臺灣博碩士論文加值系統

胃癌是全世界排名第四個最普遍被診斷的癌症，而且有將近75%的病患會有癌症轉移的現象，而腹膜轉移是腫瘤具有抗藥性、多樣性、侵略性的臨床病徵。而腫瘤幹細胞的細胞多樣性則是被認為是腫瘤具有抗藥性、預後復發的關鍵角色。根據研究指出信號傳導及轉錄激活蛋白3 (STAT3)是腫瘤細胞生存、生長、腹膜轉移的重要蛋白，並且跟原致癌基因PIM1 絲胺酸/蘇胺酸激酶 (Proto-Oncogene Serine /Threonine-Protein Kinase PIM1)的表達有密切相關。因此利用PIM1抑制劑治療胃癌異殖腫瘤，在正子電腦斷層掃描 (PET/CT)觀察給予PIM1抑制劑後四至六週腫瘤於裸鼠體內生長及轉移情形得到改善。並在分子層次利用西方墨點法 (Western blot)以及核酸干擾技術 (RNA interference)得知PIM1會調控STAT3、Smad路徑進而影響EMT。此外，從石蠟組織免疫螢光染色 (paraffin section immunohistochemistry) 及免疫組織化學染色 (immuno- histochemistry)腫瘤組織切片也能發現PIM1與腫瘤幹細胞標記之間的關聯性，利用癌症臨床用藥阿黴素 (Doxorubicin)篩選出具抗藥性及幹細胞標記的MKN45細胞並且評估其治療效果及機轉。類腫瘤胃癌幹細胞具有抗藥性、球狀聚落及高度表現腫瘤幹細胞標記 (CD133, CD44, DLL4 and LGR5)，並且利用瓊膠生長實驗 (Colony Formation Assay)、西方墨點法觀察給予PIM1抑制劑的類腫瘤胃癌幹細胞，其細胞生長明顯受到抑制，並進一步在裸鼠的腫瘤異殖動物模式中也發現PIM1抑制劑可有效抑制類腫瘤胃癌幹細胞的磷酸化STAT3以及腫瘤幹細胞標記蛋白的表現。因此可得知PIM1抑制劑不僅在一般胃癌細胞上具有治療效果，在腫瘤幹細胞的治療上也可能可針對抑制磷酸化STAT3成為具有潛力的標靶分子藥物。

Gastric cancer is the forth commonly cancer in the world, and nearly 75% of patients have cancer metastasis. Peritoneal dissemination is the key of tumor''s drug resistant, diversity and aggressiveness. The heterogeneousity of cancer stem cells have been considered as the role of tumor''s drug resistant and the prognosis of recurrence. As the study reported, STAT3 is the main protein of tumor survival, growth and peritoneal dissemination, and with highly correlation of PIM1 expression. Therefore, the treatment of PIM1 inhibitors for the gastric cancer cell xenograft mouse model will observed down-regulation of tumor growth and peritoneal dissemination after 4-6 weeks by positron emission tomography computed tomography (PET / CT) scan in nude mice. At molecular level, using western blot and RNA interference found that PIM1 effect EMT through regulated STAT3,Smad pathway. Furthermore, we also found the co-localization of PIM1 and cancer stem cell marker by histology, and indicated that PIM1 expression is associated cancer stem cell growth. So, we use the clinical drug Doxorubicin screening MKN45 and assess the effect of PIM1 inhibitor and mechanism. which have highly drug resistant, spheroid colony and over-expression cancer stem cell marker (CD133, CD44, DLL4 and LGR5). By means of colony formation assay and western blot observed that PIM1 inhibitors can suppress cancer stem-like cell MKN45 growth. And in xenograft mouse model also recognize PIM1 inhibitors has therapeutic effect not only on general gastric cancer cell, but also on cancer stem-like cell be inhibiting phosphorylation of STAT3. Thus, PIM1 inhibitor have the potential to become a molecule drugs on cancer stem cell target therapy.

中文摘要 i
英文摘要 ii
目次 iv
圖目次 vii
縮寫表 viii
第一章、前言 1
一、胃癌 (Gastric cancer). ..................................1
(一)流行病學 1
(二)治療方式 3
二、原致癌基因絲胺酸/蘇胺酸蛋白激酶 (Proto-Oncogene Serine /Threonine-Protein Kinase PIM1)........................... .........................................3
三、上皮-間質細胞轉換 (Epithelial mesenchymal transition):..............................5
四、內質網壓力 (Endoplasmic Reticulum Stress): 6
五、轉化生長因子β (Transforming growth factor beta)/SMAD信號路徑: 7
六、轉化生長作用因子 (Transforming growth-interating factor;TGIF): 8
七、類腫瘤幹細胞 (Cancer stem-like cell): 8
八、研究方向與動機:...............................................................................................9
第二章、 材料與方法 10
一、實驗儀器: 10
二、實驗材料: 10
三、實驗方法: 11
(一)細胞培養 (Cell culture) 11
(二)蛋白質萃取 (Protein extration) 11
(三)西方墨點法 (Western blot) 12
(四)免疫螢光染色法 (Immunofluorescence stain) 13
(五)免疫組織染色 (Immunohistochemistry stain) 13
(六)石蠟組織螢光染色 (paraffin section immunohistochemistry) 14
(七)核酸干擾技術 (RNA interference) 15
(八)核酸萃取 (RNA extraction) 15
(九)反轉錄聚合酶連鎖反應 (RT-PCR) 16
(十)定量即時聚合酶鏈式反應 (Quantitative PCR) 16
(十一)傷口癒合實驗 ( Wound healing assay) 17
(十二)瓊膠生長實驗 (Colony formation assay) 17
(十三)正子攝影電腦斷層掃描 (PET/CT).....................................................18
(十四)動物實驗 (Animal experiment)...........................................................18
(十五)統計 (Statistics)....................................................................................19
第三章、實驗結果 20
一、以PET/CT影像觀察PIM1抑制劑抑制胃癌細胞在裸鼠體內生長及腹膜轉移之情形 20
二、PIM1抑制劑顯著影響上皮間質細胞轉化 (Epithelial-mesenchymal transition簡稱EMT)過程以及誘發内質網壓力 21
三、PIM1抑制劑可有效抑制TGFβ及STAT3訊號路徑 22
四、在胃癌、致癌物質MNNG誘導癌化過程中，高度表現PIM1蛋白以及腫瘤幹細胞標記CD133的Co-localization 23
五、PIM1抑制劑可藉由阻斷TGFβ及STAT3訊號路徑及誘發内質網壓力抑制類腫瘤幹細胞 (Cancer stem-like cell)生長 24
第四章、討論.................................................................................................................26
一、結論.................................................................................................................33
二、未來展望.........................................................................................................34
第五章、參考文獻 35
附錄表 61

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