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研究生:蔡雅婷
研究生(外文):Ya-Ting Tsai
論文名稱:探討胸腺素β4基因在人類大腸直腸癌過量表現之機轉
論文名稱(外文):Elucidation of the mechanisms of Tβ4 upregulation in human colorectal carcinoma
指導教授:蘇瑀蘇瑀引用關係
指導教授(外文):Yeu Su
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生物藥學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:45
中文關鍵詞:大腸直腸癌胸腺素β4拷貝數甲基化
外文關鍵詞:colorectal cancerTβ4copy numbermethylation
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Thymosinβ-4 (Tβ4)為胸腺素家族成員,為一含有43個胺基酸的酸性胜肽,分子量約為5 kDa,廣泛地分佈於各組織及細胞內。在細胞內,Tβ4主要的功能是隔絕肌動蛋白(G-actin),其以1:1方式與G-actin形成複合物,而抑制G-actin聚合形成細胞骨架。近來的研究指出Tβ4的過量表現與大腸癌的惡化及遠端轉移有關,且在大腸癌患者也發現Tβ4 mRNA的量在轉移到肝臟的組織比原發組織高。本研究利用組織免疫染色發現在12位大腸癌患者中有11位其癌組織中有Tβ4高度表現的情形。由12位患者之正常與大腸癌組織抽取genomic DNA後,分別用Tβ4 X-specific引子進行聚合酶鏈鎖反應(PCR)分析或將其以限制酵素EcoR I和Pst I完全切割後,以Tβ4插入序列1為探針進行南方墨點分析(Southern blotting),發現約有50%患者其大腸癌組織之T��4基因拷貝數較正常組織高,但並無基因轉位(translocation)發生。我們也利用bisulfite修飾-PCR及定序比對,分析了8位大腸癌患者正常及癌組織中Tβ4基因的近端啟動子(-183 to +81) 及插入序列1(+344 to +766)內區域具CpG island之區域之甲基化狀況,發現啟動子區域未被甲基化,而插入序列1在3位患者的癌組織中呈高甲基化,但在1位患者呈低甲基化狀態。而以去甲基試劑5-aza-C及histone去乙醯酶抑制劑trichostatin A處理大腸直腸癌細胞株HT-29,會些微促進Tβ4的表現。有趣的是,利用報告基因(reporter gene)分析,發現插入序列1能明顯降低長度為1.5Kb之Tβ4啟動子的活性,顯示其上可能有silencer之存在。綜合以上結果,大腸癌患者之癌組織中Tβ4的過量表現應與基因拷貝數增加及其插入序列1甲基化修飾有關。
Thymosinβ-4 (Tβ4), a widely distributed small acidic polypeptide, is identified as the major G-actin sequestering protein which regulates cell shape and motility by modulating the polymerization of G-actin. Our previous work has demonstrated that Tβ4 overexpression is associated with increased invasion of SW480 colon cancer cells and the distant metastasis of human colorectal carcinoma (CRC). In the present study, by immunohistochemical staining, we found that ��90% CRC patients have Tβ4 overexpression in their tumor tissues. To elucidate the underlying mechanisms of Tβ4 upregulation in human CRC, the copy number of Tβ4 in tumor and normal tissues of 12 CRC patients was determined by PCR analysis and Southern blotting, and the latter also disclosed the arrangement of this gene. Although higher Tβ4 copy number was found in tumor tissues of 6 patients, no rearrangement or translocation of this gene was found in all tissues. In the meantime, a bisulfite-modified PCR coupled with nucleotide sequencing was used to analyze the methylation status of the CpG islands in both the promoter and intron 1 of Tβ4 in patients’ tissue samples. While no change in the methylation pattern was found in proximal the promoter region (-183 to +81) of Tβ4 gene, hypo- and hypermethylation of its intron 1 region (+344 to +766) were detected in tumor tissues of 1 and 3 patients, respectively. Accordingly, we found that Tβ4 expression could be upregulated by 5-aza-cytidine, a demethylating agent, or trichostatin A, a histone deacetylase inhibitor, in HT-29 human colon cancer cell line. Intriguingly, reporter assays showed that the intron 1 region (+332 to +756) of Tβ4 functions as a silencer when placed upstream of a 1.5-Kb Tβ4 promoter. Taken together, our results suggest that upregulation of the Tβ4 gene in human CRC may result from either an increase in its copy number or an altered methylation status in its gene, especially the first intron.
目錄………………………………………………………………1
圖次目錄…………………………………………………………2
英文摘要…………………………………………………………3
中文摘要…………………………………………………………4
緒論………………………………………………………………5
實驗材料…………………………………………………………9
實驗方法…………………………………………………………12
實驗結果…………………………………………………………20
討論………………………………………………………………24
參考文獻…………………………………………………………28
圖表………………………………………………………………32
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