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.

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