臺灣博碩士論文加值系統

人類去毒作用之機轉有三階段：第一時期是氧化 (oxidation) 作用，主要是 cytochrome p450 催化毒物或藥物呈氧化型以利於結合作用。第二時期是結合 (conjugation) 作用，主要是葡萄糖醛酸轉移使氧化的毒（藥）物具水溶性以利於排除。第三時期是運送 (transportation) 作用，由多種運送蛋白把毒（藥）物運送至肝臟或腎臟或膽汁，以進行代謝或排除。在這三種去毒作用當中，第一時期的氧化作用是被研究最多的，而目前的研究結果顯示第二時期的結合作用和第三時期的運送作用在去毒過程中扮演著把關的角色。UDP-Glucuronosyltransferase (UGT) 是一群多基因的酵素家族，主要是執行結合作用 (conjugation)，是肝臟細胞特有的酵素也是 Phase II 藥物代謝中重要的一員。已知遺傳性的 UDP-Glucuronosyltransferase (UGT) 基因變異，會影響致癌物的代謝，但 UGT 的基因變異是否與肝癌的發生具關聯性，目前仍不清楚。文獻報告指出正常的 UGT1A1 promoter 區域包含六個 TA 核甘酸重複序列，但在膽紅素代謝異常的病人中，則 TA 核甘酸的數目會發生改變，有 TA 5-8 repeats 的變異情形發生。為了解 UGT1A1 的變異是否跟肝癌有關，我們利用 PCR-RFLP 和 Cloning 技術，觀察肝癌細胞株 UGT1A1 promoter 區域和 UGT1A1 G71R (nt 211G→A) 的位置是否有基因變異。結果顯示，Hep3B 肝癌細胞株只含有四個 TA repeats 的變異情形發生，代表肝癌細胞株中 UGT1A1 promoter 的 TA 序列確實有產生變異。另外利用 RT-PCR 比較各種不同的人類肝癌細胞株 UGT1A1 之 mRNA 的表現量，結果發現肝癌細胞株中 UGT1A1 mRNA 的表現量明顯降低。綜合上述的結果 UGT1A1 promoter 區域的 TA 重複序列是否有多型性是很重要的，會直接影響 UGT1A1 基因的表現量。而在 Phase III 運送蛋白中的有機陰離子運輸蛋白 (organic anion transporter 2，OATP2) 是位於肝細胞的 basolateral membrane 處，其作用是可將血液中的有機陰離子 (organic anion) 運送至肝細胞內，其中就包括了一些內因性和外因性毒物。迄今，只有國外曾報導過 OATP2 在基因編碼過程中會呈現多型性，而引起運輸功能的喪失。為了解 OATP2 基因多型性和差異的表現與肝癌之間的關係，我們先利用 RT-PCR 把 OATP2 全長 cDNA 選殖出來，結果發現 OATP2 具有多個不同的剪接形式。定序結果，證實 OATP2 RNA 裡的剪接位置，HA22T/VGH 肝癌細胞株有最多的 OATP2 剪接形式。HepG2 肝癌細胞株不表現 OATP2 RNA，已經有文獻報告過相似的結果。然而，我們首先發現肝癌細胞 Mahlavu 不表現 OATP2 RNA。目前在全部 22 位 HCCs 病患內，包括 peritumorous non-neoplastic liver tissues (N) 和 HCC tissue (T) 樣品中，有九個有剪接情形發生，peritumorous non-neoplastic liver tissues (N) 和 HCC tissue (T) 互相比較，有八個 OATP2 的 mRNA 是明顯減少的，所以我們認為這些 OATP2 的剪接形式在這些肝癌裡是扮演一個很重要的角色。

The three phases of detoxification are oxidation (phase Ι), conjugation (phase II), and transportation (phase III). The oxidation reaction is mainly catalyzed by cytochrome P (CYP) 450, while UDP-glucuronosyltransferase (UGT) is the major enzyme in the conjugation reaction, with a number of proteins responsible for the transportation of toxic metabolites. Of these three detoxification phases, oxidation has been mostly studied, while conjugation and transportation are potential fields of research in the 21st century. UDP-Glucuronosyltransferase (UGT), exists as a superfamily enzymes, have shown glucuronidation activity which represents a major pathway in phase II drug metabolism. To date, inherited variations in genes involved in the metabolism of carcinogenesis are suggested to be associated with an increased risk of liver cancer. Recently, four UGT1A1 variations in the number of TA from five through eight repeats in the atypical TATA box region have been described. In order to figure out whether the genetic variation at UGT1A1 locus associated with liver cancer, we performed PCR-RFLP and cloning sequencing to determine the genetic variation in the UGT1A1 promoter region and nucleotide 211 site in liver cancer cell lines. Surprisingly in this study, we first report four TA repeats in the A (TA) nTAA motifs in the Hep3B liver cancer cell line, whereas six TA repeats characterize the common allele (UGT1A1*1) in other liver cancer cell lines. However, liver cancer cell lines in this study were found no polymorphism in nucleotide 211 site. Furthermore, we first investigated the expression of UGT1A1 in human liver cancer cell lines by RT-PCR. The result revealed that UGT1A1 express significantly lower than other liver cancer cell lines. Our current findings suggest that genetic polymorphism of TA repeats in the UGT1A1 promoter region is important for the regulation of UGT1A1 expression in the tumor cell. The organic anion transporter OATP2 is involved in the hepatocellular uptake of a variety of endogenous and xenobiotic substances and drugs. So far, little is known about polymorphisms in genes encoding uptake transporters and no information has been available on mutations in uptake transporter genes causing a loss of transport function. To assess the relationship between OATP2 polymorphism and expression with liver cancer, we initially performed RT-PCR to clone full-length OATP2. Unexpectedly, several splicing forms of OATP2 were originally found in this work. After sequencing, we identified the splicing sites in OATP2 RNA. Moreover, HA22T/VGH liver cancer cell line contains more splicing forms of OATP2. However, OATP2 RNA expression is not detected in HepG2 liver cancer cell line. The similar results were reported by other groups. Otherwise, we firstly found another liver cancer cell line Mahlavu neither expression OATP2. OATP2 RNA expression was present in peritumorous non-neoplastic liver tissues in all the 22 HCCs patients, while it was strongly reduced even absent in 8 in the corresponding HCC samples. There were 9 splicing forms of OATP2 observed in both peritumorous non-neoplastic and/or HCC tissues in the 22 patients. These data suggest that the splicing forms of OATP2 may play important role in liver cancer.

謝誌 i
縮寫表 (Abbreviations) iii
中文摘要 v
英文摘要 vi
第一章 簡介 (Introduction) 1
1.1文獻回顧 (Literature Review) 4
第二章 材料與方法 (Materials and Methods) 18
2.1材料 18
2.2實驗方法 20
2.2.1細胞培養 20
2.2.2解凍細胞 20
2.2.3冷凍細胞 20
2.2.4 Extraction of Cell Genomic DNA 21
2.2.5 PCR 22
2.2.6 DNA 電泳 22
2.2.7 PCR Product 濃縮與 DNA fragment elution 23
2.2.8 Transformation 24
2.2.9萃取 Mini-Plasmid 24
2.2.10 RNA Extraction 25
2.2.11 Reverse transcriptase polymerase chain reaction (RT-PCR) 26
2.2.12 PCR 26
2.2.13 RNA 電泳 27
2.2.14 Protein Extraction 27
2.2.15西方墨點法 (western blotting) 28
2.2.16 Immunofluorescence Staining 30
2.2.17 Immunohistochemistry 30
2.2.18 Differential Tissue Expression 31
2.2.19 Extraction of Tissue Genomic DNA 32
第三章 結果 (Results) 33
3.1 UGT1A1-211 polymorphisms 33
3.2 選殖不同分化程度之肝癌細胞株與正常肝細胞株的 UGT1A1 promoter 33
3.3 分析 UGT1A1 promoter 區域的變異是否會影響 UGT1A1 基因的表現差異 34
3.4 分析肝癌細胞株與正常肝細胞株其 UGT1A1 蛋白質的的表現差異 34
3.5 UGT1A1 在人類不同組織中的表現差異 35
3.6 分析肝癌病患肝組織切片與正常肝組織切片 UGT1A1 的表現差異 35
3.7 選殖肝癌病患肝組織切片與正常肝組織切片的 UGT1A1 promoter 35
3.8 選殖不同分化程度之肝癌細胞株與正常肝細胞株的 OATP2 36
3.9 不同分化程度之肝癌細胞株與正常肝細胞株的 OATP2 基因定序結果 36
3.10分析肝癌細胞株其 OATP2 蛋白質的表現差異 36
3.11 OATP2 在人類不同組織中的表現差異 37
3.12 分析肝癌病患肝組織切片與正常肝組織切片其 OATP2 的表現差異 37
3.13分析不同分化程度之肝癌細胞株與正常肝細胞株的 OATP2 表現差異以及在細胞中的表現位置 37
第四章 討論 (Discussion) 39
第五章 結論與展望 (Conclusion and Outlook) 46
第六章 參考文獻 (Reference) 47
第七章 圖表 (Figures) 64
附錄一 87
醫檢學會與期刊發表成果報告 88

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112.黃美真碩士論文，2003。台灣地區成年人未結合型高膽紅素
血症之基因變異型分析。