腺相關病毒傳送第一介白質接受器拮抗蛋白基因應用於預防眼外肌炎; Prevention of Extraocular Myositis by Recombinant Adeno-asssociated Virus Vector With Delivary Of Interleukin-1 Receptor Antagonist

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Title:	腺相關病毒傳送第一介白質接受器拮抗蛋白基因應用於預防眼外肌炎;Prevention of Extraocular Myositis by Recombinant Adeno-asssociated Virus Vector With Delivary Of Interleukin-1 Receptor Antagonist
Authors:	魏利真;Li-Chen Wei
Contributors:	中國醫藥大學：醫學研究所碩士班
Keywords:	眼肌炎;第一介白質接受器拮抗蛋白;腺相關病毒;基因治療;recombinant adeno-asssociated Virus;interleukin-1 receptor antagonist;rabbit;myositis;phorbol ester
Date:	2007-07-17
Issue Date:	2009-08-13 14:50:59 (UTC+8)
Abstract:	背景：甲狀腺眼疾及不明原因性眼肌發炎疾病的病人詳細機制尚不清楚，並無完全有效的治療方法及好的預防方法。隨著分子生物學及細胞生物學的進步，我們瞭解到人類細胞所分泌出第一介白質接受器拮抗蛋白( Interleukin-1 receptor antagonist protein) 是極有效力的抗發炎抑制劑，重組的腺相關病毒（recombinant AAV）是將病毒本身約96％的DNA序列皆移除，如此一來可防止病毒基因產生的病毒蛋白而誘發宿主強烈的免疫反應。在我們的實驗中我們嚐試以腺相關病毒載體系統(adeno-associated virus vector system)以基因傳送的方式，期使治療性基因能被有效植入組織中而且能長期自然產生治療性的物質。目的:研究以腺相關病毒載體系統(adeno-associated virus vector system)傳送第一介白質接受器拮抗蛋白基因，對預防Phobol Ester 引發白兔眼外肌炎動物模式組的預防效果。方法：實驗組為在左眼外上直肌注射rAAV-IL-1Ra劑量為20 μl；對照組於右眼外上直肌注射表現 Escherichia coli LacZ的AAV (rAAV-LacZ)。三星期後，注射致炎物質Porbol ester 0.1mg/mL 0.01ml於上直肌以引發白兔眼外肌炎動物模式。之後三星期後，以免疫化學染色及西方點默法確定第一介白質接受器拮抗蛋白的產生，預防發炎的成效評估以測量肌肉張力及檢查病理組織切片之發炎細胞數來評估。結果：經由免疫化學染色及西方點默法確定第一介白質接受器拮抗蛋白的產生。以肌肉內注射phobol ester誘發眼肌炎後7天，進行由病理組織切片之發炎細胞數評估，發現注射rAAV- LacZ組有較多的發炎細胞，而注射rAAV- IL-1Ra組發炎細胞較少(p<0.01)。肌肉張力測量發現於phobol ester誘發眼肌炎後7天於注射rAAV- LacZ組增加150.4%；於注射rAAV- IL-1Ra組增加69.4% (p<0.01)。此結果顯示先注射rAAV- IL-1Ra組較注射rAAV- LacZ組有預防眼肌發炎的情形。結論：以結合高效率又穩定的腺相關病毒載體系及有抗發炎效果的第一介白質接受器拮抗蛋白提供一個有價值的預防眼外肌炎的方法。 Purpose: To evaluate the recombinant adeno-associated virus vector encoding interleukin-1 receptor antagonist (rAAV–IL-1Ra) complementary DNA for its potential in the prevention of phorbol ester–induced extraocular myositis. Methods: Gene delivery to superior recturs muscle was achieved through intramuscular injections of rAAV-IL-1 Ra in the left eyes and AAV expressing Escherichia coli LacZ (rAAV-LacZ) in the right eyes of white rabbit. After 3 weeks, phorbol ester was used to induce myositis, and the severity of myositis was evaluated. The synthesis and accumulation of IL-1Ra within the muscle were determined using immunohistochemistry and western blot analysis three weeks after gene delivery. The preventive effects of IL-1Ra were evaluated by strain measurement, histologic changes of muscle and inflammatory cell counts one week after myositis induction. Results: Gene expression of IL-1 Ra was demonstrated through immunohistochemistry and Western blot analysis. Histology revealed more inflammatory cells were retained in the rAAV- LacZ treated muscles (2594.386±319.933 cells/mm2; n=10) than in the rAAV- IL-1 Ra treated muscles (1369.479±228.509 cells/mm2; n=10). Strain measurement increased 150.4% 1 week after injection of phorbol ester in right eye, and increased 69.4% in the left eye with previous rAAV–IL-1Ra injection (p<0.01). The myositis in rAAV- IL-1 Ra treated muscles was less severe than that in the rAAV- LacZ treated muscles. Conclusion: This gene therapy, by combining highly efficient and stable rAAV gene delivery, and the anti-inflammatory effect of IL-1Ra, provides a valuable approach for prevention of extraoculor myositis.
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