臺灣博碩士論文加值系統

根據2004年統計，乳癌 (breast Cancer) 是全世界女性最常見的惡性腫瘤，也是引起女性死亡癌症的第二名。而台灣地區，乳癌是女性十大死因的第四位，所以對於乳癌的發生、治療及術後情形的進一步了解，是刻不容緩的事情。
本實驗室多年來致力於乳癌的相關研究，尤其對於乳癌病患治療之後systemic recurrence的情形，更是我們探討的重點。在臨床上，乳癌病人接受乳房切除手術後，會分析乳房組織附近的淋巴結是否有癌細胞，來判斷病人術後復發的情形，根據本實驗室統計lymph node metastasis與 systemic recurrence關連性p值達0.00134，但lymph node negative的病人仍有6.4%的人會復發。由過去實驗室分析的結果顯示，p53 mutation和systemic recurrence有非常高的關連性，p值為0.00193，與臨床上以lymph node metastasis觀察systemic recurrence幾乎是相同重要，故p53 可以當作bio-marker輔助病人觀察術後復發的情形。但我們也發現有一些病人p53是正常的，但一樣會有術後復發的情形，因此才會引導我們想要更進一步探討p53 signaling pathway中相關分子，希望找其他的bio-marker來輔助觀察病人術後復發的情形。我們主要選擇了三個基因來分析，分別為TTK/hMps1、CHK2及Ramp/L2DTL，原因如下: CHK2是活化p53重要上游分子，而TTK/hMps1除了與細胞增生有關，最新研究更顯示此kinase可以活化CHK2;而Ramp/L2DTL也是與細胞增生有關的核蛋白，由Dr.許輝吉(台大病理科)之研究顯示，後者的過度表現與肝癌的轉移有關。希望藉由分析這三個基因，能找到輔助性的bio-marker幫助評估lymph node negative或p53正常但仍會術後復發的乳癌病人systemic recurrence的情形。
本研究利用定序分析法檢測台灣乳癌檢體中TTK/hMps1第1638nt至2645nt的變異情形，分析結果沒有發現TTK/hMps1的特殊變異﹐卻發現兩個polymorphism（Ｉ５２７Ｉ，Ｐ７８９Ｐ）;而利用semi–quantitative RT-PCR 的方法觀察乳癌檢體中TTK/hMps1表現量，經統計結果顯示TTK/hMps1高表現量與systemic recurrence有很強的關聯性(p值達0.0022)，且與p53 mutation關聯性也很高(p值達0.0001)，故TTK/hMps1表現量高時幾乎可以當作一個bio-marker來輔助臨床上觀察病人術後復發的情形。同樣利用semi–quantitative RT-PCR 的方法觀察乳癌檢體中Ramp/L2DTL表現量，結果顯示Ramp/L2DTL高表現量與systemic recurrence也有關(p值為0.0247)，但是與病人是否會術後復發的關聯性並沒有如TTK/hMps1表現量強烈。另外利用定序分析法觀察台灣乳癌檢體中CHK2核苷酸序列的變異情形，分析結果只發現CHK2 polymorphism (E84E)，由文獻已知CHK2有11種主要不同的splice variants，經分析後發現有較特殊的splice variants，但探討 CHK2 splicing異常是否與乳癌有關，尚待進一步分析。

Worldwide, breast cancer is one of the most common cancer among women, and is the second leading cause of cancer death in women as well. In Taiwan, breast cancer ranks fourth among the caures of overall female cancer-related deaths and the incidence of age in this disease tends to early onset. It is important to understand tumorgenesis and systemic recurrence in breast cancer.
Our laboratory has studied the breast carcinogenesis for few years. Lymph node metastasis is a bio-marker for poor prognosis in breast cancer. However, approximately 6.4% of lymph node negative patients that acquire systemic recurrence. We are particularly interested in setting up new supplemental bio-markers for systemic recurrence. According to the previous study, p53 mutation has shown a strong correlation with recurrence (p=0.00193). p53 is a supplemental bio-marker useful for clinical diagnosis as well as lymph node metastasis. Nevertheless, there are a few wild-type p53 patients that acquired recurrence. Therefore, we examine the status of other molecular components such as CHK2 and TTK/hMps1 in the p53 signaling pathway. Inaddition, we have also analyzed the status of Ramp/L2DTL, a nuclear protein Ramp/L2DTL has shown the correlation with early metastasis of hepatoma (personal communication with Dr. H-J Hsu, NTUH).
By sequence analysis, we identified two TTK/hMps1 polymorphism, Ｉ５２７Ｉand Ｐ７８９Ｐ. We determined the expression level of TTK/hMps1 by semi-quantitative RT-PCR. According to the statistical analysis, high expression level of TTK/hMps1 correlatd strongly both with systemic recurrence (p=0.0022) and p53 mutation (p=0.0001). TTK/hMps1 can be a supplemental bio-marker useful for clinical diagnosis. We also determined the expression level of Ramp/L2DTL by semi-quantitative RT-PCR. According to the resules of statistical analysis, high expression level of Ramp/L2DTL showns correlation with systemic recurrence (p=0.0247). We identified one CHK2 polymorphism (E84E) by sequencing analysis. According to the study by other groups, CHK2 with 11 major splice variants were detected in their tumor series. We have found some splice variants in breast cancer specimens. How the splice variants of CHK2 influence breast carcinogenesis remain to be addressed.

中文摘要 1
英文摘要 3

第一章 緒論 4
乳癌簡介 4
乳癌的病理組織分類 4
乳癌的分期 5
乳癌的治療 5
罹患乳癌之高危險族群成因探討: 6
本論文相關研究: 7
1. TTK/ hMps1蛋白激酶基因介紹 9
2. CHK2基因介紹 10
3.Ramp/L2DTL基因介紹 12
本論文研究目的 13

第二章 實驗材料與方法 14
實驗材料 14
一、實驗藥品 14
二、實驗儀器 15
三、乳癌細胞株 16
四、乳癌檢體 16
五、寡核苷酸引子 16


實驗方法 17
一、膠片電泳、製備及純化 17
1.1.5%洋菜膠(agarose gel)電泳分析與製備 15
2. 4%或6%非變性聚丙烯醯氨凝膠(natural PAGE)電泳分析及製備 18
3.自1.5%洋菜電泳膠片或4%非變性聚丙烯醯氨凝膠中純化DNA片段 19
二、實驗技術 20
1.反轉錄作用(Reverse transcription) 20
2.DNA聚合酶連鎖反應(DNA polymerase chain reaction ) 20
a) S26基因的RT-PCR analysis 20
b) TTK/hMps1基因的semi–quantitative RT-PCR analysis 21
c) TTK/hMps1基因的sequence analysis 21
d) CHK2基因的sequence analysis 21
e) CHK2基因的splice variants analysis 22
f) Ramp/L2DTL基因的semi–quantitative RT-PCR analysis 23
3.核苷酸序列分析(Direct DNA sequencing method) 24
三、生物統計 25

第三章 結果 26
1. 序列分析(sequence analysis)TTK/hMps1的第521胺基酸至第857胺基酸，發現台灣地區特有的polymorphism 26
2.在乳癌檢體中TTK/hMps1的表現量與幾個乳癌相關的重要因子有關 27
3.序列分析(sequence analysis)CHK2的第762nt至2390nt(即為coding region)，發現CHK2的一個polymorphism 28
4.在乳癌檢體中發現CHK2基因中insZ、del7、del4及sub3四種splice variant form出現的比例最高，且CHK2表現與癒後復發有關 29
5.在乳癌檢體中Ramp/L2DTL的表現量與幾個乳癌相關的重要因子有關 30

第四章 討論 32
第五章 圖表 36
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