臺灣博碩士論文加值系統

肝細胞癌（Hepatocellular carcinoma、HCC）在亞洲及非洲屬於相當常見的癌症，而在台灣也已經連續數年蟬聯十大癌症致死因第一位。近年來雖然對於其診斷與治療方式有所進展，然而病患之長期預後並未獲得明顯的改善；若能加強對肝細胞癌化之分子生物學機轉的瞭解，設法在癌化早期步驟就能夠偵測並且加以阻斷或撲殺，將能進一步減少肝癌的發生及危害。
端粒酵素(Telomerase）是一種作用於染色體端粒的核醣核酸蛋白，人類的端粒酵素由二個主要部份構成：（1）端粒酵素核醣核酸（human telomerase RNA、hTR），（2）端粒酵素反轉錄次級結構（human telomerase reverse-transcriptase subunit、hTERT），兩者均已在最近被定序完成。其作用方式為利用結合蛋白與染色體DNA尾端3’處結合，藉著hTR RNA序列（CCCUAA）為模板，經由反轉錄酵素來進行端粒的延長。能將逐漸縮短之DNA端粒序列補足，以防止細胞老化死亡 (senescence)。在人體內除了生殖細胞、幹細胞外，端粒酵素罕見於正常體細胞中，然而在大於百分之九十的惡性腫瘤，如乳癌、胃癌、子宮頸癌、肝癌、肺癌、前列腺癌等均已證實其存在。本論文之目的旨於探討在肝癌上端粒活性之相關調控機轉。
我們首先偵測25個肝癌組織標本，其中21個（84％）表現出不同強度的端粒酵素活性，而在相同樣本之非腫瘤組織上，僅有五個表現出較弱的端粒酵素活性。基於端粒酵素與細胞分裂有著密不可分的關係，接著我們利用化學藥物將肝癌細胞株（J5）及非惡性之肝細胞株（Chang-Liver，nonmalignant immortalized）進行細胞週期同步化，以分析細胞週期與端粒酵素活性的關係，結果發現在J5與C-L細胞株中，其端粒酵素活性於四個週期皆有表現，而又以M週期表現量最高。再利用西方點墨法（Western blot），分析細胞週期調控因子與端粒酵素活性的相關性，實驗發現除了p21外，其他細胞週期調控因子之蛋白質表現量，與端粒酵素活性並無明顯關聯性。然後我們以反轉錄聚合酵素連鎖反應（Reverse Transcriptase Polymerase Chain Reaction）分析端粒酵素組成部分hTR、hTERT之mRNA表現量與端粒酵素活性的關係，結果顯示hTR在四個週期皆有表現，而hTERT在J5細胞株中，四個週期皆有表現，且於M週期表現量最高，與端粒酵素活性呈一正向關係，至於C-L細胞株中，其hTERT在四個週期的表現量則皆相似。由實驗結果我們發現在肝癌上，腫瘤與非腫瘤組織之間端粒酵素活性表現出明顯的不同，可以視為一項腫瘤標記。在細胞週期調控因子中，以p21與端粒酵素活性最具有關聯性，同時肝癌細胞株中端粒酵素活性與hTERT mRNA在整個細胞週期都有表現，而以M週期表現量最高，有著正向的關係，顯示出轉錄層面（Transcription Level）在端粒酵素活性的調控佔有重要的角色。
為了解決手術取得之肝癌組織，其成份大多是異質性，混雜著肝硬化或發炎部分，我們利用原位雜交染色法（in-situ hybridization），準確地偵測肝癌組織切片中hTERT mRNA表現的量及細胞位置，將探討方向放在轉錄層面上，並且與c-Myc蛋白質表現加以比較，分析其在肝癌癌化上的佔有之地位。然而由c-Myc組織免疫染色結果與hTERT mRNA之表現，無法確認之間的相關性，於是進一步藉建立長度不等的hTERT 啟動子表現載體建購物，利用轉染（transfection）方式，以探討端粒酵素活性其cis-element 與trans-acting之間的調控機制；，並進一步分析hTERT啟動子之sequence，發現在肝癌組織中端粒酵素活性與c-Myc的調控關係並非如此單純，c-Myc與hTERT之間的調控，可能只是質（即開或關－on or off）而非量（多寡）的關係。

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, with its highest prevalence occurring in Asia and Africa. Despite some progress in its clinical management in recent years, HCC remains a disease having a poor prognosis. It is important to improve the early diagnosis and treatment strategy of HCC by better understanding the molecular events involved tumor development and progression.
Telomerase, a ribonucleoprotein with two components required for core enzyme activity: telomerase RNA (hTR) and a telomerase reverse transcriptase protein (hTERT). It compensates for the continued shortening telomere length by adding hexameric (TTAGGG) repeat to the telomeric ends of the chromosomes. Telomerase activity (TA) is expressed in almost all malignant tumors, but is rarely detected in normal somatic cells.
At first stage, we analyzed the TA by PCR-based telomerase repeat amplification protocol assay in the tissues of 25 HCC. The fraction of telomerase positive tumors was 84 % (21/25) and only five non-tumorous tissues had very weak TA. To explore the possible regulatory mechanisms of telomerase, we examined the TA, expression of human telomerase RNA (hTR), human telomerase reverse transcriptase (hTERT) mRNA isoforms and cell cycle modulators in HCC cell lines (J5) and a normal human immortalized hepatic epithelial cell line (Chang-liver). The cell lines were chemically synchronized in G1, G1/S, G2/M or M phases. The hTR and hTERT mRNA levels were analyzed by reverse transcriptase polymerase chain reaction. Western blotting was used to assay the cell cycle modulators.
The TA of J5 and Chang-liver cell lines tested was highest in M phase.The expression level of hTERT mRNA associated with the highest TA detected in the M phase of HCC cell lines. Chang-liver expressed markedly less TA and hTERT mRNA than J5. The elevated TA and expression of hTERT mRNA in M phase of HCC cell lines did not significantly correlate with that of the cell cycle modulators except p21.
There is significant different expression level of TA between tumor part and non-tumor part of HCC that suggests the analysis of TA in specimens might contribute to assessment of the severity of HCC. The highest TA in the M phase suggested that there were other unidentified factors which controlled the progressive cell cycle of tumor and might be correlated with the telomerase activity. Moreover, the result implicates that regulation of TA is related to hTERT mRNA isoform expression.

目 錄
目錄 I
表目錄 III
圖目錄 IV
中文摘要 VI
英文摘要 X
緒論 01
實驗材料與方法 12
壹、肝癌組織標本的收集 12
貳、細胞株培養 12
參、端粒活性的測定 15
肆、端粒相關基因hTR、hTERT 在不同週期之表現 21
伍、西方點墨法：細胞調節因子的檢測 25
陸、影像密度分析 27
柒、RNA原位雜交染色法 28
捌、免疫組織化學染色 36
玖、啟動子分析 37
實驗結果 42
壹、端粒活性之測定結果 42
貳、化學藥物細胞週期同步化結果 47
參、偵測hTR、hTERT在不同週期之表現結果 58
肆、檢測不同細胞週期中細胞調控因子之表現結果 61
伍、hTERT mRNA原位雜交染色法結果 64
陸、啟動子分析結果 69
討論 71
結論 91
參考文獻 94
表 目 錄
表1、 真核細胞生物之端粒重複序列 04
表2、 肝癌標本之基本資料與端粒活性 43
表3、 腫瘤大小與端粒活性之關係 47
表4、 病毒標記與端粒活性之關係 47
表5、 肝癌標本端粒活性與hTERT mRNA表現量的比較 64
表6、 肝癌標本hTERT mRNA表現量與肝癌分化程度的比較 65
表7、 肝癌標本之hTERT mRNA與c-Myc兩者表現量的比較 66
表8、 肝癌標本之c-Myc與肝癌分化程度的比較 66
表9 不同固定方式會影響c-Myc之組織免疫染色表現位置 86
圖 目 錄
圖1、 DNA末端複製問題 04
圖2、 端粒假說與少數特例 05
圖3、 端粒之作用方式 07
圖4、 TRAP方法原理 08
圖5、 端粒反轉錄次級結構要素（hTERT motifs） 24
圖6、 端粒活性電泳膠片結果 45
圖7、 端粒活性電泳膠片結果 46
圖8、 化學藥物對肝癌細胞株之毒性測試 49
圖9、 J5細胞株在化學藥物處理下之細胞同步化 50
圖10、 C-L細胞株在化學藥物處理下之細胞同步化 51
圖11、 J5細胞在不同蛋白質濃度中之端粒活性表現 53
圖12、 影像密度分析不同濃度蛋白質中之端粒活性表現 54
圖13、 J5及C-L細胞受不同藥物處理之端粒活性影響 55
圖14、 J5及C-L細胞在不同細胞週期中端粒之活性 57
圖15、 影像密度分析不同細胞週期之端粒活性表現情形 58
圖16、 端粒hTR mRNA於不同細胞週期之表現 59
圖17、 端粒hTERT1 mRNA於不同細胞週期之表現 60
圖18、 端粒hTERT2 mRNA於不同細胞週期之表現 60
圖19、 細胞週期調控因子於不同細胞週期之表現 62
圖20、 影像密度分析細胞週期調控因子於不同細胞週期之表現 63
圖21 肝癌標本之hTERT mRNA原位雜交染色結果 67
圖22 肝癌標本之hTERT mRNA與c-Myc表現量之比較 68
圖23、 肝癌細胞株螢光活性的偵測 69
圖24 不同長短端粒啟動子建購物螢光活性的比較 70

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