根據行政院衛生署統計，肝癌為台灣男性十大癌症死因的第一位，女性的第二位。本研究室先前根據 DNA 微矩陣資料的探勘 (data-mining) 與分析，發現 STMN1 基因在肝臟腫瘤細胞中有高度表現的情形。本研究主要探討 STMN1 基因表現量與肝臟腫瘤細胞以及其他癌症細胞株的相關性。人類 STMN1 基因全長為 1.5 Kbp，位在染色體 1p35-p36.1，轉譯出的蛋白質分子為 18 KDa。先前的研究指出， STMN1 蛋白質主要為促進微小管 (microtubule) 的分解，且在細胞週期時，促進紡錘絲 (spindle) 的組成。另外，也有研究指出，STMN1為一輔因子，可調控血管新生、細胞移動或是細胞侵犯的能力。在肝癌開始癌化或是癌化的過程中， STMN1 基因所扮演的角色仍不明確。在此試驗中，我們首先藉由即時定量反轉錄聚合酵素連鎖反應 (quantitative RT-PCR) 分析，建立肝腫瘤細胞株與其他癌症細胞株中 STMN1 mRNA 的表現量變曲線 (expression profile)。為進一步了解在肝腫瘤病患之腫瘤組織中，與其相對應正常肝臟組織基因體中STMN1 之基因體 (genomic STMN1) 是否有擴增 (amplification) 與否以及 mRNA 與蛋白質表現的情形，本研究收集台南奇美醫學中心 (Chi-Mei Medical Center, Tainan) 的 62 對樣本進行分析。在細胞株的試驗中，六個肝癌細胞株：HA22T、HCC36、Hep3B、Huh7、Malaru、Sk-hep1，一個乳癌細胞株：BCM1，以及一個胃癌細胞株：SCM1，其 STMN1 mRNA 均有高度表現。在肝組織試驗中， STMN1 mRNA 的表現量在 47對肝癌組織中明顯比正常肝臟組織高 (p<0.01)。此外，在 27 對肝癌組織中， STMN1 基因體有擴增的情形。因此接著，利用西方點墨法 (Western blotting) 來分析 STMN1 蛋白質在肝癌組織以及正常組織中的表現，結果發現在 30 對肝臟樣本中 STMN1 蛋白質的表現在癌組織比正常組織還來的高。此結果與即時定量 PCR 的結果有 84% 一致性。我們進一步從肝腫瘤細胞株 Sk-hep1 中選殖出 STMN1 基因完整的 cDNA (CDs)，並將選殖出來的 STMN1 cDNA 轉接入可表現蛋白質的不同載體上以表現蛋白質並進行細胞轉染 (transfection)，藉以檢視 STMN1 蛋白質在 HeLa與Sk-hep1 細胞中表現的次級位置 (subcellular localization)。經由免疫螢光染色的技術，我們發現 STMN1 蛋白質主要表現在細胞核與細胞質的位置，與先前報導 STMN1 蛋白質只表現在細胞質不同。另一方面，在肝腫瘤細胞株 Sk-hep1中有 7% 的細胞， STMN1 蛋白質僅表現在細胞核中。

Hepatocellular carcinoma (HCC) is now the first leading cause of male and the second of female cancer mortality in Taiwan. In a preliminary microarray data-mining, we identified that STMN1 was up-regulated in HCCs. This study was aimed to establish the STMN1 expression profiles in assorted cancer cell lines and HCC tissues. The STMN1 genomic DNA is 1.5 Kbp in length, mapped to the 1p36.1-p35 and encodes for an 18 KDa polypeptide in the human. STMN1 is a ubiquitous phosphoprotein that promotes microtubule catastrophe and spindle assembly during cell cycle. In addition, STMN1 protein is also a cofactor to regulate the angiogenesis, cell migration and invasion. The role of STMN1 in the onset or progression of HCC is still not clear. In this study, we firstly examined the STMN1 mRNAs expression profiles in several cancer cell lines and found its significant up-regulation in six HCC cell lines, HA22T, HCC36, Hep3B, Huh7, Malaru, SK-hep1, one breast tumor, BCM1 and one stomach tumor, SCM1. To further understand if any amplifications of genomic STMN1, the expression profile of STMN1 mRNA and STMN1 protein in HCC tissue specimens, 62 HCC tissues, in pair, that collected from Chi-Mei Medical Center (Tainan) were analyzed. Among 62 pairs of HCC tissues analyzed, 47 STMN1 mRNAs were significant higher in HCC tissues than in their normal counterparts. On the other hand, the STMN1 genomic DNA was amplification in 27 HCC tissues. Then, we have determined and compared the STMN1 protein in both HCC tissues and normal tissues by the Western blotting analysis. STMN1 protein expression was found to be higher in 30 tumor tissues than in their corresponding normal tissues. This result was 84% consistent with the quantitative RT-PCR results. Furthermore, STMN1 CDs were cloned from HCC cell line (Sk-hep1) and subcloned into various protein expression vectors for further examining STMN1 subcellular localization in HeLa and Sk-hep1 cell lines. The STMN1 protein locates in both cytoplasm and nucleus by immunofluorescence analysis. This result was different from the previously report that STMN1 located in cytoplasm. Furthermore, there were 7% of Sk-hep1 cells which STMN1 proteins were only located in nucleus.

Chinese Abstract………………………………………………………...Ⅰ
Abstract………………………………………………….………………Ⅲ
Abbreviations………………………………………………………...…Ⅴ
Introduction……………………………………………..…….………….1
Materials and Methods…………………………………….………..……9
Results……………………………………………….…………….……19
Discussion……………………………………………..………………...41
References……………………………………………………………....48
Appendix……………………………………………………………..…54

行政院衛生署 (2004) 台灣地區主要癌症死亡原因。
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