臺灣博碩士論文加值系統

根據行政院衛生署所發表之台灣地區癌症登記調查報告，膀胱癌是常見的泌尿系統癌症。目前被認為最有效的膀胱癌檢查方法為膀胱內視鏡檢查，並且透過內視鏡摘取可疑檢體送病理部化驗。然而，該檢查方法的過程是屬於侵犯性，因此會令人感覺不舒服。所以目前研究較傾向於利用尿液排出之膀胱上皮細胞之DNA修飾分析來檢測膀胱癌的情形，而偵測特定基因之DNA甲基化程度被認為可以提供膀胱癌診斷與預測的訊息。例如DAPK, RARβ、E-cadherin與p16基因被發現，在膀胱癌形成初期有高度DNA甲基化的情形。根據我們先前的研究顯示，在處理能夠引發膀胱癌的亞硝酸胺類致癌劑BBN餵食後的小鼠，利用2D電泳分析其膀胱黏膜層蛋白質，發現某些顯著下降的蛋白質，其中包括去毒性蛋白Glutathione-S-transferase Mu1 (GSTM1)等。而基因下調的機轉是否為透過GSTM1基因上之5’-CpG島的DNA甲基化這條路徑？為了探討此點，我們設計了細胞實驗與動物實驗來證明。因此，在本實驗中利用RT-PCR及Western Blotting技術觀察在去甲基化試劑5-aza-2’-deoxycytidine或zebularine處理下7株不同癌化程度之膀胱癌上皮細胞株其GSTM1基因表現情形，發現J82膀胱癌上皮細胞株其GSTM1基因表現有明顯增加的情形，表示在J82細胞株中可能經由DNA甲基化下調節GSTM1基因表現。此外，我們也分析6株不同癌化程度之人類膀胱癌細胞株及比較控制組和處理BBN誘導膀胱癌的小鼠模式其膀胱上皮細胞genomic DNA之DNA甲基化情形，結果發現在人類膀胱癌細胞株中5’-CpG島表現高程度的DNA甲基化現象，除了T24細胞株。此外，我們也分析在去甲基化試劑5-aza-2’-deoxycytidine處理下GSTM1基因表現上調節的J82細胞株其DNA甲基化情形，結果顯示J82細胞株在去甲基化試劑處理下GSTM1 DNA甲基化程度下降的幅度不大(約13.5%)，而合用組蛋白乙醯化抑制劑TSA藥物雖然增加J82細胞株GSTM1基因表現，但其DNA甲基化程度與單獨使用5-aza-2’-deoxycytidine處理差異不大。此外，在小鼠膀胱癌細胞MB49中GSTM1基因表現受DNA甲基化影響並不大，但合用組蛋白乙醯化抑制劑Trichostatin A藥物處理可協助5-aza-2’-deoxycytidine去甲基化作用使得GSTM1基因表現量增加。而動物模型中BBN水處理組的小鼠膀胱上皮細胞其GSTM1基因甲基化程度並不高，但GSTM1基因/promoter區域之平均DNA甲基化程度在處理BBN的小鼠膀胱黏膜層細胞中 (1.32 %) 略高於控制組的小鼠膀胱黏膜層細胞 (0.5 %)，推測DNA甲基化所造成的基因關閉在小鼠模式中造成GSTM1蛋白質下降的影響不大。而在我們測試的6株人類膀胱癌細胞株中有5株有高程度的GSTM1基因/promoter DNA甲基化現象，因此GSTM1基因的甲基化或許可作為人類膀胱癌檢測的參考，但不適用於BBN所誘導的小鼠膀胱癌。

According to the statistics of Taiwan Cancer Registration, bladder cancer is one of the common urinary system cancers. The most efficient method for diagnosing cancer of the bladder is cystoscopy along with biopsy of suspicious lesions. However, the process of examination is invasive and uncomfortable. Many studies of bladder cancer detection tend to focus on DNA modification in the voiding bladder epithelial cells. Detection of specific DNA methylation can be a diagnostic and prognostic information of bladder cancer. For example, DAPK, RARβ, E-cadherin and p16 genes have been shown to have a high degree of methylation in the early stage of bladder cancer. In our previous study, we found that the glutathione-S-transferase Mu1 (GSTM1) protein expression is significantly down-regulated in carcinogen N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-treated mice bladder. In order to study whether the down-regulated GSTM1 protein was due to gene methylated on its 5' CpG islands or not, we designed experiments in vitro and in vivo. In vitro study, we observed that the mRNA and protein expression of GSTM1 in 7 human bladder cancer cell lines, which are identified in tumor grade I~III. It showed that GSTM1 gene expression was up-regulated under a treatment of demethylation drug 5-aza-2’-deoxycytidine or zebularine in J82 cells. The data suggested that GSTM1 may be down-regulated by DNA methylation in this cell line. Next, we used bisulfite conversion and PCR followed by T&;A cloning and sequencing to analyze the CpG islands methylation site of GSTM1 gene promoter in 6 human bladder cancer cell lines. The results revealed that high DNA methylation level was present in human GSTM1 5' CpG islands in all cell lines except T24. Besides, the DNA methylation level of J82 GSTM1 gene was also studied after demethylation agent treatment. The result revealed that DNA methylation level decreases slightly in demethylation agent-treated J82 cells (about 13.5%). Although the GSTM1 gene expression of J82 cell lines up-regulated under the treatment of 5-aza-2’-deoxycytidine and trichostatin A, the GSTM1 DNA methylation level decreased a little. Otherwise, GSTM1 gene expression in mice bladder cancer cell lines MB49 was affected slightly by DNA methylation. But GSTM1 gene expression increased under the treatment of 5-aza-2’-deoxycytidine and trichostatin A combination more than 5-aza-2’-deoxycytidine alone. In animal model study, we observed the GSTM1 protein expression are down-regulated obviously in BBN-treated mice. However, the DNA methylation level of mice GSTM1 gene CpG island was always low. The average of GSTM1 methylation level was a little increased in BBN-treated mice (1.32%) compared to control mice (0.5%). Based on these findings, it suggested that GSTM1 protein down-regulation was mediated by DNA methylation in a little part in BBN-treated mice. In human bladder cancer cells, 5 of 6 cell lines have high methylation level in GSTM1 gene CpG island. Therefore, GSTM1 gene methylation level maybe a diagnostic target in human bladder cancer, but not in BBN-induced mice bladder cancer.

Abstract ................................................................................................................... 5
中文摘要 ................................................................................................................. 7
第一章、 序論...................................................................................................... 10
第一節、 膀胱癌 (Bladder cancer) ................................................................. 10
一、 膀胱之功能與結構 ............................................................................. 10
二、 膀胱癌之流行病學及種類 ................................................................. 11
三、 膀胱癌之分期 ..................................................................................... 14
四、膀胱癌之治療及預後 ............................................................................ 15
第二節、 N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) ............................. 17
一、 BBN介紹 ............................................................................................ 17
二、 BBN代謝途徑及相關機轉 ................................................................ 18
第三節、 DNA甲基化與癌症 ........................................................................ 20
一、 表觀遺傳學 ......................................................................................... 20
二、 與膀胱癌相關之DNA甲基化研究 ................................................... 25
第四節、 穀胱甘肽 S-轉移酶(Glutathione-S-transferase，GSTs) ................ 27
一、 GST gene family .................................................................................. 27
二、 GSTM family與基因多型性 .............................................................. 29
三、 GSTM1與膀胱癌相關之研究 ........................................................... 30
第五節、 實驗目的 .......................................................................................... 32
第二章、 材料與方法 ......................................................................................... 33
一、材料............................................................................................................ 33
(一)人類細胞膀胱癌上皮細胞株.................................................................. 33
(二)細胞實驗 .................................................................................................. 33
(三)逆轉錄聚合酶鍊式反應 .......................................................................... 34
(四)西方墨點法 .............................................................................................. 35
3
(五)Bisulfite Specific PCR and T&;A clone ................................................... 36
二、方法............................................................................................................ 36
(一)細胞培養 .................................................................................................. 36
(二)細胞RNA萃取 ....................................................................................... 39
(三)逆轉錄聚合酶鍊式反應 .......................................................................... 39
(四)西方墨點法 .............................................................................................. 41
(五) 抽取細胞之genomic DNA ................................................................... 43
(六) Bisulfite conversion ................................................................................ 44
(七) Bisulfite conversion後之PCR設計 ...................................................... 45
(八) Bisulfite conversion後之T&;A cloning及定序分析............................ 46
(九) 小鼠實驗設計 ........................................................................................ 47
第三章、 結果...................................................................................................... 48
第四章、 討論...................................................................................................... 59
第五章、 結論...................................................................................................... 66
第六章、 參考文獻 ............................................................................................. 67
第七章、 未來計劃 ............................................................................................. 90

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