利用JC病毒殼體輸送RNAi以抑制IL-10之表達

Suppression of IL-10 expression by IL-10 RNAi using JC virus like particles as a gene transfer vector

周孟穎(Meng-Ing Chou)

指導教授 ： 蔡嘉哲

中山醫學大學/醫學院/醫學研究所/碩士(2007年)

摘要

自體免疫疾病是由於免疫系統失調，而產生不正常的免疫細胞、細胞激素及過量的自體抗體，影響自身的組織器官，造成病變。Interleukin-10(IL-10)在systemic lupus erythematosus(SLE)中異常的表現早已廣泛的被學者研究討論，在lupus大量表現的IL-10被認為與疾病的嚴重程度有著密切的關係。以siRNA來治療自體免疫疾病帶來了新希望，此法有很多優點，包括特異性高，不會造成免疫抗原性。由於RNAi作用必須在細胞質中進行，如何將人工化學合成的siRNA送入特定人體組織細胞中，是臨床應用上面臨的重要課題。JC病毒殼體已被研究當作一種基因載體，利用其殼體蛋白VP1可攜帶基因輸送至各種細胞，此法簡單且易操作。以JC病毒殼體攜帶IL-10之shRNA為主要目標，在RAW264.7細胞中測其抑制細胞激素產生之能力。首先將病毒顆粒以滲透壓震盪法（osmotic shock）包裝外源性核酸，再將病毒顆粒送入哺乳類細胞，表達或是抑制特定蛋白質，以影響細胞生理。以TR-PCR及Realtime-PCR的方式測量IL-10 shRNA對於抑制細胞中IL-10 mRNA表達的效率。以JC 病毒殼體攜帶shRNA輸送至細胞及動物，這種治療模式將有機會應用到治療人類自體免疫疾病。

關鍵字

介白素-10 ； RNA干擾現象 ； JC病毒

並列摘要

The immunological abnormality presentation of interleukin-10 (IL-10) in systemic lupus erythematosus (SLE) has been extensively studied. IL-10 is over-expressed in lupus patients and correlates with order of disease severity. RNA interference (RNAi) is a powerful tool to silence gene expression post-transcriptionally. There are considerable excitements about its potential therapeutic applications in human diseases. RNAi offers the prospects of higher specificity, lower immunogenicity, and greater disease modification than current antibody therapies for systemic autoimmune diseases (AID). The major challenge in turning RNA interference into an effective therapeutic strategy is the delivery system. The aim of this study is to develop RNAi against IL-10 for the clinical application using JCV VLPs (virus-like particles) as a gene delivery vector. JCV VLPs were generated by recombinant JCV VP1 protein in yeast expression system. DNA fragment containing IL-10 shRNA template driven by HU6 promoter was packaged into VLPs using osmotic shock. The VLPs containing IL-10 shRNA templates entered RAW264.7 cells presumably by phagocytosis. The effectiveness of IL-10 shRNA in suppression of IL-10 mRNA was analyzed by RT realtime PCR. Pseudoinfection efficiency of VLPs was determined by delivery of the reporter plasmid pEGFP. Nearly all RAW264.7 cells were pseudoinfected by VLPs and expressed green fluorescence. Two IL-10 shRNA templates, IL-10i-1, IL-10i-2, were packed into VLPs separately. The presence of IL-10 shRNA template packed in VLPs was confirmed by PCR. The IL-10i-2 shRNA was found to reduce IL-10 expression about 80%. JC VLPs is a competent vector to deliver the valuable gene in this case. IL-10 shRNA template delivered by VLPs can interfere with IL-10 expression in RAW264.7 cells. The cytokines RNAi have potential to be used in clinical therapy for autoimmune disease in the future.

並列關鍵字

IL-10 ； RNAi ； JCV

參考文獻

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