及菌種………………………………………………………………12
二、實驗藥品試劑……………………………………………………………12
三、實驗常用儀器……………………………………………………………14
伍、實驗方法………………………………………………………………………16
一、人類鮫鯊烯合成酶（hSQS）基因之選殖………………………………16
二、Total RNA純化……………………………………………………………17
三、反轉錄聚合酶連鎖反應……………………………………………………17
四、人類鮫鯊烯合成酶表現載體之構築………………………………………19
五、人類鮫鯊烯合成酶蛋白質在大腸桿菌系統中之大量表達………………23
六、鮫鯊烯合成酶活性測定……………………………………………………26
陸、結果………………………………………………………………………………27
一、人類鮫鯊烯合成酶…………………………………………………………27
二、Total RNA純化……………………………………………………………27
三、反轉錄聚合酶連鎖反應…………………………………………………27
四、人類鮫鯊烯合成酶表現載體之構築……………………………………27
五、人類鮫鯊烯合成酶蛋白質在大腸桿菌系統中之大量表達……………29
六、人類鮫鯊烯合成酶之純化………………………………………………29

七、hSQS酵素的最適反應濃度及時間………………………………………30
八、E.coli系統中hSQS酵素之分佈及其活性分析…………………………30
九、分析中草藥萃取物、化合物、醫學用藥品對人類鮫鯊烯合成酶
酵素活性的影響…………………………………………………………31
柒、討論……………………………………………………………………………33
捌、結論……………………………………………………………………………33
玖、圖表………………………………………………………………………………37
拾、參考文獻…………………………………………………………………………61
拾壹、附錄……………………………………………………………………………69

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