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研究生:張祐瑞
研究生(外文):Yu-Jui Chang
論文名稱:探討轉移相關蛋白2與腦癌之關連性
論文名稱(外文):Investigation of metastasis-associated protein 2 is associated with brain tumor
指導教授:謝逸憲
學位類別:碩士
校院名稱:中山醫學大學
系所名稱:醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:39
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惡性腦瘤,主要以嬰孩兒常見之兒童神經母細胞瘤和成人之多型性神經膠質母細胞瘤,目前仍屬人類最具致命性的腫瘤疾病。因此,為了了解整個腦瘤的分子機轉,尋找具代表性的目標基因是必要的。轉移相關蛋白(metastasis-associated protein)是一種快速生長的新穎基因,目前研究顯示此轉移相關蛋白的家族成員有三種, MTA1、MTA2和MTA3 且為Mi2/NuRD ( nucleosome remodeling histone deacetylase ) complex的組成物,它可以藉由HDAC (histone deacetylase)將histone tail 上的lysine residue進行去乙醯作用 (deacetylation),進而改變chromatin的結構因而抑制轉錄作用,同時也調控不同腫瘤細胞生長和侵襲轉移的過程,MTA2過度表達在不同腫瘤細胞,例如卵巢癌、乳癌、肝癌和肺癌等。但是,目前MTA2在人類腦癌細胞的角色,至今仍然未知。
本研究主要探討MTA2在人類惡性腦瘤組織和細胞之表現量。利用免疫化學組織染色證實MTA2會大量表達在腦癌組織,且與腦癌臨床病人資料的年齡、腫瘤分化程度和腫瘤期數有正相關 (P<0.001)。利用五株腦癌細胞(GBM8401、Hs68、M059K、M059J和U-87MG)證實GBM8401和Hs68細胞內MTA2表現量高於M059K、M059J和U-87MG細胞,接下來採用RNAi系統證實shRNA-MTA2#2和shRNA-MTA2#4抑制GBM8401細胞內MTA2的表現最為明顯,因此為了將採用shRNA-MTA2#2和shRNA-MTA2#4片段來瞭解MTA2在腦癌細胞的生物功能和基因調控機制,希望MTA2未來可以當作一個腦癌腫瘤指標蛋白。


Malignant brain tumors, including childhood neuroblastoma and adult glioblastoma multiforme (GBM), are among the most fatal human cancer diseases. Thus, the find to candidate target genes may be necessary to understand the molecular mechanism of malignant transformation of human brain tumors. Metastasis-associated genes (MTAs) represent a rapidly growing novel gene family. At present, there are three different known genes (MTA1, MTA2, and MTA3). These are components of the NuRD (nucleosome remodeling histone deacetylation) complex, which represses transcription by altering chromatin conformation through the histone deacetyaltion. However, histone methylation is also involved in repression of transcription, and regulation of tumor cell proliferation and metastasis. MTA2 overexpression is closely correlated with cell proliferation and metastasis in several human carcinomas, such as ovarian cancer, breast cancer HCC and lung cancer. However, the roles of MTA2 in brain tumor progression is presently unknown.
The thesis is to study the role of MTA2 in human malignant brain tumors. Using immunohistochemical staining from brain cancer tissue array to found that MTA2 protein expression levels were both significantly upregulated in brain tumor tissues compared with normal brain tissues, and was positively correlated with age (P<0.007), differentiation (P<0.001) and tumor stage (P<0.002). We found that MTA2 protein expression levels was significantly upregulated in GBM8401 and Hs68 cells compared with M059K、M059J and U-87MG cells. Inhibition of endogenous MTA2 protein expression by using of shRNA technologies, and we found that shRNA-MTA2#2 and shRNA-MTA2#4 have high efficiency to inhibits the MTA2 protein expression in GBM8401 cells. However, we will use this shRNA-MTA2 to study the biological function and gene regulation of MTA2 in brain tumor. The result will be providing the overexpression of MTA2 may play an important role in the progression of brain tumor and MTA2 expression may serve as a tumor marker for poor prognosis for brain tumor.

壹.中文摘要…………………………………………………… 1
貳.英文摘要…………………………………………………… 2
参.縮寫表……………………………………………………… 4
肆.前言………………………………………………………… 5
一.人類腦瘤(Human Brain Tumors)………………………… 5
1.腦瘤的流行病學…………………………………………… 5
2.腦瘤的介紹………………………………………………… 5
3.誘發腦瘤的危險因子……………………………………… 8
二.轉移相關基因家族(Metastasis-associated gene family)………………………………………………………………… 8
三.轉移相關基因-2 (Metastasis-associated gene 2;MTA2)………………………………………………………………… 10
四.MTA2與癌症之關係………………………………………… 11
伍.實驗動機…………………………………………………… 13
陸.實驗材料與方法…………………………………………… 14
一.實驗材料…………………………………………………… 14
1.細胞株資料………………………………………………… 14
2.實驗試劑…………………………………………………… 14
3.實驗儀器…………………………………………………… 15
二.實驗方法…………………………………………………… 16
1.細胞解凍…………………………………………………… 16
2.腦癌檢體…………………………………………………… 16
3.腦癌細胞繼代培養………………………………………… 16
4.免疫組織染色……………………………………………… 17
5.西方墨點法(Western Blotting)………………………… 18
6.RNAi核心設施(RNAi core)……………………………… 19
7.統計分析…………………………………………………… 23
柒.實驗結果…………………………………………………… 24
一.人類腦癌生物晶片的臨床病人資料……………………… 24
二.MTA2在人類腦癌細胞和正常腦組織的表現…………… 24
三.MTA2與腦癌病人臨床資料之相關性…………………… 24
四.MTA2與人類腦癌細胞的關連性………………………… 25
五.利用shRNA方式來測試抑制MTA2的效率……………… 25
捌.討論………………………………………………………… 27
玖.參考文獻…………………………………………………… 30
拾.圖表與圖表說明…………………………………………… 34


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