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研究生:朱智傑
研究生(外文):Chu, Chih-Chieh
論文名稱:建構細胞色素P450表現之細胞株:異質物代謝系統的最佳化
論文名稱(外文):Generation and Characterizations of a Cell System Expressing Recombinant Cytochrome P450: Implications for Optimization of Xenobiotics Metabolism System
指導教授:宋信文徐祖安
指導教授(外文):Sung, Hsing-WenHsu, Tsu-An
口試委員:吳宗遠姚賢宗劉玉麗宋信文徐祖安
口試日期:2011-7-25
學位類別:博士
校院名稱:國立清華大學
系所名稱:化學工程學系
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:87
中文關鍵詞:細胞色素 P450藥物代謝細胞毒性
外文關鍵詞:Cytochrome P450Drug metabolismCytotoxicity
相關次數:
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  • 收藏至我的研究室書目清單書目收藏:1
肝臟是人體主要進行藥物代謝的器官。在肝臟細胞中,細胞色素P450 (Cytochrome P450,CYP450) 參與了大部份藥物代謝過程的第一步。本研究成功架構CYP450重組慢病毒顆粒,經轉導Huh7細胞後,挑選出CYP450表現量高的細胞株:表現CYP1A2的Huh7-1A2-I-E、表現CYP2E1的Huh7-2E1和表現CYP3A4的Huh7-3A4-I-E的細胞株,其中表現CYP1A2和CYP3A4細胞藉由IRES (Internal ribosome entry site) 調控綠螢光基因,可同時於細胞中表現CYP450和綠螢光蛋白質。穩定表現CYP450的細胞株可作為高速篩選系統的工具,用於研究CYP450抑制劑、探討CYP450於細胞內的代謝功能。為了探討Huh7-1A2-I-E細胞和Huh7-3A4-I-E細胞的效用,本研究選擇會經CYP450作用而產生對細胞更具毒性代謝產物的黃麴毒素 (aflatoxin B1) 來驗證。當Huh7-1A2-I-E或Huh7-3A4-I-E細胞於培養基中添加不同濃度的aflatoxin B1後,其細胞活性下降的比率較對照組 (Huh7-E細胞於培養基中添加aflatoxin B1) 多,然而此現象會因CYP450抑制劑 (CYP1A2:furafylline;CYP3A4:ketoconazole) 的加入而消失,因此,aflatoxin B1極可能會分別經過CYP1A2或CYP3A4代謝成毒性代謝產物,對細胞造成傷害。在Huh7-2E1細胞處理乙醯胺酚 (acetaminophen) 的實驗中也可得到相似的結果。另一方面,我們也證實穩定表現CYP450的細胞可被使用在藥物代謝的研究,接近200個抗癌藥物經CYP450代謝後的結果,代謝產物可被分類成不影響細胞活性、降低細胞活性,和提升細胞活性等三大類;此外,一些目前未定義的CYP1A2抑制劑,像是evoxine和berberine,亦已於此研究所建構的系統中被篩選出來。因此,由蛋白質表現、酵素活性測量、以及毒性代謝物代謝後對細胞的影響等方面,皆能驗證本研究所發展的細胞株的確具有CYP450的功能。為了提高細胞內CYP450的酵素活性,我們嘗試了以基因工程改良細胞本身與以外加藥物於培養基調控細胞內的蛋白質表現量等方式進行探討。在基因層級 (genetic level) 方面,本研究已探討細胞色素b5、細胞色素b5還原酶、細胞色素P450還原酶、和桿狀病毒IE2 (the activator of CMV promoter),對酵素活性的影響。結果得知,細胞色素b5和桿狀病毒IE2能分別在非肝臟細胞 (293FT和Vero) 中提高CYP450酵素活性,但對Huh7細胞則沒有效果,而細胞色素b5還原酶和細胞色素P450還原酶對兩種類型的細胞都沒有幫助。除此之外,由外加藥物調控胞內蛋白表現量的研究中發現,valproic acid (VPA) 能於細胞中增進CYP1A2蛋白質表現,進而提升其酵素活性。本研究中所建構的穩定表現CYP450細胞模式,應能真實的在以細胞為基礎 (cell-based) 的系統中呈現肝臟代謝藥物的結果。此CYP450表現系統的建立與實際經驗 (know-how) 對人類醫藥使用的安全和類肝細胞 (hepatocyte-like cells) 建構的發展,應能提供一個更有效率的研究工具。
Liver plays a major role in drug metabolism that was carried out by a family of critical enzymes; cytochrome P450 (CYP450). In this study, cell lines with high expression level of CYP450 are established based on Huh7 cells: Huh7-1A2-I-E cells expressing CYP1A2, Huh7-2E1 cells expressing CYP2E1, and Huh7-3A4-I-E cells expressing CYP3A4. To achieve this, we constructed recombinant lentiviral particles, containing a single promoter encoding CYP1A2 or CYP3A4 followed by an internal ribosome entry site (IRES) to permit the translation of enhanced green fluorescence protein (EGFP). Such a design has greatly facilitated the selection of stable cell lines because the translations of CYP450 and EGFP proteins would be based on a single bi-cistronic mRNA. The stable CYP450-expressing cells were evaluated as a cell-based model for identification of CYP450 inhibitors and for studies of cytotoxicity resulted from CYP-mediated drug metabolism. Treatment of stable cells with aflatoxin B1 showed that cells with CYP1A2 or CYP3A4 expression were much sensitive to aflatoxin B1 and the cellular toxicity of aflatoxin B1 in cells was prevented by CYP450 inhibitors (CYP1A2: furafylline; CYP3A4: ketoconazol). In addition, acetaminophen was also shown as a toxic substrate in Huh7-2E1 cells. Thus, we confirm that the CYP450-expressing cells have CYP450 characteristics based on the expressed protein level, the detectable enzyme activity, and the CYP450-mediated cytotoxicity study. Furthermore, a collection of approximately 200 drugs were screened using this system, and them could be separated into active metabolites, toxic metabolites, and inactive metabolites according to the cell-based screening based on the cells carrying the CYP450 metabolic activity. Several previously unidentified CYP1A2 inhibitors such as evoxine and berberine were also identified in this study. To enhance the intercellular CYP450 enzyme activity of stable cells, we attempted to modify the cellular characteristics using the genetic engineering approach and to improve the protein expression with the help of the small molecular compounds in culture medium. Cytochrome b5, cytochrome b5 reductase, cytochrome P450 reductase, and baculovirus IE2 (the activator of CMV promoter) were used to increase the enzyme activity. Results showed that cytochrome b5 and baculovirus IE2 enhanced the CYP450 enzyme activity in non-liver cells (293FT and Vero), but it was ineffective in Huh7 cells. Neither did cytochrome b5 reductase nor cytochrome P450 reductase help to enhance the enzymatic activity of CYP450 in liver and non-liver cells. Besides, valproic acid could increase the CYP450 enzyme activity when it was included in the culture medium. Overall, results from this thesis showed that the CYP450-expressing system can be effectively employed to study drug metabolism and to facilitate the establishment of hepatocyte-like cells in the future.
目 錄
第一章 緒論 ………………………………………...…………….…..…………... 1
第二章 文獻回顧 …………………………………………..………………….….. 5
2.1 細胞色素P450的歷史緣由、命名與人體分佈 ……….……………….….. 5
2.2 細胞色素P450在人體中的催化反應機制 …….……………………...….. 7
2.3 細胞色素P450的基因多樣性 (genetic polymorphism) 對藥物代謝的影響
- 以細胞色素P450 1A2為例 ………………………………...…………… 9
2.4 誘導與抑制細胞色素P450活性對於人體的影響 ……………...…….… 10
2.5 黃麴毒素 (aflatoxin B1) 和乙醯胺酚 (acetaminophen) 經過細胞色素
P450代謝後,細胞毒性的產生 ……………………………………...…… 12
2.6 細胞色素P450研究模式的探討 …………………………………….... 12
第三章 實驗材料與方法 ………………………………………………………... 23
3.1 實驗材料 ………………………………………..……………………..…. 23
3.1.1 化學品與試劑 ………………………………………...…………… 23
3.1.2 細胞色素P450基因 …………………………………..…………… 23
3.2 實驗方法 ……………………………………………………………...….. 23
3.2.1 重組慢病毒質體的架構 ……………..……………….…………… 23
3.2.2 細胞培養與轉染 (transfection) ……………….……...…………… 24
3.2.3 重組慢病毒的製備 …………………………………..……………. 24
3.2.4 重組慢病毒的濃縮 …………………...…………………………… 25
3.2.5 病毒效價 (viral titer) 的測定………………………..……………. 25
3.2.6 病毒轉導 (transduction) 與穩定表現細胞色素P450基因之細胞轉
殖株的建立 …………………………………………..………….… 27
3.2.7 西方墨點轉漬法 (western blot) ……………………..……….…… 27
3.2.8 細胞微粒體的製備 …………………………………..……………. 28
3.2.9 連續波長雙掃描盤式螢光暨UV/Vis分析儀 (SpectraMax M2) 測量
完整細胞之細胞色素P450活性 …………………………….……. 28
3.2.10 流式細胞儀偵測細胞內螢光值 ………………………………..... 29
3.2.11 藥物篩選與細胞活性測定 (MTS分析) …………………….....… 29
3.2.12 評估高速藥物篩選的統計學參數 - S/N、S/B和Z’ factor .....…… 30
3.2.13 細胞色素P450 1A2活性抑制分析 ……………………………. 30
第四章 實驗結果 ………………………………………………………………... 33
4.1 細胞色素P450重組類慢病毒質體的架構與病毒顆粒的製備 ……….. 33
4.1.1 細胞色素P450質體的架構 …………………………………..…… 33
4.1.2 細胞色素P450多株抗血清 (polyclonal antisera) 的製備 ………. 33
4.1.3 細胞色素P450蛋白質的生成確認 ………………………...…….. 34
4.1.4 類慢病毒顆粒的濃縮 ………………………………………..….… 34
4.1.5 短暫重組質體轉染後,細胞色素P450酵素活性偵測 ………..… 35
4.1.6 細胞色素P450還原酶和細胞色素b5對細胞色素P450 1A2和3A4
酵素活性的影響 …………………………………………….…..… 36
4.2 穩定表現細胞色素P450細胞株的建立 ……………………….……….. 37
4.2.1 利用短暫轉染Huh7細胞,評估慢病毒雙效 (bi-cistronic) 表現載體
的功能 …………………………………………………………… 37
4.2.2 穩定表現細胞株的建立 (Huh7-1A2-I-E、Huh7-2E1、Huh7-3A4-I-E
和Huh7-E細胞株) ………………………………………………..... 38
4.2.3 穩定表現細胞色素P450和綠螢光的Huh7細胞株的細胞特性比
較 …………………………………………………………………. 38
4.2.4 評估在穩定表現細胞色素P450的Huh7細胞中,已知的毒性代謝
物所造成的細胞毒性 …………………………….……………….. 40
4.3 細胞色素P450藥物毒性篩選系統的建立 ……………………………. 41
4.3.1 具有生物活性的化合物經過細胞色素P450 1A2代謝所造成的細胞
毒性 …………………………………………………………...…… 41
4.3.2 經細胞色素P450 1A2代謝後,代謝產物造成細胞毒性的未知化合
物 (thimerosal和evoxine) 的發現 ………………………..…….. 42
4.3.3 經細胞色素P450 2E1或3A4代謝的抗癌藥物,對細胞活性的影
響 …………………………………………………………………... 42
4.3.4 未知的細胞色素P450 1A2抑制劑 (inhibitors) 的發現 ………. 43
4.4 穩定雙效表現細胞色素P450細胞株的酵素活性提升 ………………. 44
4.4.1 Huh7-1A2-I-E和Huh7-3A4-I-E細胞微粒體與人類肝臟微粒體,在
細胞色素P450 1A2和3A4酵素活性的比較……………………. 44
4.4.2 探討桿狀病毒IE2 (immediate-early gene 2) 和hr1 (homologous
region 1) 對細胞色素P450 1A2酵素活性的影響 ………….……. 44
4.4.3 組織蛋白去乙醯基酶抑制劑 (histone deacetylase inhibitors,HDACi)
對細胞色素P450 1A2酵素活性的影響 ………………………..… 45
第五章 研究討論與結論 ………………………………………………………... 70
第六章 未來展望 ……………………………………………………………...… 77
參考文獻 ………………………………………………………………………...… 80


圖 目 錄
圖 1 藥物經過細胞色素P450代謝的結果 ……………………..……………… 4
圖 2.1 酵素催化市售藥物的比例 ………………………………….………..…. 19
圖 2.2 美國200個處方藥排出體外的路徑與催化代謝的酵素 ………………. 19
圖 2.3 基本的細胞色素P450催化反應循環 (catalytic cycle) …………………. 20
圖 2.4 黃麴毒素經細胞色素P450代謝後,代謝產物與DNA結合形成的加合物
(adduct) …………………………………………………..……………… 20
圖 2.5 乙醯胺酚在肝臟中的代謝 ………………………………..….…………. 21
圖 2.6 間葉幹細胞經過誘導分化成類肝細胞後,其肝細胞特性比較 …….…. 22
圖 4.1 重組慢病毒表現質體架構圖 …………………………………………… 49
圖 4.2 細胞色素P450的polyclonal antisera對於細胞色素P450微粒體的免疫測
試 …………………………………………………………………….…. 50
圖 4.3 細胞色素P450的多株抗血清對於胞內細胞色素P450蛋白質的免疫測
試 ............................................................................................................... 51
圖 4.4 293FT細胞短暫轉染 (transient transfection) 細胞色素P450 1A2或3A4
質體後的綠螢光表現圖 …………………………………………...…… 52
圖 4.5 短暫轉染細胞色素P450基因 (CYP1A2、CYP2E1和CYP3A4) 的293FT
細胞代謝反應受質,其代謝產物生成速率 ………………...………… 53
圖 4.6 於293FT和Huh7細胞中比較細胞色素P450 1A2的酵素活性 ……….. 54
圖 4.7 細胞色素P450還原酶對細胞色素P450 1A2和3A4微粒體的影響 ….. 54
圖 4.8 Huh7細胞短暫轉染細胞色素P450 1A2質體後的綠螢光表現圖與酵素活
性測量 ……………………………………...…………………………… 55
圖 4.9 穩定表現細胞色素P450的細胞株,其酵素活性和蛋白質表現的測量 . 56
圖 4.10 穩定表現細胞色素P450 1A2和綠螢光的Huh7細胞株的細胞特性比較
…………………………………………………………………………. 58
圖 4.11 穩定表現細胞色素P450細胞株處理已知毒性代謝物後,代謝產物對細
胞活性的影響 …………………….…………………………………… 61
圖 4.12 藥物經細胞色素P450 1A2代謝後,代謝產物造成細胞毒性的相關評估
………………………………………………………………………… 63
圖 4.13 藥物經細胞色素P450代謝後,代謝產物對細胞活性的影響 ……… 65
圖 4.14 利用完整細胞,篩選細胞色素P450 1A2酵素活性抑制劑 ………..… 66
圖 4.15 穩定表現細胞色素P450的細胞株,其細胞微粒體與人類肝臟細胞微粒
體在酵素活性與細胞色素P450蛋白質表現的比較 ….……....………. 67
圖 4.16 昆蟲桿狀病毒的hr1和IE2對細胞色素P450 1A2酵素活性的影響 .... 68
圖 4.17 Valproic acid (VPA)對Huh7-1A2-I-E細胞株的細胞色素P450 1A2酵素
活性和蛋白質表現的影響 ………….......…………………..…..……… 69

表 目 錄
表 2.1 體內藥物經過Phase I酵素和PhaseII酵素的代謝反應 ………...……… 16
表 2.2 人體器官表現細胞色素P450基因的分類 ………………....………..…. 16
表 2.3 細胞色素P450的反應受質 (substrates)、誘導劑 (inducers) 和抑制劑
(inhibitors) ………………………………………………………...…..…. 17
表 2.4 目前細胞色素P450研究系統的優缺點 ………………………………. 18
表 3.1 引子名稱、genebank accession number與序列 …………………………. 31
表 3.2 細胞色素P450的多株抗血清 (polyclonal antisera) 使用之胜肽 (peptide)
序列 ………………………………………………………………….…. 31
表 3.3 細胞色素P450活性偵測所使用的反應受質、代謝產物 (metabolite) 之激
發/偵測 (Ex/Em) 波長設定值 ………………………………..…...……. 32
表 3.4 Z’ factor 計算公式以及相對值的統計學意義 ………………..……… 32
表 4.1 超高速離心法與PEG-it Virus precipitation方法對類慢病毒顆粒濃縮後,
所偵測的病毒效價 …………………………………………………….. 47
表 4.2 篩選得到的細胞色素P450 2E1代謝物,其代謝產物對細胞活性的影
響 ……………………………………………………………………….. 47
表4.3 篩選得到的細胞色素P450 3A4代謝物,其代謝產物對細胞活性的影
響 ………………………………………………………………………... 47
表4.4 新穎和已知的細胞色素P450 1A2抑制劑 …….……………………….... 48


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