臺灣博碩士論文加值系統

根據過去的研究成果發現，紫外光 (ultraviolet, UV) 是造成皮膚癌的重要危險因子 (risk factor)。照射 UV主要會造成 cyclobutane–pyrimidine dimmers (CPDs) 以及6–4 photoproducts (6–4PPs) 等DNA損害。在台灣，皮膚癌名列十大癌症發生率第九，而與皮膚癌相關功能性蛋白的研究，仍然有許多值得探討的地方，尤其是DNA修補機制 (DNA repair system) 方面，故本研究探討人類人類皮膚表皮細胞株(HaCaT) 以及皮膚基底層癌細胞株 (BCC) 中DNA修補機制與皮膚細胞癌化的相關性。我們利用人類皮膚表皮細胞株 (HaCaT) 以及皮膚基底層癌細胞株 (BCC) ，分析給予UVB後對細胞之生長曲線 (Growth curve) 之影響 ；並以即時定量反轉錄聚合酶連鎖反應 (q-PCR) 、微陣列分析 (Microarray) 、西方墨點法 (Western blot) 等，對於HaCaT細胞株與BCC細胞株給予UVB之後，分析在DNA修補機制 (DNA repair system) 的基因表現 (Gene expression) 及蛋白質表現 (Protein expression) 的差異
從UVB 對HaCaT細胞株與BCC細胞株生長影響的實驗結果發現，HaCaT細胞株與BCC細胞株在給予100 mJ/cm2 UVB劑量時，其細胞生長曲線 (Growth curve) 有明顯差異，我們可以得知BCC
細胞株對於UVB的傷害有較高的易感受性。 我們也從不同的DNA修補機制中，篩選了9個DNA修補基因，包括OGG1、MGMT、MPG、TDG、UNG、XPA、XPC、RAD51、XRCC4等。由基因表現及蛋白質表現分析發現，主要與UVB相關之DNA修補機制- base excision repair (BER) ，雖然在BCC細胞株中表現量高於HaCaT細胞株，但在UVB誘導下，HaCaT細胞株中BER的誘導程度遠高於BCC細胞株。我們的實驗結果證明以BER為主的DNA修補機制可能在UVB誘導皮膚癌化的過程 (carcinogenesis) 中，扮演一個重要的角色。
另外，我們的實驗結果也發現，HaCaT細胞株與BCC細胞株若預先照射UVB，可以增加細胞對游離輻射的易感受性，且實驗結果與細胞癌化程度呈現正相關。我們的實驗結果證明，癌細胞若預先照射UVB，只需要約十分之一目前臨床所使用的輻射劑量 (約60 Gy) ，便可以有效抑制癌細胞的生長。 此實驗結果，對未來皮膚癌的臨床治療策略上，可能可以提供一個全新的思考方向。

Exposition of solar ultraviolet (UV) radiation has been identified as one of the major risk factors for skin cancer development in Americans. UV radiation induces two of the most abundant mutagenic and cytotoxic DNA lesions such as cyclobutane–pyrimidine dimmers (CPDs) and 6-4 photoproducts (6-4PPs) and their Dewar valence isomers. However, cells have developed a number of repair mechanisms to counteract the DNA damage caused by UV. Although skin cancer is a common in Taiwan, the pathogenesis is still incompletely studied. In this proposal, we would like to study the role of DNA repair mechanisms in skin tumorgenesis pathway by exposing UV radiation.
Our preliminary results showed that differential susceptibility to UV radiation exposure between a spontaneously immortalized human keratinocyte HaCaT cell line and a human skin basal cell carcinoma (BCC) cell line. We investigated numerous candidate genes in different DNA repair mechanisms affected by UV radiation. Including of OGG1, MGMT, MPG, TDG, UNG, XPA, XPC, RAD51, XRCC4 from different DNA repair mechanisms .Our data indicated that treatment with UVB in HaCaT cells base excision repair (BER) is higher than BCC cells. Similarly results were also observed in mRNA, measured by Real-time polymerase chain reaction (Real-time PCR) method.
Our data suggest that BER may be involved in the development of skin carcinogenesis. Furthermore, UV-B phototherapy maybe is a helpful strategy to skin cancer patients as a pretreatment for radiation therapy. This study is valuable for the understanding the role of functional protein plays in the progression of skin cancer and for the development of new therapeutic modality in the future.

目錄
誌謝 I-II
中文摘要 III-IV
Abstract V-VI
壹、前言 1-12
一、皮膚癌之流行病學 1
二、皮膚癌種類 3
三、皮膚癌發生原因 5
四、皮膚癌之治療方式 6
五、DNA修補機制與皮膚癌之關係 7
六、DNA鹼基刪除修補機制 8
七、DNA 核酸切割修補 9
八、DNA雙股斷裂同源基因重組及非同源基因重組修補機制 11
貳、研究動機與研究假說 13
參、實驗方法 14-26
一、人類皮膚細胞株之來源 14
二、人類皮膚細胞株培養 14
2.1細胞培養 14
2.2細胞繼代 16
2.3冷凍細胞 16
2.4解凍細胞 16
2.5 細胞數之計算 17
三、UV照射 17
四、游離輻射照射 18
五、西方墨點法 18
5.1 製備細胞均質液 18
5.2 蛋白定量分析 19
5.3 SDS-PAGE的製備 20
5.4 SDS-PAGE電泳操作及轉漬 21
5.5 抗體作用及偵測方法 21
六、即時定量反轉錄聚合酶連鎖反應應 22
6.1 細胞處理 22
6.2 萃取RNA 23
6.3 定量RNA 23
6.4 反轉錄反應 23
6.5 即時定量聚合酶連鎖反應 24
6.6 實驗數據分析 25
七、Affymetrix 微陣列分析 25
肆、實驗結果 27-51
一、分析不同之UVB劑量對於人類皮膚表皮細胞株 (HaCaT) 及皮膚基底層癌細胞株 (BCC) 生長之影響 27
二、UVB在人類皮膚表皮細胞株 (HaCaT) 及皮膚基底層癌細胞株 (BCC) 中對DNA修補基因群之基因表現之影響 31
三、UVB誘導之DNA修補基因在人類皮膚表皮細胞株 (HaCaT) 及皮膚基底層癌細胞株 (BCC) 之的蛋白質表現之差異 36
四、以Affymetrix microarray analysis微陣列分析 (Affymetrix DNA microarray analysis) 在人類皮膚表皮細胞株 (HaCaT) 及皮膚基底層癌細胞株 (BCC) 中UVB對細胞全基因體表現之影響 43
五、UVB在人類皮膚表皮細胞株 (HaCaT) 及皮膚基底層癌細胞株 (BCC) 中對細胞凋亡之影響 47
六、預處理UVB後分析游離輻射對人類皮膚表皮細胞株 (HaCaT) 及皮膚基底層癌細胞株 (BCC)之敏感度之影響 49
伍、討論 52-63
陸、圖目錄 64-89
圖一、分析不同劑量之UVB照射對於人類皮膚表皮細胞株 (HaCaT)及皮膚基底層癌細胞株 (BCC) 生長之影響 64
圖二、給予單一劑量UVB (100mJ/cm2) 照射對於人類皮膚表皮細胞株(HaCaT) 及皮膚基底層癌細胞株 (BCC) 細胞型態之影響 65
圖三、利用影像學定量分析人類皮膚表皮細胞株 (HaCaT) 及皮膚基底層癌細胞株 (BCC) 在單一劑量UVB (100mJ/cm2) 照射下對細胞存活率之影響 66
圖四、UVB照射後人類皮膚表皮細胞株 (HaCaT) 及皮膚基底層癌細胞株 (BCC) 中OGG1之蛋白質表現 69
圖五、UVB照射後人類皮膚表皮細胞株 (HaCaT) 及皮膚基底層癌細胞株 (BCC) 中MGMT之蛋白質表現 70
圖六、UVB照射後人類皮膚表皮細胞株 (HaCaT) 及皮膚基底層癌細胞株 (BCC) 中MPG之蛋白質表現 71
圖七、UVB照射後人類皮膚表皮細胞株 (HaCaT) 及皮膚基底層癌細胞株 (BCC) 中TDG之蛋白質表現 72
圖八、UVB照射後人類皮膚表皮細胞株 (HaCaT) 及皮膚基底層癌細胞株 (BCC) 中UNG之蛋白質表現 73
圖九、UVB照射後人類皮膚表皮細胞株 (HaCaT) 及皮膚基底層癌細胞株 (BCC) 中XPA之蛋白質表現 74
圖十、UVB照射後人類皮膚表皮細胞株(HaCaT) 及皮膚基底層癌細胞株(BCC) 中XPC之蛋白質表現 75
圖十一、UVB照射後人類皮膚表皮細胞株 (HaCaT) 及皮膚基底層癌細胞株(BCC) 中RAD51之蛋白質表現 76
圖十二、UVB照射後人類皮膚表皮細胞株 (HaCaT) 及皮膚基底層癌細胞株(BCC) 中XRCC4之蛋白質表現 77
圖十三、以Affymetrix 微陣列分析(Affymetrix DNA microarray analysis)人類皮膚表皮細胞株 (HaCaT) 及皮膚基底層癌細胞株 (BCC) 中UVB照射對細胞全基因體表現之影響 80
圖十四、依基因功能性分析，Affymetrix 微陣列實驗結果中，UVB照射對人類皮膚表皮細胞株 (HaCaT) 及皮膚基底層癌細胞株 (BCC) 功能性基因之影響 81
圖十五、以Affymetrix 微陣列分析(Affymetrix DNA microarray analysis) 人類皮膚表皮細胞株 (HaCaT) 及皮膚基底層癌細胞株 (BCC) 中UVB對DNA修補基因群表現之影響 82
圖十六、以Affymetrix 微陣列分析(Affymetrix DNA microarray analysis)並比較人類皮膚表皮細胞株 (HaCaT) 及皮膚基底層癌細胞株 (BCC) 中UVB對DNA修補基因群表現之差異 83
圖十七、UVB照射後人類皮膚表皮細胞株 (HaCaT) 及皮膚基底層癌細胞株(BCC) 中P53之蛋白質表現 84
圖十八、UVB照射後人類皮膚表皮細胞株(HaCaT) 及皮膚基底層癌細胞株(BCC) 中CDK4之蛋白質表現 85
圖十九、UVB照射後人類皮膚表皮細胞株 (HaCaT) 及皮膚基底層癌細胞株(BCC) 中Caspase-12之蛋白質表現 86
圖二十、UVB照射後人類皮膚表皮細胞株 (HaCaT) 及皮膚基底層癌細胞株(BCC) 中Caspase-12之蛋白質表現之定量分析 87
圖二十一、預處理UVB後再給單一劑量之予游離輻射 (ionizing radiation) 對人類皮膚表皮細胞株 (HaCaT) 及皮膚基底層癌細胞株 (BCC) 細胞存活與型態之影響 88
圖二十二、預處理UVB後再給予不同劑量之游離輻射 (ionizing radiation) 在人類皮膚表皮細胞株 (HaCaT) 及皮膚基底層癌細胞株 (BCC) 之細胞存活影像定量分析 89
柒、表目錄 67-79
表一、DNA修補基因群在人類皮膚表皮細胞株 (HaCaT) 及皮膚基底層癌細胞株 (BCC) 中基因表現之差異 67
表二、UVB在人類皮膚表皮細胞株 (HaCaT) 及皮膚基底層癌細胞株 (BCC) 中影響DNA修補基因群基因表現之差異 68
表三、DNA修補基因群在人類皮膚表皮細胞株 (HaCaT) 及皮膚基底層癌細胞株 (BCC) 中蛋白質表現之差異 78
表四、UVB在人類皮膚表皮細胞株 (HaCaT) 及皮膚基底層癌細胞株 (BCC) 中影響DNA修補基因群蛋白質表現之差異 79
捌、附表及附圖 90-101
附表一、引子序列 90
附表二、行政院衛生署民國100年發布之全國癌症發生率之統計 91
附圖一、皮膚之真皮層構造圖 92
附圖二、基底細胞癌之症狀示意圖 93
附圖三、上皮細胞癌之症狀示意圖 93
附圖四、黑色素細胞癌之症狀示意圖 94
附圖五、UVB造成DNA損害及其分子途徑機制簡圖 95
附圖六、UVB造成細胞週期停滯及細胞凋亡路徑之簡圖 96
附圖七、DNA鹼基刪除修補機制之代謝途徑 97
附圖八、DNA核酸切割修補之代謝途徑 98
附圖九、DNA雙股斷裂同源基因重組之代謝途徑 99
附圖十、DNA非同源基因重組修補機制之代謝途徑 100
附圖十一、細胞凋亡之代謝途徑 101
玖、參考文獻 102-108

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