博碩士論文 103826004 詳細資訊




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姓名 王松年(Sung-Nine Wang)  查詢紙本館藏   畢業系所 系統生物與生物資訊研究所
論文名稱 偵測微型核糖核酸 miR-524-5p表現量利用原位雜交染色法來作為輔助診斷惡性黑色素瘤的生物標記之研究
(Develop the miR-524-5p by in situ hybridization as an ancillary biomarker in melanoma diagnosis)
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摘要(中) 本實驗室先前的研究證明,過度表達的miR-524-5p在黑色素瘤中具有抑制細胞增殖、遷移和小鼠腫瘤生長的能力,此外miR-524-5p在黑色素瘤細胞中透過直接抑制BRAF以及ERK2,來抑制MAPK /ERK的訊號。並且在黑色素瘤細胞中, miR-524-5p的表達與MAPK/ ERK訊息傳遞途徑的活性,呈現反比的關聯性。但是目前miR-524-5p在病人中的表達程度是未知的。因此我們的研究目的,在調查人體組織中miR-524-5p的分布狀況是否可以準確地辨別惡性黑色素瘤組織以及良性痣組織,這些結果可以協助確定黑色素瘤病理學在惡性黑色素瘤診斷上的輔助生物標記。我們利用台灣醫院的惡性黑色素瘤組織檢體以及良性痣的組織檢體,一共有58位病人,以及商業化的黑色素瘤組織陣列,總共含有99個黑色素瘤組織和良性痣組織,來研究miR-524-5p和MAPK/ERK訊息傳遞途徑的活性。藉由原位雜交法 (In Situ Hybridization, ISH) 分析miR-524-5p的表達,和免疫組織化學染色 (Immunohistochemistry, IHC) 來偵測MAPK/ ERK訊息傳遞途徑的活性 (透過評估phospho-MEK) 和BRAF的表達。我們的研究結果顯示,這些結果可以協助病理上對惡性黑色素瘤的認定,或更進一步應用於生物標記物用來診斷惡性黑色素瘤組織。
摘要(英) Our previous study demonstrated that over-expression of miR-524-5p suppresses cells proliferation, migration and tumor growth of melanoma in mice. In addition, miR-524-5p plays an important function in regulating MAPK/ERK signaling through directly suppressing BRAF and ERK2 in melanoma cells. In previous study, the expression of miR-524-5p is inversely associated with the activity of the MAPK/ERK pathway in melanoma cells. However, the expression level of miR-524-5p in melanoma patients is unknown and if there are similar inversely expression pattern between MAPK pathway activity and miR-524-5p in human tissue. We investigated the expression of miR-524-5p and MAPK activity using total 58 tissues from melanoma and nevus from Taiwan patients and total 99 melanoma and nevus tissues from commercial tissue array. The expression of miR-524-5p was detected by in situ hybridization (ISH) and the activity of MAPK/ERK pathway (assessed by phosph-MEK) and BRAF expression were measured by immunohistochemistry (IHC). These results could assist to determine the pathology for melanoma or further applied in the biomarker of melanoma diagnosis.
關鍵字(中) ★ 微型核糖核酸 關鍵字(英) ★ miR-524-5p
論文目次 中文摘要 i
Abstract ii
目錄 iii
一、 緒論 1
1-1 黑色素瘤 1
1-2 黑色素瘤發生原因 2
1-3 黑色素瘤的診斷方式 2
1-4 黑色素細胞分化標記物 3
1-5 惡性黑色素瘤診斷之生物標記物 5
1-6 微型 RNA 在黑色素瘤細胞株的調控機制 6
1-7 研究目的 7
二、 實驗材料及方法 9
2-1 實驗材料 (Material) : 9
2-1-1 癌症病患組織 (Patient Tissues) 9
2-1-2 商業化之黑色素瘤組織陣列 (Commercial Melanoma Tissue Array) 9
2-1-3 分析軟體 (Software) 9
2-2 實驗方法 (Methods) : 9
2-2-1 免疫組織化學染色法 (Immunohistochemistry;IHC) 9
2-2-2 原位雜交染色法 (In Situ Hybridization;ISH) 10
2-2-3 Allred 評分系統 (Allred Scoring System) 11
2-2-4 資料分析 (Data Analysis) 12
2-2-4-1 長條圖分析 (Bar Plot Analysis) 12
2-2-4-2 點散圖分析 (Scatter Blot Analysis) 12
2-2-4-3 盒鬚圖分析 (Box Plot Analysis) 12
2-2-4-4 接收者操作特徵曲線分析 (Receiver Operating Characteristic curve analysis;ROC curve analysis) 13
三、 實驗結果 14
3-1 組織切片檢體資料 14
3-2 黑色素漂白之黑色素瘤的免疫組織化學染色分析 14
3-3 miR-524-5p 在良性痣以及黑色素瘤患者中的表現量狀況分析 15
3-4 BRAF蛋白在良性痣以及黑色素瘤患者中的表現量狀況分析 15
3-5 phospho-MEK 蛋白在良性痣以及黑色素瘤患者中的表現量狀況分析 16
3-6 miR-524-5p / BRAF / phospho-MEK在良性痣以及黑色素瘤之表現量關聯性分析 16
3-7 miR-524-5p / BRAF / phospho-MEK在非轉移性以及轉移性黑色素瘤之表現量關聯性分析 17
3-8 miR-524-5p在不同期別的黑色素瘤組織中的表現量差異分析 18
3-9 miR-524-5p / BRAF / phospho-MEK在不同性別中的良性痣以及黑色素瘤之表現量關聯性分析 18
3-10 miR-524-5p / BRAF / phospho-MEK 接收者操作特徵曲線 (ROC) 分析 19
四、 問題與討論 20
4-1 miR-524-5p 在不同程度中的惡性黑色素瘤之表現差異 20
4-2 惡性黑色素瘤患者之男女差異 20
4-3 miR-524-5p 在黑色素細胞中當作用以診斷惡性黑色素瘤之生物標記的潛力 21
4-4 miR-524-p 在其它癌症的發展狀況 22
五、 結論 23
六、 參考資料與參考文獻 24
七、 圖 32
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指導教授 馬念涵(Nian-Han Ma) 審核日期 2016-7-27
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