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研究生:劉葦君
研究生(外文):LIU,WEI-CHUN
論文名稱:IL-6受體抗體克服K-Ras / Raf 突變並抑制體內直腸癌腫瘤生長
論文名稱(外文):IL-6 Receptor Antibody Overcome K-Ras/Raf mutation and inhibites CRC Tumor Growth In Vivo.
指導教授:徐治平徐治平引用關係
指導教授(外文):HSU, CHIH-PING
口試委員:鍾元強賴銘淙徐治平高婷玉
口試委員(外文):CHUBG, YUAN-CHIANGLAI, MING-TSUNGHSU, CHIH-PINGKAO, TING-YUH
口試日期:2019-07-17
學位類別:碩士
校院名稱:元培醫事科技大學
系所名稱:醫學檢驗生物技術系碩士班
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2020
畢業學年度:107
語文別:中文
論文頁數:67
中文關鍵詞:介白素-6受體抗體結腸直腸癌K-Ras突變Raf突變
外文關鍵詞:IL-6R Abcolorectal cancerK-Ras mutantRaf mutant
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研究目的:介白素-6受體抗體(IL-6R Ab)已被應用於治療類風濕性關節炎。先前細胞實驗證實,此一抗體可於體外試驗抑制K-Ras基因突變之大腸腫瘤生長。然而,在體內是否有同樣效果,以及與其中之訊號傳遞分子活性變化仍不清楚。因此,想了解介白素-6受體抗體對結腸癌細胞的影響。方法: SW480(K-Ras突變型)和 HT-29(K-Ras野生型/Raf突變型)細胞分別皮下接種於nu / nu小鼠。每週兩次,在小鼠腹腔注射介白素-6受體抗體 1.0毫克/公斤。於三週後,取下腫瘤。經組織固定、切片後,以H&E和免疫組織化學法染色,並進行評估。
結果: 經過介白素-6受體抗體治療的小鼠,兩種細胞株之移植腫瘤其腫瘤生長均被抑制。H & E染色顯示抑制其細胞分裂與腫瘤侵犯周邊組織。在信號蛋白表現量上,STAT-3 、AKT、ERK-1/2的表現量及其磷酸化均有變化;同時,在腫瘤侵犯轉移相關的MMP-2、MMP-9蛋白,皆低於未注射介白素-6受體抗體的對照組。綜合以上結果,介白素-6受體抗體是可以克服K-Ras/ Raf突變並抑制大腸直腸癌體內腫瘤生長與侵犯性,因此可做為帶有這兩種突變的大腸癌另一種標靶治療的藥物。

Objective: Interleukin-6 receptor antibody(IL-6R Ab)has been used to treat Rheumatoid arthritis. Previously, cell experiments confirmed that this antibody can be used to test the growth of colorectal tumors that inhibit K-Ras gene mutation in vitro. However, whether it has the same effect and the signal pathway molecules and molecular activity remain unclear in vivo. Therefore, we would like to know the effect of interleukin-6 receptor antibody on colon cancer cells.
Methods: SW480(K-Ras mutant)and HT-29(K-Ras wild-type/Raf mutant)cells were subcutaneously inoculated into nu/nu mice, respectively. Twice a week, intraperitoneal injection of interleukin-6 receptor antibody in mice 1.0 mg /kg. After three weeks, the tumor was removed. The tumor was fixed and sectioned. It was stained and evaluated with H&E and immunohistochemistry.
RESULTS: In mice treated with interleukin-6 receptor antibody, tumor growth in both cell line transplants was inhibited. H&E staining showed inhibition of cell division and tumor invasion of surrounding tissues. The expression levels of STAT-3, AKT, ERK-1/2 and their phosphorylation were all altered in signal protein expression. At the same time, MMP-2 and MMP-9 proteins are associated with tumor invasion and metastasis. Both were lower than the control group that did not receive the interleukin-6 receptor antibody. Comprehensive result, interleukin-6 receptor antibody can overcome K-Ras/Raf mutation and inhibit tumor growth and invasion in colorectal cancer in vivo. Therefore, it can be applied to colorectal cancer with these two mutations and another target therapeutic drug.

目 錄
論文口試委員審定書I
誌謝 II
中文摘要 III
英文摘要IV
目錄 VI
圖目 錄 X
表目 錄 XII
第一章緒 論
第一節 癌症是全球第二大死亡原因........................................................ 1
1.1.1 癌症............................................................................................................. 1
1.1.2 癌症管理與方案..................................................................................... 1
1.1.3 衛生福利部國民健康署癌症登記之整體發現............................. 2
1.1.4 六個癌症標誌.......................................................................................... 2
第二節 大腸直腸癌......................................................................................6
1.2.1 大腸直腸癌流行病學............................................................................ 6
1.2.2. 大腸直腸癌之區域、年齡與性別.................................................... 6
1.2.3 大腸直腸癌之病理期別,有左側和右側的差異......................... 6
1.2.4 大腸直腸癌的分期與預後................................................................... 7
1.2.5 大腸直腸癌臨床表現和風險原因..................................................... 8
1.2.6 大腸直腸癌致病機轉............................................................................ 9
1.2.7 K-Ras 突變與大腸直腸癌症期別................................................... 10
1.2.8 B-Raf 突變與大腸直腸癌.................................................................. 11
1.2.9 K-Ras/ Raf 訊號傳遞路徑與大腸直腸癌進展的關係............. 11
1.2.10 EGFR 訊號傳遞途徑在癌症中的應用限制.............................. 13
第三節 IL–6 與大腸直腸癌治療.............................................................14
1.3.1 IL–6 細胞因子家族.............................................................................. 14
1.3.2 IL–6 的生理功能.................................................................................. 14
1.3.3 IL-6 與腫瘤微環境、大腸直腸癌病理變化................................ 15
1.3.4 IL-6 訊號傳遞途徑.............................................................................. 15
1.3.5 IL-6 受體抗體與疾病治療的關係.................................................. 18
第四節 研究假設與研究目的....................................................................19
1.4.1 研究假設................................................................................................. 19
1.4.2 研究目的................................................................................................. 20
第二章 研究材料與方法21
第一節 研究材料與設備............................................................................21
2.1.1 實驗用人類大腸癌細胞株................................................................. 21
2.1.2 實驗用小鼠............................................................................................. 21
2.1.3 一般器材................................................................................................. 21
2.1.4 試劑........................................................................................................... 22
2.1.5 抗體........................................................................................................... 23
2.1.6 儀器........................................................................................................... 23
第二節 研究方法......................................................................................24
2.2.1 解凍細胞................................................................................................. 25
2.2.2 凍存細胞................................................................................................. 25
2.2.3 繼代培養細胞........................................................................................ 26
2.2.4 細胞存活率分析(Trypan Blue Dye Exclusion Assay) ......... 26
2.2.5 動物實驗 ................................................................................................. 27
2.2.6 腫瘤組織處理流程與切片................................................................. 28
2.2.7 H&E 染色................................................................................................ 28
2.2.8 免疫組織化學染色(IHC) ............................................................. 29
第三節研究流程圖及步驟........................................................................31
2.3.1 細胞培養及異種移植.......................................................................... 31
2.3.2 組織處理與切片染色.......................................................................... 32
2.3.3 免疫組織化學染色............................................................................... 32
第四節免疫組織化學染色半定量分析....................................................34
2.4.1 比例分值(Proportion Score) ........................................................ 34
2.4.2 強度評分(Intensity Score)............................................................ 34
2.4.3 總體評分(Total Score)................................................................... 35
第三章 結 果36
3.1 比較注射IL-6 受體抗體前/後裸鼠移植腫瘤生長速度............. 36
3.2 IL-6 受體抗體處理後的大腸直腸癌異種移植腫瘤組織顯微影像變化... 36
3.3 比較注射IL-6 受體抗體前/後 IL-6 受體表現............................. 37
3.4 比較注射IL-6 受體抗體前/後裸鼠移植腫瘤細胞增殖............. 37
3.5 比較注射IL-6 受體抗體前/後裸鼠移植腫瘤MMP-2、-9 的表現量...... 37
3.6 比較注射 IL-6 受體抗體前/後裸鼠移植腫瘤血管新生作用.... 38
3.7 比較注射IL-6 受體抗體前/後裸鼠移植腫瘤訊號傳遞ERK / p-ERK..... 39
3.8 比較注射IL-6 受體抗體前/後裸鼠移植腫瘤訊號傳遞AKT / p-AKT .... 39
3.9 比較注射IL-6 受體抗體前/後裸鼠移植腫瘤訊號傳遞STAT3/p-STAT3 40
第四章 討 論41
4.1 注射 IL-6 受體抗體後裸鼠移植腫瘤大小受抑制。.................... 41
4.2 注射IL-6 受體抗體後裸鼠移植腫瘤轉移、侵犯受限制........... 42
4.3 注射IL-6 受體抗體下調訊息ERK/p-ERK 情形.......................... 44
4.4 注射 IL-6 受體抗體下調訊息AKT/p-AKT 與STAT3/p-STAT3 情形........ 44
4.6 注射IL-6 受體抗體裸鼠活動情形..................................................... 45
第五章 結 論46
第六章 圖 表47
第七章 附 錄63
第八章 參考文獻64
一、中文部分
1. 衛生福利部國民健康署癌症登記年報 中華民國107年12月出版
二、英文部分
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