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博碩士論文 etd-0731103-110240 詳細資訊
Title page for etd-0731103-110240
論文名稱
Title
PTEN 基因傳送對肝癌細胞之效應
none
系所名稱
Department
畢業學年期
Year, semester
語文別
Language
學位類別
Degree
頁數
Number of pages
51
研究生
Author
指導教授
Advisor
召集委員
Convenor
口試委員
Advisory Committee
口試日期
Date of Exam
2003-06-06
繳交日期
Date of Submission
2003-07-31
關鍵字
Keywords
腫瘤致病性、肝癌、細胞死亡
hepatocellular carcinoma, cell motility, tumorigenicity, PTEN
統計
Statistics
本論文已被瀏覽 5645 次,被下載 7188
The thesis/dissertation has been browsed 5645 times, has been downloaded 7188 times.
中文摘要
肝癌(hepatocellular carcinoma,HCC)在世界是常見的癌症之一,肝癌的致病機轉,被認為是多因素和多步驟過程的機制。包含致癌基因的活化或是腫瘤抑制基因的不活化。腫瘤抑制基因PTEN(或稱MMAC或TEP1)位於人類染色體10q23的位置。PTEN為403胺基酸之蛋白質具有雙重去磷酸酵素活性,PTEN基因突變常發生於許多末期癌症。在我們先前以免疫組織染色分析的研究中,發現罹患肝癌病人的檢體中接近40%有PTEN表現減少的情形。因此,在PTEN不活化的Mahlavu肝癌細胞,以腺病毒載入PTEN基因恢復PTEN的表現方式進行in vitro 和 in vivo的研究。從研究的結果顯示PTEN基因傳送可以有效的抑制Mahlavu肝癌細胞致癌性。
在研究報告中,主要是想以PTEN基因傳送的方式在能有PTEN功能表現的肝癌細胞觀察是否有抑制腫瘤的特性,由PTEN的表現和基因定序分析,確認人類SK-Hep1肝癌細胞對PTEN基因傳送能夠有所表現。以腺病毒感染SK-Hep1肝癌細胞的最佳條件為multiplicity of infection 50~100(M.O.I)。由SK-Hep1細胞能有效傳達外送的PTEN基因,利用腺病毒載體外送PTEN基因增加PTEN表現的狀況下,並不會改變在SK-Hep1細胞中Akt-p的狀況,但是可以減少SK-Hep1細胞的增生約~20%。另外相對於對照組,經PTEN基因傳送的SK-Hep1細胞能顯示出有意義降低細胞的生成。利用西方墨點法分析可以推測經PTEN基因傳送的肝癌細胞移行數目的減少,可能是因降低了FAK磷酸化的緣故所致。經PTEN基因傳送的SK-Hep1肝癌細胞在瓊脂膠上也可以發現細胞群落形成的減少。但經PTEN基因傳送的SK-Hep1肝癌細胞卻不會影響到MMP( matrix metallo-proteinase )的釋放。而這些in vitro實驗發現的結果仍有待未來做動物實驗來確認。總結上是希望利用PTEN基因傳送的方式在有PTEN功能肝癌細胞中提供一個有效治療肝癌的方法。
Abstract
Hepatocellular carcinoma (HCC) is one of the most prevalent cancers worldwide. Hepatocarcinogenesis is considered a multifactorial and mulitstep process that involves the activation of oncogenes or the inactivation of tumor suppressor genes. Tumor suppressor gene PTEN (also known as MMAC or TEP1) is located on human chromosome 10q23. The 403–amino acid PTEN protein encodes dual specificity protein phosphatases. Mutation of the PTEN is a common event in advanced stage of diverse human cancers. In our previous studies, immunohistochemical analysis indicated that reduced PTEN expression was found in nearly 40% of HCC specimens. Furthermore, restored PTEN expression by adenovirus gene delivery effectively inhibited the in vitro and in vivo tumorigenicity of Mahlavu cells, a human HCC cell line with PTEN inactivation. In the present study, we further characterize whether PTEN gene delivery still suppressed the oncogenic potential in HCC cell lines with functional PTEN. By expression and sequencing analysis, we identified human SK-Hep-1 cells as the hepatoma cell line with functional PTEN expression. The optimal condition for adenovirus vector to infect SK-Hep-1 cells was determined at the multiplicity of infection (MOI) of 50-100. Tough SK-Hep-1 cells were effectively transduced with exogenous PTEN gene, the enhanced PTEN expression by adenovirus gene delivery did not alter the phosphoryation extent of Akt in SK-Hep1 cells. Nevertheless, PTEN gene delivery reduced the proliferation of SK-Hep-1 cells by ~20%. In addition, the motility of PTEN-transduced SK-Hep-1 cells significantly decreased comparing to cells of control groups. Western blot analysis suggested the decreased cell motility might be attributed to the reduced phosphorylation of focal adhesion kinase (FAK) by PTEN gene delivery. Above all, PTEN gene delivery profoundly reduced the colony formation of SK-Hep-1 cells in soft-agar. However, PTEN gene delivery did not affect the secretion of matrix metallo-proteinases (MMPs) release. Animal studies will be carried out in the future to validate the present in vitro findings. In summary, PTEN gene delivery holds promise for treatment of HCC even when the hepatoma cells possess functional PTEN gene.
目次 Table of Contents
目錄 1
中文摘要 4
英文摘要 6
介紹 9
一、肝癌 9
二、腫瘤抑制基因PTEN 9
三、肝癌與腫瘤抑制基因PTEN 10
四、實驗背景 11
五、實驗目的和設計 12
材料和實驗方法 13
一、細胞培養 13
二、cDNA定序分析 13
三、細胞數目之測定 14
四、Gelatin zymorgraphy 15
五、細胞移行分析-chemotaxis assay 15
六、蛋白質濃度分析 16
七、蛋白質電泳 17
八、細胞增生分析 17
九、細胞群落形成分析 18
十、西方墨點法 18
結果 18
SK-Hep1 肝癌細胞株PTEN基因序列的測試 19
測試腺病毒感染SK-Hep1肝癌細胞之最佳條件 19
Western blot 確認PTEN於SK-Hep1肝癌細胞的表現 19
PTEN基因傳送對SK-Hep1肝癌細胞之增生效應 20
PTEN基因傳送對SK-Hep1肝癌細胞之移行效應 20
PTEN基因傳送對SK-Hep1肝癌細胞之致癌性表現 21
PTEN基因傳送對SK-Hep1肝癌細胞釋放MMP9之影響 21
PTEN基因傳送對MAPK/Erk之表現 22
討論 22
參考文獻 29
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