1,721,102 research outputs found
The mechanism of targeting CD47 regulates gastric cancer cell peritoneal dissemination
No full text胃癌的全球死亡率高居不下，除了胃癌難以根治，更重要的是術後會有轉移的高風險，因此積極尋找一個新的治療策略顯得至關重要，而在過去的文獻指出CD47在癌細胞逃脫吞噬作用和轉移扮演一個重要角色，且在眾多癌症中已被發現是過度表達，而中和性抗體CD47能有效抑制黑色素瘤、乳癌等腫瘤生長，但在胃癌的角色及機轉尚待釐清。在本篇研究中，我們證明使用中和性抗體治療，標靶分子CD47可以抗腫瘤生長和腹膜轉移。利用西方點墨法和免疫染色法證明在人類胃癌細胞與致癌物質MNNG誘導老鼠腫瘤發生中，發現CD47高度表現。在裸鼠腫瘤異體移植試驗中，經由正子電腦斷層掃描(PET/CT)也清楚驗證CD47抗體有效的減少腫瘤在腹膜中的擴散轉移。更重要的是CD47抗體有效抑制芳香烴受體（AhR）和Snail，且增加上皮細胞指標蛋白，如细胞角蛋白-18（Cytokeratin 18），另外活化內質網壓力（ER stress）指標蛋白Calpain-10。透過免疫沈澱法證明CD47抗體治療提升AhR和Calpain-10交互作用。從電泳遷移率實驗(EMSA)的實驗結果我們更進一步證實CD47抗體有效降低轉錄因子Snail和Cytokeratin 18的轉錄作用並減少兩個蛋白間的交互作用。此外，從共軛焦顯微鏡影像圖也證實CD47抗體治療會增加Calpain-10和Cytokeratin 18。另一方面，由正子電腦斷層掃描(PET/CT)有效證明CD47抗體抑制大腸癌、肺癌、乳癌細胞所引起的腹膜轉移。綜合以上的實驗結果得知，使用CD47抗體治療可以透過活化ER stress和抑制EMT，進而降低胃癌的腫瘤生長和腹膜轉移，顯示CD47抗體可以作為未來治療胃癌及其腹膜轉移的良好策略之一。CD47 (Integrin-associated protein, IAP) participates in evade phagocytosis and metastasis but its role in gastric cancer is unclear. We set out to elucidate the expression profile and function of CD47 antibody therapy during gastric antitumor growth and antiperitoneal dissemination effects. Immunoblot and immunohistochemical studies revealed elevated CD47 expression in human gastric cancer cell lines and carcinogen MNNG-induced gastric cancer tissues in animal model. Treated CD47 antibody significantly reduced peritoneal dissemination in a mouse model by positron emission tomography/computed tomography (PET/CT) imaging. Simultaneously, therapeutic antibodies down-regulated AhR, Snail expression in tumor nodules, increased epithelial signatures such as cytokeratin-18 (CK 18), ER stress marker and calpain-10 activation by western blotting. Immuno-precipitation assay, proved adding CD47 therapy promoted AhR/calpain-10 interaction. CD47 therapy efficiently abolished physical interaction and mutual functional between Snail and CK 18 by EMSA. Confocal microscope image demonstrated that CD47 therapy-induced up-regulation of CK 18 translocation was abrogated by calpain-10 blocked. Treated neutralize CD47 antibody efficiently reduced all of peritoneal carcinomatosis such as colon, lung and breast cancer. Taken together, our results suggest that the therapeutic CD47 antibody suppresses both gastric tumor growth and peritoneal dissemination by inducing ER stress and inhibiting EMT.中文摘要 ............................................................ i
英文摘要 ........................................................... ii
目次 .............................................................. iii
圖目次 .............................................................. v
縮寫表 ............................................................. vi
第一章、前言 ........................................................ 1
一、胃癌(Gastric cancer): ....................................................................................... 1
(一)流行病學: .................................................................................................. 1
(二)治療方式: .................................................................................................. 2
二、整合素相關蛋白(Integrin-associated protein,IAP 或 CD47): ......................... 2
三、上皮-間質細胞轉換 (Epithelial mesenchymal transition): ............................ 3
四、細胞角蛋白 18 (Cytokeratin 18): ................................................................... 4
五、芳香烴受體 (Aryl hydrocarbon receptor)： ................................................. 4
六、內質網壓力(Endoplasmic Reticulum Stress): ................................................. 4
(一)ER Stress 的分子機轉 ............................................................................... 5
(二)ER Stress 和 Calpain ................................................................................. 6
七、研究方向與動機: ............................................................................................. 6
第二章、 材料與方法 ................................................. 7
一、實驗儀器: ......................................................................................................... 7
二、實驗材料: ......................................................................................................... 7
三、實驗方法: ......................................................................................................... 7
(一)細胞培養(Cell culture) .............................................................................. 7
(二)人類臍帶靜脈內皮細胞初代培養(Primary HUVEC culture) ................. 8
(三)蛋白質萃取(Protein extraction) ................................................................ 8
(四)西方墨點法(Western Blot) ........................................................................ 8
(五)免疫螢光染色法(Immunofluorescence stain)........................................... 9
(六)免疫組織染色(Immunohistochemistry stain) ........................................... 9
(七)免疫沉澱法(Immunoprecipitation) ......................................................... 10
(八)核酸干擾技術(RNA interference) .......................................................... 10
(九)傷口癒合試驗(Wound Healing Assay) ................................................... 10
(十)動物實驗(Animal experiment) ................................................................ 10
(十一)電泳移動率試驗(Electrophoretic Mobility Shift Assay, EMSA)....... 11
(十二)統計 ..................................................................................................... 11
第三章、實驗結果 ................................................... 12
一、在胃癌、致癌物質 MNNG 誘導癌化過程中，高度表現 CD47 的蛋白質量........................................................................................................................ 12
二、以 PET/CT 影像觀察 N/A CD47 抑制胃癌細胞在裸鼠體內生長和腹膜轉移
之情形 ............................................................................................................ 12
三、N/A CD47 減緩上皮間質細胞轉化(Epithelial-mesenchymal transition 簡稱
EMT)過程 ...................................................................................................... 12
四、N/A CD47 在轉錄層次上透過 Snail 及 AhR 有效抑胃癌細胞中 Cytokeratin
18 的轉錄表現 ............................................................................................... 13
五、N/A CD47 誘導內質網壓力的形成且降低 AhR 的表現量，促進胃癌細胞
中的 Calpain 10 與 AhR 的交互作用 ........................................................... 13
六、N/A CD47 會抑制由癌細胞所引起的腹膜轉移 .......................................... 14
七、結論 ................................................................................................................ 14
第四章、討論 ....................................................... 15
第五章、參考文獻 ................................................... 17
附錄表 ............................................................. 34
附圖一 ............................................................. 41
附圖二 ............................................................. 4
Sequential FDG-PET/CT as a Biomarker of Response to Thalidomide in Peritoneal Gastric Cancer Dissemination in Mice
No full textBackground and Purpose: A clinical positron emission tomography imaging (Postrion Emission Tomography/Computed Tomography Images) applied to the mice, a new architecture that combines the adjustment of the hardware and software on the construction of clinical positron emission tomography scanning can be applied to tumors in micedetection by the image showing the tumor cells to give the number of assessments and effective construction of a tumor suppressor effect of drug screening platform. In this paper, the use of drugs as thalidomide, thalidomide has been pointed out with anti-angiogenesis function, however, its mechanism has not yet fully clarify, in the basic experimental section, we use in vivo and in vitro tests to analyze and vascular newborn associated protein of PPAR-γ and transcription factors of the CEBP-β in the inhibition of metastasis of gastric cancer on the feasibility and mechanisms. And combined with the assessment of molecular imaging, in order to reduce the sacrifice mice in the experiment.
Methods: A total of four gastric cancer cell experiment, given first of all by the three different tracer to confirm the future of drug to give way. Then scanned using positron emission tomography assessment required to give the number of tumor cells, tumor cells in mice on growth of and monitoring by image, select the best given the time of the tumor suppressor drugs, re-use continuous video monitoring drug effects. Western blot and immunohistochemical staining of the tumor in nude mice peritoneal PPAR-γ and transcription factor of the CEBP-β expression.
Results: PPAR-γ and CEBP-β decreased expression in gastric cancer tissues and cells. The Salle sinus step is to influence the phosphorylation of the CEBP-β through the activation of Effect of Calpain I, reducing the expression of PPAR-γ and the CEBP-β, thereby inhibiting tumor metastasis. In addition, thalidomide effective inhibition of gastric cancer cells in peritoneal metastasis in nude mice, and in accordance with the results of image analysis.
Conclusions: 1. The clinical positron emission tomography imaging as a biomarker to detecte growth and metabolism of cancer cells in mice. 2. Thalidomide effectively inhibited the metastasis of gastric cancer, this show has potential anti-cancer drugs in the treatment of gastric cancer.背景及目的：本文提出臨床正子電腦斷層影像（ Postrion Emission Tomography/Computed Tomography Images）應用於實驗小鼠的新架構，此架構結合了硬體上的調整及軟體上的建構，使臨床用正子電腦斷層掃瞄可應用於小鼠腫瘤偵測上，藉由影像呈現進行腫瘤細胞給予定性質定量上的評估及有效建構一個抑癌藥物效果的篩選平台。下一段本文主要使用藥物為沙利竇邁(Thalidomide)，沙利竇邁已有研究指出具有抗血管新生之功用，然而其機轉至今尚未完全釐清。因此在基礎實驗部分，我們利用體內及體外試驗來分析與血管新生相關蛋白 PPAR-γ及轉錄因子 CEBP-β 在抑制胃癌轉移上的可行性及其機轉。並同時結合分子影像的評估，以減少實驗過程中小鼠的犧牲。
實驗方法：共使用三株胃癌細胞(AGS、SCM-1、MKN45)進行實驗，首先藉由三種不同給予示蹤劑方式確認日後藥物給予方式。接著利用正子電腦斷層掃描評估所需給予腫瘤細胞數量，並藉由影像監控腫瘤細胞於小鼠體內成長情形，選取最佳給予抑癌藥物的時間點，再利用持續影像監控藥物之效果。西方點墨法及免疫組織化學染色法分析裸鼠腹膜內腫瘤組織 PPAR-γ 及轉錄因子 CEBP-β 的表現量。
實驗結果：PPAR-γ 及 CEBP-β 均於胃癌組織及細胞內降低表達。沙利竇邁乃透過活化 Calpain I 來影響CEBP-β 的磷酸化，降低 PPAR-γ 及 CEBP-β 的表現量，進而抑制腫瘤轉移。另外，於裸鼠體內試驗中也發現沙利竇邁可有效抑制胃癌細胞腹膜內的轉移，且符合影像分析結果。
結論：1.以臨床用正子電腦斷層掃描影像評估應用於生物醫學基礎研究偵測癌細胞在小鼠體內的生長代謝是可行的。2.腹腔注射沙利竇邁可有效抑制胃癌轉移，此顯示在胃癌治療上是具有潛力的抗癌藥物。中文摘要…………………………………………………………….i
英文摘要……………………………………………………………….ii
目次………………………………………………………………....iii
圖目次…………………………………………………………...vi
縮寫表………………………………………………………………...vii
第一章 緒論……………………………………………………..1
1. 正子電腦斷層掃描…………………….…………………........1
1.1 成像原理……………………..……………………………...1
1.2 臨床應用…………..………………………………………...1
2. 氟化去氧葡萄糖(FDG)……………….…………………………1
3. 胃癌………………………………………………………………2
3.1 流行病學………………………….………………………….2
3.2 臨床治療…………………………….………………………….3
4. 鈣蛋白酶 (Calpain)……………..…………………………….3
4.1 Calpain family…………………………………………………3
4.2 Calpain I 的功能…………………………………………3
4.3 Calpain I 與癌症……………………………………………4
5. C/EBPβ(CCAAT-enhancer-binding protein β)………………4
5.1 C/EBPβfamily…………………………………….4
5.2 C/EBPβ的功能……………………………………….4
5.3 C/EBPβ與癌症…………………….…………………………5
6. PPARγ(Peroxisome proliferator- activated receptor gamma)5
6.1 PPARγfamily……………….……………………….5
6.2 PPARγ的功能………………….…………………………….5
6.3 PPARγ與癌症…………….…………………………………….5
7. 內質網壓力 …………………………………………….5
7.1 內質網壓力……………….………………………………….5
7.2 內質網壓力與癌症…………….…………………………….5
8. 沙利竇邁(Thalidmoide)……………………………………….6
9. 研究方向及動機………………………………………………………6
9.1 基礎實驗 ……………………………………………………….6
9.2 分子影像實驗………………………………………………….7
第二章 材料與方法 ………..………………………………………...8
1. 實驗儀器……….…………………………………..……………...8
1.1 基礎實驗 ………………………….…………………………….8
1.2 分子影像實驗……………………….………………………….8
2. 實驗材料…………………………..………………………........8
2.1 基礎實驗 ………………………….…………………………….8
2.2 分子影像實驗……………….………………………………….8
3. 實驗方法………………………………………..……….………...8
3.1 基礎實驗 ………………………….………………………….8
3.2 分子影像實驗………………….………………………….11
第三章 實驗結果 …………………………………………………………...15
1. 臨床用正子電腦斷層掃描可應用於小鼠的偵測………………...15
2. 口服方式給予示蹤劑可取代尾靜脈注射方式………….……...15
3. 給予示蹤劑後第四個小時為小鼠造影之最適時機……….……16
4. 臨床用正子電腦斷層掃描可有效監控小鼠體內之腫瘤生長……16
5. 胃癌細胞株(MKN45)於小鼠體內生長所需最低細胞數量為5x10..16
6. Thalidomide 有效延緩胃癌細胞在裸鼠體內的生長 …………16
7. Thalidomide有效降低C/EBPβ、PPARγ及COX2在胃癌細胞內蛋白表現量……… 17
8. Thalidomide可誘導胃癌細胞內質網壓力的形成……………….17
9. Thalidomide可誘導內質網壓力的形成在胃癌腫瘤組織的表現.18
10. Thalidomide透過活化CalpainI，促使C/EBPβ在細胞內之表現降低…………18
11. Thalidomide有效抑制胃癌細胞腹膜轉移的現象………………18
12. Thalidomide降低VEGFR2、ERK及uPAR在胃癌細胞中的表現..…19
13. Thalidomide可降低基質金屬蛋白酶(MMP)的表現及活性……..19
14. 臨床用正子電腦斷層掃描可架構為抗腫瘤藥物的篩選平台.20
15. 臨床用正子電腦斷層掃描可運用於癌變過程監控平台………..20
16. 臨床用正子電腦斷層掃描可運用於原位癌化過程監控平台… 20
17.總結……………………………….…………………………...…20
第四章 討論 ………………………………………………………22
第五章 未來展望………………………………………………….....24
參考文獻…………………………………………………….…….….25
實驗結果圖表……………………………………………………….….29
附錄……………………………………….………………………..…5
The Mechanisms of PIM1 Inhibitor Thwarts Peritoneal Dissemination and Gastric Cancer Stem-like Cells Proliferation
No full text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 6
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Eugenol suppresses gastric tumor growth and peritoneal dissemination by increasing ER stress in an orthotopic model
No full text丁香酚(4-allyl-2-methoxyphenol)已經知道具有抗發炎的功用, 但是在抑制癌症以及抑制腹膜轉移的機制尚未清楚。在本研究中，我們的目的是評估在體內和體外的抗腫瘤生長和抗轉移的潛力。我們的結果發現，經由丁香酚處理原位植入MKN45胃癌細胞的小鼠不管在腫瘤的生長以及腹膜傳播和肝/肺轉移有顯著減少，並且伴隨著誘導細胞凋亡。此外，在給予丁香酚後，在胃癌細胞中發現相對增加內質網壓力的標記蛋白，GADD153，IRE1a，P-PERK，P-elf2a，Caspase7等蛋白表達。另外芳香烴受體（AHR）和COX-2是環境致癌物的活化轉錄因子與腫瘤發生和轉移因子。同時，丁香酚處理也減少了AhR/RelA相互作用和COX-2 基因的表達。如同上述，我們使用AGS SCM-1和N87胃癌細胞在體外處理皆可發現相同結果。因此進一步在細胞凋亡的部分我們發現在給予丁香酚後，胃癌細胞增加內質網壓力的標記蛋白GADD153和死亡受體5主要是經由增加（CHOP）/ DR5上游啟動子結合活性，但不是死亡受體4。此外，丁香酚處理後的胃癌細胞中AhR的降解和在內質網產生壓力有相關聯性，如同上述內質網產生壓力時相對產生特定鈣蛋白酶-10和AhR的相互作用增加，促使將AhR蛋白的降解，但不是鈣蛋白酶-1或鈣蛋白酶-2。沉默鈣蛋白酶-10可以發現相對應的抑制由丁香酚治療的生物效應。而不管在隨著時間或濃度劑量效應都可以發現丁香酚處理後AhR/RelA相互作用減少也會相對影響核易位和在腫瘤細胞中的DNA結合活性並且活化鈣蛋白酶-10將AhR降解。兩者合計，我們的研究結果表明，丁香酚抑制胃腫瘤的生長和腹膜轉移是經由激活內質網壓力和誘導細胞凋亡。Eugenol (4-allyl-2-methoxyphenol) is known to suppress the inflammation; however, its anti-tumor growth, anti-peritoneal dissemination effects have not been studied so far in orthotopic mouse model. In the present study, we aimed to evaluate the anti-tumor growth and anti-metastatic potential of Eugenol in vivo and in vitro. Our results demonstrate that tumor growth, peritoneal dissemination and liver/lung metastasis of orthotopically implanted MKN45 cells were significantly reduced in Eugenol-treated mice along with the induction of apoptosis. Furthermore, Eugenol-treated tumors showed increased ER stress signature such as increased expression of IRE1a, GADD153, p-PERK, p-elf2a, Caspase7. The aryl hydrocarbon receptor (AhR) and COX-2 is an environmental carcinogen-activated transcription factor associated with tumorigenesis and metastasis. Simultaneously, Eugenol-treated also decreased cooperation of AhR/NF-kB/RelA and COX-2 expression. Similar observations were made when SCM-1, AGS and N87 cells were treated in vitro. Eugeno-induced upregulation of death receptor 5 by increase GADD153(CHOP)/DR5 binding activity but not death receptor 4. Moreover, AhR was down-regulated and cleavaged in the ER fraction of Eugenol-treated cells, as indicated by increased interaction of specific Calpain-10, but not Calpain-1 or Calpain-2. Silence Calpain-10 was abrogated by Eugenol treatment biological effects. In addition, Eugenol inhibited AhR/NF-kB/RelA interaction, nuclear translocation and DNA binding activity in cancer cells in time course and dose-dependent manner by Calpain-10 activation. Taken together, our results suggest that Eugenol suppresses both gastric tumor growth and peritoneal dissemination by inducing apoptosis and activating ER stress.目次
中文摘要....................................................i
英文摘要...................................................ii
目次.....................................................iii
圖目次.....................................................vi
縮寫表....................................................vii
第一章、 前言................................................1
一、 丁香酚(Eugenol) ...................................1
二、 胃癌(Gastric cancer)...............................1
(一) 流行病學...........................................1
1. 遺傳因素...........................................1
2. 性別與年齡.........................................1
3. 飲食生活習慣........................................2
4. 惡性貧血...........................................2
5. 胃部病變及胃切除手術.................................2
6. 幽門螺旋桿菌感染.....................................2
(二) 治療方式...........................................2
三、 COX-2 (Cyclooxygenase-2) .........................3
(一) COX的結構與功能.....................................3
(二) COX-2與癌症........................................3
四、 AhR (aryl hydrocarbon receptor)....................4
(一) AhR與癌症..........................................4
五、 內質網壓力(Endoplasmic Reticulum Stress) ...........4
(一) ER Stress的分子機轉................................4
(二) ER Stress和Calpain................................5
六、 上皮-間質細胞轉換(epithelial-mesenchymal transition 簡稱
EMT).......................................................5
七、 研究方向與動機.......................................6
第二章、 材料與方法...........................................8
一、 實驗儀器............................................8
二、 實驗材料............................................8
(一) 常用緩衝溶液: 附表一.................................8
(二) 實驗試劑: 附表二....................................8
(三) 實驗藥品: 附表三....................................8
(四) 實驗抗體: 附表四....................................8
(五) PCR Primer: 附表五.................................8
三、 實驗方法............................................8
(一) 細胞培養(Cell culture).............................8
(二) 人類臍靜脈內皮細胞初代培養(Primary HUVEC culture).....9
(三) 蛋白質萃取(Protein extraction).....................9
(四) 西方墨點法(Western Blot)...........................9
(五) 核酸萃取(RNA extraction)..........................10
(六) 反轉錄聚合酶連鎖效應(Reverse Transcription-PCR).....10
(七) 聚合酶連鎖效應(Polymerase Chain Reaction)..........10
(八) 免疫螢光染色(Immunofluorescence stain).............11
(九) 免疫組織染色(Immunohistochemistry stain)...........11
(十) 瓊膠生長實驗(Colony Formation Assay)...............11
(十一) 流氏細胞儀分析(Flowcytometry)......................12
(十二) 人類臍靜脈內皮細胞血管新生實驗(HUVEC Tube
Formation).........................................12
(十三) 大鼠主動脈環試驗(Rat Aorta Ring Assay)..............12
(十四) 酶譜分析(Gelatin Zymography)......................12
(十五) 酵素活性分析(Enzyme Activity)......................13
(十六) 細胞增生試驗(MTT Assay)............................13
(十七) 細胞核質蛋白分離萃取(Nuclear/ Cytosol protein
extraction)........................................13
(十八) 免疫沉澱法(Immunoprecipitation)...................13
(十九) 核酸干擾技術(RNA interference).....................14
(二十) 傷口癒合試驗(Wound Healing Assay)..................14
(二十一) 動物實驗(Animal experiment).......................14
(二十二) 動物組織蛋白萃取....................................15
(二十三) 電泳移動率試驗(Electrophoretic Mobility Shift
Assay, EMSA)....................................15
(二十四) 染色質免疫沉澱法(Chromatin Immunoprecipitation,
CHIP Assay)......................................15
(二十五) 雞胚胎絨毛膜試驗(Chicken chorioallantoic membrane assays，CAM)..............................................16
(二十六) 彗星試驗(Comet assay)..............................16
(二十七) 分離內質網蛋白(Isolation of ER).....................16
第三章、 實驗結果............................................18
一、 胃癌癌化過程中顯著增加小鼠胃部組織中AhR的蛋白表現
量........................................................18
二、 Eugenol在轉錄層次上透過NFkB及AhR有效抑制胃癌細胞中COX-2的轉錄表現.....................................................18
三、 Eugenol有效抑制NFkB-p65和AhR在胃癌細胞中內生性交
互作用.....................................................18
四、 Eugenol降低AhR在胃癌細胞中的表現......................19
五、 Eugenol誘導胃癌細胞內質網壓力的形成....................19
六、 Eugenol活化胃癌細胞內質網壓力進而活化Calpain的蛋白表現及活性........................................................20
七、 Calpain抑制劑有效恢復胃癌細胞中由Eugenol所減少AhR的表現........................................................20
八、 Eugenol抑制胃癌細胞增生並促進細胞走向死亡...............21
九、 Eugenol促進細胞走向死亡並造成細胞產生DNA碎片............22
十、 以PET/CT影像觀察建立的原位胃癌的情形...................22
十一、 Eugenol抑制胃癌細胞在裸鼠體內生長和擴散之情形............23
十二、 Eugenol有效抑制胃癌細胞在裸鼠皮下及體外試驗之生長........23
十三、 Eugenol有效抑制胃癌細胞的移動性和血管新生作用............24
十四、 Eugenol調控胃癌細胞在上皮-間質細胞轉換(epithelial-mesenchymal transition 簡稱EMT)現象........................25
十五、 總結..............................................26
第四章、討論................................................27
第五章、參考文獻.............................................31
附錄表.....................................................6
The Role of Ciglitazone Inhibits Tumor Growth in Human Glioblastoma Multiforme
No full text多型惡性神經膠細胞瘤(glioblastoma multiforme, GBM)是大腦最常見的惡性腫瘤，生長快速也具侵襲性，治療方法多為手術、放射性以及化學治療。給予手術後放射性再加上化療，中期存活將延長至一年，但是五年存活率仍是非常的低。PPAR活化劑thiazolidinediones為臨床使用治療第二型糖尿病的藥物，已有文獻指出其抗腫瘤生長的效果，但其作用機制尚未完全釐清，因此本實驗使用PPARγ活化劑來檢測其對GBM抑制生長的功用。在體外試驗中，首先在軟瓊膠生長實驗中顯示PPARγ活化劑會抑制GBM細胞生長，接著利用西方墨點法檢測與腫瘤生長和血管新生相關的蛋白質STAT3，PPARγ活化劑會抑制STAT3於tyrosine 705位點的磷酸化。抑制STAT3磷酸化，主要是透過誘導SHP-2蛋白質增加及活性增加，並以免疫沉澱法及免疫螢光染色觀察SHP-2與STAT3的交互作用。PPARγ活化劑也會抑制細胞週期相關蛋白質Cyclin D1、Cyclin E、CDK2及CDK4的表現，同時會活化蛋白酶體(proteasome)及溶酶體(lysosome)來降解細胞週期相關蛋白質。另外，本實驗中PPARγ活化劑會誘導自噬(autophagy)標記LC3表現量增加及acidic vesicular oranganelles(AVOs)產生。傷口癒合試驗及酶譜分析顯示PPARγ活化劑會抑制GBM細胞移動活性及MMP2與MMP9的活性。此外，在動物體內試驗中顯示，PPARγ活化劑會抑制皮下腫瘤生長與血管新生。本研究顯示，PPARγ活化劑可以有效的抑制GBM腫瘤生長，可成為具有開發潛力的GBM抗癌藥物。Gioblastoma multiforme (GBM) is the most common and most aggressive malignant tumor in brain. The three most common patient's treatments for GBM include surgery, radiation therapy, and chemotherapy. The survival can be prolong from a range of 3 to 4 months to a range of 7 to 12 months when combine the treatment surgery, radiation therapy, and chemotherapy. But GBM is still one of the worst 5-year survival rates among all human cancers. PPARγ agonists are used widely in patients with type 2 diabetes mellitus. Previous studies showed that the potent anticancer effects of PPARγ agonists but the mechanisms are not well understood. This study examed the anticancer effects on GBM. We showed that the inhibitory effect of PPARγ agonists on tumor cells in soft agar assay. Our experiments demonstrated that ciglitazone induced SHP-2 protein expression and dephosphorylation of STAT3 at tyrosine 705 then by using immunoprecipitation and immunofluorescence stain to exam the interaction and co-localization of SHP-2 and phospho-STAT3 (Tyr705). In addition, ciglitazone decreased the protein expression of Cyclin D1, Cyclin E, CDK2, and CDK4 by activated proteasome and lysosome to degrade cell cycle related protein. Ciglitazone also induced autophagy marker LC3 expression and acidic vesicular organelles (AVOs) in this study. We used wound healing assay and zymography to test the inhibitory effects of ciglitazone, the results showed ciglitazone inhibited cell mobility and activity of MMP2 and MMP9. In animal experiment, PPARγ agonists suppressed tumor growth and angiogenesis. The overall findings indicated that PPARγ agonists could be a promising drug candidate in treat gioblastoma multiforme.中文摘要 i
Abstract ii
目次 iii
圖目次 vi
縮寫表 viii
第壹章、 前言 1
一、 多型惡性神經膠細胞瘤 (Glioblastoma multiforme, GBM) 1
（一） 神經膠細胞瘤(Astrocytoma)與分類 1
（二） 多型惡性神經膠細胞瘤 1
（三） 多型惡性神經膠細胞瘤發生原因 1
（四） 多型惡性神經膠細胞瘤致癌分子機制 1
（五） 治療方式 2
二、 轉錄訊息傳遞及活化子蛋白質 (Signal transducer and activator of 2
transcription, STAT) 2
（一） STAT家族 2
（二） STAT訊息調控與結構 2
（三） STAT3致癌能力 3
（四） STAT3與多型惡性神經膠細胞瘤 4
三、 細胞週期 (Cell cycle) 4
（一） 簡述細胞週期 4
（二） GBM與細胞週期 4
四、 過氧化體增生劑活化受體γ (Peroxisome proliferator-activated receptors, PPARγ) 5
（一） 過氧化體增生劑活化受體 5
（二） PPAR作用機制 5
（三） PPARγ與癌症 5
（四） PPARγ配體 6
五、 研究方向及動機 6
第貳章、 材料與方法 7
一、 實驗儀器： 7
二、 實驗材料： 7
（一） 常用緩衝溶液：附表一 7
（二） 實驗試劑：附表二 7
（三） 實驗藥品：附表三 7
（四） 實驗抗體：附表四 7
三、 實驗方法： 7
（一） 細胞培養(Cell culture) 7
（二） 蛋白質萃取(Protein extraction) 7
（三） 西方墨點法(Western blot) 8
（四） 免疫沉澱法(Immunoprecipitation) 8
（五） 免疫螢光染色(Immunofluorescence stain) 8
（六） 酪胺酸去磷酸酶活性分析(Protein tyrosine phosphatase activity assay) 9
（七） 免疫組織染色(Immunohistochemistry stain) 9
（八） 細胞存活率分析(MTT assay) 9
（九） 流式細胞儀(Flow cytometry) 10
（十） 丫啶橙染色(Acridine orange stain) 10
（十一） 大鼠主動脈環試驗(Rat aortic ring assay) 10
（十二） 傷口癒合試驗(Wound healing assay) 10
（十三） 軟瓊膠生長實驗(Soft agar assay) 10
（十四） 酶譜分析(Zymography) 11
（十五） 皮下異種腫瘤移植(Subcutaneous xenograft) 11
（十六） 小鼠皮下血管新生Matrigel Plug試驗(Matrigel plug assay) 11
（十七） 統計分析(Statistical analyses) 11
第參章、 實驗結果 12
一、 PPARγ活化劑抑制神經膠細胞瘤在裸鼠體內生長 12
二、 PPARγ活化劑抑制神經膠細胞瘤在軟瓊脂凝膠中生長 12
三、 Ciglitazone抑制STAT3在tyrosine位置的磷酸化 12
四、 Ciglitazone 誘導酪胺酸去磷酸酶SHP-2蛋白質的表現量而非SHP-1 13
五、 Ciglitazone誘導U-87 MG細胞中p-STAT3和SHP-2交互作用 13
六、 Ciglitazone 活化酪胺酸去磷酸酶SHP-2活性及誘導SHP-2蛋白質在GBM腫瘤組織中的表現 14
七、 Ciglitazone抑制細胞週期蛋白質表現 14
八、 Ciglitazone誘導蛋白酶體(proteasome)及溶酶體(lysosome)的形成 14
九、 蛋白酶體及溶酶體的抑制劑回復Ciglitazone所抑制細胞週期蛋白質表現 14
十、 Ciglitazone處理U-87 MG細胞以細胞存活率分析和流式細胞儀分析並無顯著差異 15
十一、 Ciglitazone誘導參與自噬(autophagy)的蛋白質LC3表現量增加 15
十二、 Ciglitazone抑制血管新生 16
十三、 PPARγ活化劑抑制細胞移動能力 16
十四、 PPARγ活化劑抑制基質金屬蛋白酶MMP2和MMP9的活性 16
十五、 總結 17
第肆章、 討論 18
第伍章、 參考文獻 21
結果圖表 27
附錄表 43
附錄圖 5
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use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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