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研究生:陳芃瑋
研究生(外文):Peng-Wei Chen
論文名稱:靈芝多醣體萃取物,RF3,對於人類多發性硬化症動物模式EAE的免疫調控影響
論文名稱(外文):The immuno-modulatory effect of Reishi polysaccharide, RF3, in Experimental Autoimmune Encephalomyelitis mice
指導教授:黃智生黃智生引用關係
指導教授(外文):Jason C. Huang
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:醫學生物技術暨檢驗學系暨研究所
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:2009
畢業學年度:97
語文別:中文
論文頁數:55
中文關鍵詞:多發性硬化症靈芝多醣體
外文關鍵詞:EAE
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EAE (Experimental autoimmune encephalomyelitis)為人類多發性硬化症的實驗動物模式,是ㄧ種免疫反應導致神經髓鞘受損的自體免疫疾病,主要發生在中樞神經系統。EAE的實驗是利用人工合成的髓鞘蛋白胜肽混合佐劑乳化後,打至小鼠體內,可誘發和人類多發性硬化症類似的症狀,例如:尾巴癱瘓、後肢無力等等,因此常被用來做為研究人類多發性硬化症的相關研究。之前的研究顯示CD4+ T 細胞,尤其是Th1的T細胞對於髓鞘的受損扮演重要的角色。這些細胞分泌的細胞激素例如IFN-�蛂BIL-2等等,都和此病的病程進展有很大的關係。但是在近期內的研究也發現,調節性T細胞與Th17細胞都和EAE的病程發展有密切的影響。調節性T細胞能夠抑制過多的免疫反應,降低發炎的現象,因此可以降低EAE的嚴重性,而Th17所分泌IL-17也被研究出與EAE有重要的關聯,可使得病情變得嚴重。
 Ganoderma lucidum (Lingzhi or Reishi),俗稱靈芝,靈芝多醣體為靈芝的有效成份之一,在之前的研究已經顯示,靈芝多醣體具有調節免疫功能的效果。例如能夠促進抗原呈現細胞的能力、增強單核球或吞噬細胞的效果以及細胞/體液免疫力。但另外一方面,靈芝多醣體也能夠有抗發炎的效果。能夠降低發炎反應、降低自體免疫疾病、以及抑制多餘的免疫反應。
此研究我們在探討靈芝多醣體(F3)對於誘發EAE的小鼠體內如何影響免疫系統。我們發現餵食的餵食F3時,能降低疾病的病程,但是腹腔注射對病情沒有太大的影響。而兩種方式對於調節性T 細胞與Th17細胞的數量都沒有影響。此外在腹腔注射靈芝多醣體的組別,血清中髓鞘蛋白專一性抗體比沒有施與的小鼠來得低,但是餵食靈芝多醣體的組別的血清抗體卻比較高。而在兩組的細胞激素也有不同。我們認為經由這些實驗,可以了解到靈芝多醣體在自體免疫疾病中所扮演的角色,以及F3將來應用在治療上的可能性以及其所產生的影響。
Experimental autoimmune encephalomyelitis (EAE) is an animal model of human multiple sclerosis (MS), an inflammatory demyelinating disease that affects the central nervous systems (CNS). EAE is induced by emulsion containing myelin proteins and adjuvants, and is a useful model for MS studies because they share similar disease characteristics. Pervious studies have shown that CD4+ T cells, especially Th1 T cell, mediate the destruction of myelin. Those cells secrete many kinds of cytokines such as IL-2 and IFN-�� which are related to the disease induction. But recent studies have also shown that regulatory T cells (Tregs) and Th17 cells were also involved in myelin destruction. Tregs reduce the inflammation and severity of EAE, but Th17 make disease more severe.
Ganoderma lucidum (Lingzhi or Reishi) has been used for a long time in China to prevent and treat various diseases. The polysaccharides extracts from G. lucidum are one of efficacious ingredients of G. lucidum. Many previous studies demonstrated that these polysaccharides possess immuno-modulatory activities. For example, these polysaccharides can enhance the function of antigen presenting cell (APC), the mononuclear phygocytic system and the humoral/cellular immunity. On the other hand, the polysaccharides also mediate anti-inflammatory function. They downregulate the inflammatory effect, slow autoimmune diseases progression and inhibit the over-reactive immunity.
My current specific aim is to treat EAE mice with the Reishi polysaccharides (F3) in various ways and investigate their immuno-modulatory function. We found that the disease score decreased when mice were given RF3 by oral treatment, but intraperitoneal (i.p) treatment had no effects on the disease. There were no differences between EAE mice treated by oral or i.p routes in the cell number of Tregs and Th17 of splenocytes. However, i.p treatment of RF3 can reduce serum myelin-specific antibody but oral treatment increases the serum myelin-specific antibody. Our current results indicate that the study of RF3 in the treatment of EAE mice may shed light on the treatment of F3 in autoimmune diseases in the future.
中英對照縮寫表                      4
中文摘要 6

英文摘要 7

第一章 緒論

第一節 多發性硬化症

ㄧ、多發性硬化症之病症定義 8
二、多發性硬化症之臨床病程 8
三、多發性硬化症之致病因素 9
四、研究多發性硬化症之動物模式─實驗性自體免疫性腦炎 10
五、髓鞘蛋白與實驗性自體免疫型腦炎之誘發 11
六、實驗性自體免疫型腦炎與多發性硬化症之致病機制 12
七、實驗性自體免疫型腦炎老鼠對於治療多發性硬化症之貢獻 13
第二節 靈芝多醣體萃取物
一、 靈芝(Ganoderma lucidum)簡介 13
二、 靈芝多醣體(Reishi, F3)的成份與結構 14
三、 靈芝多醣體萃取物在免疫方面的影響 15
第三節 研究動機與目的 16

第二章 材料與方法
第一節 實驗材料
一、 實驗小鼠品系 17

二、 人工合成胜肽 17
三、 動物實驗注射藥品 17
四、 商業試劑套組 17
五、 細胞培養液 18
六、 引子清單 19
七、 抗體清單 19
八、 緩衝液 19

第二節 實驗方法

一、 實驗性自體免疫型腦炎之動物模式建立
A. 實驗性自體免疫型腦炎小鼠之誘發 22
B. 靈芝多醣體之施打 22
a. 腹腔注射
b. 口腔灌食
C. 分離脾臟及淋巴結細胞 23
D. 細胞增生之分析 23
E. 細胞激素之分析 24
F. 血清抗體之分析 25
G. 細胞表面及細胞內激素染色 25
a. 體外細胞培養
b. 細胞表面及細胞內激素染色
H. 即時定量聚合酶連鎖反應 27
a. 體外細胞培養
b. CD4+ T 細胞分離
c. RNA 萃取
d. 反轉錄聚合酶鍊式反應
e . 即時定量聚合酶連鎖反應
I. 組織切片染色分析 30
第三章 實驗結果 31
第四章 實驗討論 35
第五章 參考文獻 40
第六章 結果圖表 46
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