臺灣博碩士論文加值系統

第一章 中文摘要
由於分子生物學的進步，有些在已往不知道其致病機轉的疾病，在目前利用分子生物學的方法可以證明是由於某些物質之過份分泌所造成。其中一部份的這類物質被稱做為細胞激素（cytokines），因為它們是由某些細胞所合成分泌並作用在本身或其他的細胞產生反應。在實驗動物或人體中抑制某些這類的分泌物可以有效的預防或治療因為這些分泌物所造成的疾病。本論文主要報告了兩部份的實驗：第一個是利用特別設計合成的反意寡核酸（antisense oligonucleo-tide，antisense OLIGO）以抑制白血球受細菌內毒素刺激後腫瘤壞死因子之分泌；另一個是研究肝細胞生長因子在肝癌病人手術前後的變化以及在阻塞性黃疸病人的變化。
在第一個實驗中，我們所要針對的目標是腫瘤壞死因子。腫瘤壞死因子雖然已經被發現相當久的時間，但是它在病態生理所扮演的角色直到最近才逐漸被明瞭。在急性感染症所引起的敗血症之中，腫瘤壞死因子是至目前能夠測到之細胞激素中相當早`出現的。其次才是介白質-1，介白質-6等等。單純給健康人或實驗動物注射腫瘤壞死因子可以引發與敗血症相同的臨床症狀，而且所引發的種種次級細胞激素也幾乎和敗血症完全一樣。在動物實驗中以中和抗體對抗敗血症初期所分泌之腫瘤壞死因子可以有效的提高動物存活率。由此可見腫瘤壞死因子在敗血症中所扮演的重要角色。另外，在一些慢性發炎疾病如類風溼性關節炎時，腫瘤壞死因子也是造成慢性疼痛相當重要的原因。利用抗體吸附的方法去除血中的腫瘤壞死因子可以有效的改善病人的疼痛症狀。因此，如何阻止或減少腫瘤壞死因子的分泌在某些疾病的處置上是相當重要而且是具有潛力的治療方式。
想要抑制或減少人體中一種蛋白質之作用有相當多的方法。目前最普遍使用的就是使用中和抗體使這種蛋白質失去作用。然而這些抗體均是由其他動物所和成之生物製劑，重覆使用不但會失效並且可能會產生過敏反應，因此並不是很理想的治療方式。反意寡核酸的生化特性自從於1978年由Zamecnik及Stephenson所提出之後，近年來已有了相當程度的深入瞭解與發展，並且開始使用於一些分子生物學及生理學之研究工作。反意寡核酸可以有效及專一的抑制某一種蛋白質的合成。它不具有生物毒性，它分解後之成份可以被身體再利用。由於具有上述之種種優點，反意寡核酸理論上可以算是一種相當理想的『藥物』。當然目前的研究離實際使用還有一段距離。
本次的實驗利用體外血液培養的敗血症模式進行反意寡核酸抑制細胞合成腫瘤壞死因子的實驗。利用含腫瘤壞死因子蛋白質基因啟始符號在內之一段反意寡核酸，將其中磷酸根上的一個非鍵結性氧原子以硫原子取代，並在5’端予以螢光標記。在正常狀況時，血液中的白血球受1μg/ml之細菌內毒素刺激後在短時間之內即會分泌大量之腫瘤壞死因子到血漿中。若在加入細菌內毒素之前先加入1μM之反意寡核酸，則可以抑制掉百分之六十三至百分之六十九腫瘤壞死因子之分泌。此種抑制作用在增加反意寡核酸之濃度時會變得更明顯。在同樣的實驗中，介白質-6的分泌則完全不受所加入之反意寡核酸之影響。若加入的是scrambler在低濃度下亦沒有抑制的作用。由此顯示反意寡核酸對於腫瘤壞死因子分泌的抑制作用是有其專一性的。
在第二部份的實驗當中，我們探討了肝細胞生長因子在肝癌手術前後以及阻塞性黃疸病人所扮演的角色。我們在研究中發現肝癌病人血液中之肝細胞生長因子以及介白質-6均比正常人為高。在肝癌手術切除之後會出現一個肝細胞生長因子、介白質-6、以及C-反應蛋白質濃度的高峰。肝細胞生長因子及介白質-6的高峰在手術後第五日即降回到手術前的基礎值；但是C-反應蛋白質的高峰在手術後兩星期仍未恢復手術前的濃度。而此三種細胞激素的分泌分別與不同的臨床變項有關聯。由這些結果我們可以引申出下列的推論：（一）肝細胞生長因子、介白質-6、以及C-反應蛋白質都不是由肝癌細胞所分泌；（二）此三種細胞激素分別反應肝臟受刺激之後不同層面的反應。另外，我們在阻塞性黃疸的病人研究發現病人膽汁中的肝細胞生長因子減少、血中肝細胞生長因子濃度升高、每日自膽汁中所排出的肝細胞生長因子濃度亦減少。我們得到的結論是：肝臟是人體主要處理及排出肝細胞生長因子之主要器官。在阻塞性黃疸時，膽汁中排出的肝細胞生長因子減少，因而使得血液中的肝細胞生長因子濃度升高。膽汁中高濃度之肝細胞生長因子以及肝細胞生長因子未能自膽汁中順利排出或許是阻塞性黃疸病人以及新生兒膽道閉鎖症病人在短時間之內會演變為膽汁鬱積性肝硬化的原因之一。
由我們的幾個實驗及他人的種種報告可知：細胞激素的分泌異常是很多臨床疾病的原因之一。利用種種的方法來調控細胞激素的分泌是治療或防此類疾病的方法之一。反意寡核酸之使用可以有效的調控標的細胞激素的分泌。這在當今醫學中仍是一個極具發展潛力之領域。

第六章 英文摘要
Following the progress in molecular biology, the mechanism of some diseases of which were not known before are demonstrated now due to the dysregulation of some biological molecules. Some of these molecules are called cytokines because they are synthesized in the cell and are secreted out of the cell to induce reactions of self or other cells. Inhibition of the secretion and/or function of these molecules may sometimes prevent or treat certain diseases effectively.
The pathogenesis of sepsis has becoming more clearly understood recently. The role of tumor necrosis factor plays in the pathogenesis is well-documented. Tumor necrosis factor is one of the initiating and key factor in the disease process of sepsis. There were some measures suggested to suppress the biosynthesis or the secretion of tumor necrosis factor to treat sepsis. But none of them proved to be effective clinically. Antisense DNA technique has been developed since more than ten years ago. It had remarkable progress in both clinical utilizations and laboratory researches. So, in the first part of our experiment, we tried to utilize antisense DNA technique to suppress TNF biosynthesis from white blood cells under the stimulation of endotoxin.
Hepatocyte growth factor is one of the most potent growth—stimulating molecules up to now. Though it has been found for about ten years, but the profile of hepatocyte growth factor in certain diseases are still not clearly studied. The profile of hepatocyte growth factor in patients with hepatocellular carcinoma has not been reported in detail. So it is of interest to investigate the profiles of hepatocyte growth factor and other acute phase proteins in patients with hepatocellular carcinoma, which is the most frequent cancer death in Taiwan, before and after surgical resection. Besides, we were also interested in understanding the way from which endogenous hepatocyte growth factor was cleared from circulation. So in the second part of our study we studied the profiles of three acute phase proteins, i.e. hepatocyte growth factor, interleukin-6 and C-reactive protein, in patients undertaken resection for their hepatocellular carcinoma.
In the first study, the target of our experiment was TNF. Though TNF has been discovered for a long time, but the clinical significance of TNF in pathophysiology was not well demonstrated until recently. In sepsis, TNF is one of the most early synthesized and secreted cytokines that can be detected in circulation. It then activates a cascade of downstream cytokines such as interleukin-1, interleukin-4, interleukin-6, etc.. Injection of TNF into healthy volunteer or experimental animal could induce the symptoms and signs of sepsis, which are not distinguishable from sepsis due to infection. Neutralizing antibody to TNF also showed promising effects in animal to improve survival in experimental bacteremia and endotoxemia. Besides, TNF also played important roles in some chronic inflammatory diseases such as rheumatoid arthritis. Application of TNF can improve clinical symptoms of these patients. It is then rational that suppression of TNF synthesis can prevent or even treat very early stage of sepsis.
In this study, we used the specific characters of antisense OLIGO to suppress TNF synthesis and secretion from white blood cells after the stimulation by lipopolysaccharide. The 16mer antisense oligomer was such designed to target the translation initiation region of TNF gene. The backbone of this oligomer was modified to phosphorothioate to combat the digestive effect of nuclease in serum or cytoplasm. A sense and a 50% matched scrambler were also synthesized and used in this experiment as two controls. In our experiment, the antisense oligomer can effectively suppress the synthesis and secretion of TNF by WBC under the stimulation of lipopolysaccharide. The suppressive effect was shown to be cytokine-specific, dose-effect dependent and nucleotide sequence specific. So, we draw a conclusion that this designed antisense oligomer is a potentially effective “information” drug and had the potential to proceed in vivo experiment to prevent such diseases resulted from persistently high TNF secretion.
In the second study, we demonstrated that the baseline concentrations of hepatocyte growth factor, interleukin-6 were significantly higher than those of normal person. There were surges of these three acute phase proteins after resection for their hepatocellular carcinoma. The concentrations of hepatocyte growth factor and interleukin-6 returned to the preoperative level at the fifth postoperative day. But the concentration of C-reactive protein did not return to preoperative value in two weeks. In this study, we proved indirectly that these three acute phase proteins were not produced by tumor cell per se, but were secreted from the non-tumor part of the liver. These three acute phase proteins reflected different aspects of reaction of the liver after surgical resection. In a second part this study, we demonstrated that liver is a main organ which processes and excretes endogenous hepatocyte growth factor. In patients with obstructive jaundice, the concentration of hepatocyte growth factor in the bile decreased. This implied that the higher than normal concentration of hepatocyte growth factor in patients with obstructive jaundice was due to decreased excretion of hepatocyte growth factor into bile.
In conclusion, as the progress in molecular biology, there are many strategies which may be used to suppress the effects of some cytokines through the many potential mechanisms. Some of these theories have been used clinically and some of them are still under investigation. This anti-cytokine therapy is a brand new and wide field of modern and future medicines.

IV 總目錄
第一章 中文摘要-------------------------------------------- 1
第二章 緒論 ----------------------------------------------- 6
第三章以antisense OLIGO抑制血中白血球受細菌內毒
素刺激時腫瘤壞死因子之分泌----------------------------------19
第一節 antisense OLIGO之選擇-------------------------------19
第二節 antisense OLIGO 長度的選擇--------------------------21
第三節 antisense OLIGO之修飾（modification）----------------22
第四節 核酸溶液之配置---------------------------------------23
第五節 實驗材料--------------------------------------------23
第六節 主體實驗--------------------------------------------25
六、一 血液體外培養對腫瘤壞死因子分泌之影響----------------25
六、二 細菌內毒素對血球細胞腫瘤壞死因子分泌之刺激-----------27
六、三受細菌內毒素刺激後會有持續之腫瘤壞死因子製造----------28
六、四 血中之腫瘤壞死因子是新合成的------------------------29
六、五antisense OLIGO 對白血球腫瘤壞死因子分泌之影響--------30
六、六以dose-effect特性來證明此種抑制作用之特異性-----------31
六、七測量不同之細胞激素以證明antisense OLIGO之作用只針對腫瘤壞死因子有特異性----------------------------------------------33
六、七、一 血液培養中加入細菌內毒素後IL-6之分泌-------------33
六、七、二 血液培養以細菌內毒素刺激後IL-6之持續性分泌-------33
六、七、三 antisense OLIGO 對白血球受刺激後IL-6分泌之影響--34
六、七、四 不同濃度之antisense OLIGO 對血液受刺激後IL-6 分泌之影響--------------------------------------------------------35
六、八 以含56% GC之隨意排列OLIGO(scrambler)為對照組測試antisense OLIGO作用之特異性---------------------------------36
六、九 以senseOLIGO為對照組測試antisense OLIGO作用之特異性--------------------------------------------------37
第七節 討論 -----------------------------------------------38
第四章 肝細胞生長因子(hepatocyte growth factor, HGF)等在肝癌病人以及阻塞性黃疸病人之表現------------------------------------47
第一節 肝癌病人手術前後血中肝細胞生長因子、介白值-6、及C-反應性蛋白質之變化
一、一簡介------------------------------------------------47
一、二病人與方法------------------------------------------49
一、三結果-----------------------------------------------51
一、四討論-----------------------------------------------53
第二節 阻塞性黃疸病人其血中及膽汁中肝細胞生長因子之變化
二、一 簡介------------------------------------------------ 56
二、二 病人與方法------------------------------------------ 57
二、三 結果----------------------------------------------- 59
二、四 討論------------------------------------------------61
第五章 展望-------------------------------------------------66
第六章 英文摘要---------------------------------------------78
參考文獻 ---------------------------------------------------83
圖表-------------------------------------------------------109
發表之相關論文---------------------------------------------129
V 圖目錄
圖一: 細菌內毒素之結構-------------------------------------109
圖二: 人類腫瘤壞死因子基因之核酸排列順序-------------------110
圖三: 核酸之基本構造及其骨架之數種可能之修飾---------------111
圖四: 單純的血液培養並不會刺激腫瘤壞死因子之分泌-----------112
圖五: 細菌內毒素會造成血球細胞大量分泌腫瘤壞死因子---------113
圖六: 受細菌內毒素刺激後血球細胞會持續分泌腫瘤壞死因子-----114
圖七: 本實驗進行時程之圖示流程-----------------------------115
圖八: Cycloheximide 會完全抑制血球細胞受細菌內毒素刺激後腫瘤壞死因子之分泌-------------------------------------------------116
圖九: 腫瘤壞死因子antisense OLIGO會抑制腫瘤壞死因子之分泌--117
圖十: 不同濃度antisense OLIGO對腫瘤壞死因子分
泌有不同程度之抑制作用---------------------------------118
圖十一: 受細菌內毒素刺激後血中會有持續性介白質-6 之分泌----119
圖十二: 白血球受細菌內毒素刺激後會有持續性介白質-6 之分泌--120
圖十三: Cycloheximide 完全抑制介白質-6 的合成--------------121
圖十四: 在有TNF antisense OLIGO存在時介白質-6 之分泌-------122
圖十五: 在不同濃度之TNF antisense OLIGO下介白質-6之分泌----123
圖十六: 高濃度之scrambler沒有抑制腫瘤壞死因子分泌之效果----124
圖十七: 高濃度之sense OLIGO沒有抑制腫瘤壞死因子分泌之效果--125
圖十八: 肝癌切除手術前後血中肝細胞生長因子濃度之變化-------126
圖十九: 肝癌切除手術前後血中介白質-6濃度之變化-------------127
圖二十: 肝癌切除手術前後血中C-反應蛋白質濃度之變化---------128
VI 表目錄
表一：在各種不同濃度之反意寡核酸、sense oligo、scrambler 下白血球受細菌內毒素刺激後培養液中腫瘤壞死因子之濃度-------------129
表二：在各種不同濃度之反意寡核酸、sense oligo、scrambler 下白血球受細菌內毒素刺激後培養液中介白質-6之濃度-----------------130
表三：手術前肝細胞生長因子、介白質-6、與C-反應蛋白質之濃度和各種臨床變異項目之關係-----------------------------------------131
表四：手術前肝細胞生長因子、介白質-6、與C-反應蛋白質之濃度和各種臨床變異項目之關係---------------------------------------- 132
表五：阻塞性黃疸病人肝細胞生長因子之各種動態指數與臨床變異項目間之關係-----------------------------------------------------134
表六：阻塞性黃疸病人其各種肝細胞生長因子彼此間之相關關係---135

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