210 research outputs found
sj-pdf-2-whs-10.1177_21650799221082304 – Supplemental material for Field Test of an m-Health Worksite Health Promotion Program to Increase Physical Activity in Taiwanese Employees: A Cluster-Randomized Controlled Trial
No full textSupplemental material, sj-pdf-2-whs-10.1177_21650799221082304 for Field Test of an m-Health Worksite Health Promotion Program to Increase Physical Activity in Taiwanese Employees: A Cluster-Randomized Controlled Trial by Sheu-jen Huang, Wen-chi Hung, Meei-Ling Shyu, Tzren-ru Chou, Kuo-chen Chang and Jackson P. Wai in Workplace Health & Safety</p
sj-docx-1-whs-10.1177_21650799221082304 – Supplemental material for Field Test of an m-Health Worksite Health Promotion Program to Increase Physical Activity in Taiwanese Employees: A Cluster-Randomized Controlled Trial
No full textSupplemental material, sj-docx-1-whs-10.1177_21650799221082304 for Field Test of an m-Health Worksite Health Promotion Program to Increase Physical Activity in Taiwanese Employees: A Cluster-Randomized Controlled Trial by Sheu-jen Huang, Wen-chi Hung, Meei-Ling Shyu, Tzren-ru Chou, Kuo-chen Chang and Jackson P. Wai in Workplace Health & Safety</p
Studies on the cellular signaling pathways in hyperglycemia-induced cell proliferation and apoptosis
No full text糖尿病為一常見的內分泌代謝疾病，目前已經成為公共衛生問題的一部份。在世界各地隨著肥胖人數日驅增加，糖尿病的人數也隨之顯著提高。而糖尿病對身體造成最大的問題是對實質性組織器官的傷害；尤其是心血管系統以及腎臟的影響。也由於在世界各地糖尿病的病人愈來愈多，其導致的複雜變化以及合併病發症更值得我們去重視。
糖尿病病人體內血糖過高的訊息是不可逆的並且最後導致組織器官的功能障礙或損傷。由過去的文獻報導以及臨床病人報告中已知：糖尿病的病理過程可引起血管內皮細胞功能缺失，造成的血管功能障礙、加速冠狀動脈硬化並對預後不佳。然而進一步地導致血管內皮細胞凋亡的機制至今仍不十分清楚。探討此一異常訊息是如何造成人類血管內皮細胞凋亡？其機制為何？進而如何避免糖尿病在人類心血管系統功能障礙及合併症的產生，以及病程的惡化是十分重要的課題。於本論文第一部分的研究主要為探討高血糖的狀態下誘導人類血管內皮細胞凋亡之機制，以及PI3K所調控之環氧化蛋白酶表現在其中的角色。本實驗發現暴露於高血糖的環境中皆可測得早期血管內皮細胞凋亡訊號。在細胞凋亡的形態學上以Hoechst染色觀察細胞形態以及以Annexin V/Propidium Iodide偵測早期細胞凋亡訊號。高血糖狀態也會誘導環氧化蛋白酶的表現增加，同時也誘導環氧化蛋白酶的下游產物PGE2生成，進而促使凋亡酶-3 (caspase-3)活性增強而導致細胞走向凋亡。出乎意外地，給予PI3K抑制劑皆可有效地抑制高血糖狀態之環氧化蛋白酶蛋白表現、環氧化蛋白酶的下游產物PGE2生成、凋亡酶-3活性和細胞凋亡。高血糖誘導PI3K活化也依序促使Akt的磷酸化。更進一步地，高血糖引發氧化自由基的生成和NFDiabetes has become a public health crisis. With the incidence of obesity rising in the world, the number of diabetics will grow considerably. Of greatest concern is the impact this trend will have on damage to the kidneys and cardiovascular disease. The incidence of diabetes is increasing worldwide, with subsequent increase in the incidence of diabetic complication. The hyperglycemic signal exchange occurs ubiquitously and irreversibly in patients with diabetes mellitus, and its consequences are especially relevant to organ dysfunctions. In type-2 diabetes, a greater proportion of patients have overt nephropathy at shortly and vascular complications after diagnosis of diabetes. In this thesis, the studies are divided into two parts for description. Diabetes has been demonstrated to accelerate vascular dysfunction, coronary atherosclerosis, and the prognosis were worse following cardiac events. Therefore, the part I will investigate the regulation of abnormal signalings that promote human umbilical vein endothelial cells apoptosis under high glucose condition and relevant in understanding the intracellular signaling associated with vascular disease and preventing the development of vascular complication in diabetics, principally the evolution of this practice from its beginning until cell death. Diabetes mellitus causes endothelial dysfunction. The precise molecular mechanisms by which hyperglycemia causes apoptosis in endothelial cells are not yet well understood. The aim of this study was to explore the role of cyclooxygenases-2 (COX-2) and the possible involvement of phosphoinositide 3-kinase (PI3K) signaling in high glucose (HG)-induced apoptosis in human umbilical vein endothelial cells (HUVECs). For detection of apoptosis, the morphological Hoechst staining and Annexin V/Propidium Iodide staining were used. Glucose up-regulated COX-2 protein expression, which was associated with the induction of prostaglandin E2 (PGE2), caspase-3 activity and apoptosis. Unexpectedly, we found that PI3K inhibitors could suppress COX-2 expression, PGE2 production, caspase-3 activity, and the subsequent apoptosis under HG condition. Glucose-induced activation of PI3K resulted in the down stream effector Akt phosphorylation. PI3K inhibitors effectively attenuated the intracellular reactive oxygen species (ROS) generation and NF-TABLE OF CONTENTS
中文摘要 1
Abstract 5
List of abbreviations 9
PART I
CHAPTER 1 Introduction
1.1 Diabetes "Basics" 11
1.2 Classification 11
1.3 Signs and complications 14
1.4 Endothelium cellS in cardiovascular system 16
1.5 The role of transcription factors in human umbilical vein endothelial cells pathophysiology of diabetes 17
1.6 The role of cyclooxygenase-2 (COX-2) in human umbilical
vein endothelial cells pathophysiology of diabetes 18
1.7 The functions of glomerular mesangial cell 20
1.8 The role of glomerular mesangial cell in pathophysiology
of diabetes 20
1.9 The role of COX-2 in glomerular mesangial cell 22
1.10 The role of NF- B in glomerular mesangial cell 24
1.11 The role of PI3K in glomerular mesangial cell 25
1.12 Purpose and rationales of present research 27
CHAPTER 2 Materials and Methods 28
2.1 Primary culture HUVECs 29
2.2 Apoptosis and Caspase-3/cpp32 activity 29
2.3 Phosphoinositide 3-kinase (PI3K) activity 30
2.4 Immunoblotting 30
2.5 Electrophoretic mobility shift assay (EMSA) 31
2.6 Reactive oxygen species production 31
2.7 PGE2 production 32
2.8 Mesangial cell culture 32
2.9 Cell proliferation assay
MTS Assay 33
[3H]Thymidine incorporation 33
2.10 RT-PCR for COX-2 expression 34
2.11 Preparation of nuclear extracts 35
2.12 Transient transfection with dominant-negative vectors-DN-P85 and DN-Akt 35
2.13 Immunofluorescence staining for NF- B 36
2.14 Statistical analyses 36
CHAPTER 3 Results in endothelial cells 38
3.1 COX-2 protein expression and involvement in high glucose (HG)-induced apoptosis 38
3.2 PI3K activity and involvement in HG-induced COX-2 protein expression and apoptosis 38
3.3 Effects of PI3K inhibitors on HG-induced NF
Aggravation of pulmonary fibrosis after knocking down the Aryl hydrocarbon receptor in the Insulin-like growth factor 1 receptor pathway
No full textVasorelaxing Effect in Rat Thoracic Aorta Caused by Fraxinellone and Dictamine Isolated from the Chinese Herb Dictamnus Dasycarpus Turcz:Comparison with Cromakalim and Ca2+-Channel Blockers
No full textThe 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
Dicentrine, a Novel Antiplatelet Agent Inhibiting Thromboxane Formation and Increasing the Cyclic Amp Level of Rabbit Platelets
No full textSequential 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
Honokiol, a Small Molecular Weight Natural Product, Alleviates Experimental Mesangial Proliferative Glomerulonephritis
No full textGlomerulonephritis (GN) is still the most common cause of end-stage renal disease. Accumulation of glomerular macrophages, proliferation of mesangial cells, and deposition of extracellular matrix proteins are pathobiological hallmarks of GN. Pharmacological interventions that can inhibit these insults may be beneficial in the retardation of the progression of GN. Honokiol originally isolated from Magnolia officinalis, shows antioxidative, anti-inflammatory, and antiproliferative activities in a variety of inflammation models. In this study, we first investigated the in vivo effects of honokiol on rat anti-Thy1 nephritis. Anti-Thy1 nephritis was induced in Wistar rats by injecting mouse anti -rat Thy1 antibodies intravenously. Nephritic rats were randomly assigned to receive honokiol (2.5mg/kg, twice a day ) or vehicle and were killed at various time points. Glomerular histology and immunohistopathology and urine protein excretion were studied. Western blotting was conducted for markers of proliferation. Adhesion molecules, chemokine, and extracellular matrix gene expression were evaluated by Northern blotting. Honokiol-treated nephritic rats excreted less urinary protein and had lower glomerular cellularity and sclerosis. The increased intraglomerular proliferating cell nuclear antigen and Akt phosphorylation in nephritic rats could be abolished by the treatment of honokiol. Honokiol also alleviated glomerular monocyte chemoattractant protein-1 and intracellular adhesion molecule-1, similar to type I (alpha 1) collagen and fibronectin mRNA levels of nephritic rats. These results indicate that honokiol may have therapeutic potential in mesangial proliferative GN
- …
