TLR2適體-干擾RNA嵌合物於抑制免疫之研究

Abstract

TLR 接受器在先天免疫反應中占有重要的一環。主要會辨識外來病原如細菌與病毒。這些病原具有稱為病原相關分子結構(Pathogen associated molecular pattern, PAMP)的特殊結構。 在一般狀況下，這些病原相關分子結構會刺激TLR接受器使其活化進而使免疫細胞分泌大量的細胞激素(Cytokines)，產生發炎反應，以消滅入侵的病原菌。 雖然TLR 接受器在人體對抗外來疾病佔有非常重要的角色，但近期研究中發現，在TLR接受器過度活化下，將造成許多臨床症狀的產生，例如關節炎(arthritis)、動脈硬化(arteriosclerosis)、癌症甚至是敗血症的發生。因此發展能夠調節TLR 接受器功能的藥物將是一個十分重要的目標。適體是由一段DNA 、RNA分子所構成的特殊序列結構，因為DNA 、RNA分子會因序列的不同而產生各式各樣可能的二級或三級結構，這些特殊結構經由SELEX (Systematic evolution of ligands by exponential enrichment)方式篩選，便可篩選出會與目標物有專一性結合的序列，由於舊式SELEX方式對於篩選針對細胞膜上的目標物具有一定的困難，因此本論文利用三種SELEX方法，分別是IP-SELEX、cell-based SELEX以及endocytic SELEX來篩選對TLR2接受器具專一性並可藉此進入細胞的適體。實驗結果成功獲得專一辨識並拮抗TLR2接受器之適體。將適體與siRNA連結後，適體可以將siRNA帶進細胞內，並更進一步抑制LTA所造成的NF-κB活性。本篇研究提供一個完整的實驗流程方法，從如何篩選針對細胞膜上TLR2接受器並進入細胞中之適體，進一步找出具有功能的適體序列並對適體做定量以及定性的分析，最後將適體接上siRNA進行免疫反應抑制實驗。同樣的流程亦可套用在其他TLR接受器上，配合接上抑制不同發炎相關基因的siRNA，來達到調節免疫反應的作用，這些適體與siRNA的嵌合體在針對發炎相關疾病的治療上，具有相當大的潛力。

Toll-like receptors (TLRs) play important roles in the innate immunity against invading microorganisms. They recognize pathogen-associated molecular patterns (PAMPs) that are expressed on pathogens, and mediate the production of cytokines necessary for triggering effective immunity. Although TLRs are critical for immune responses, it can cause severe clinical manifestation such as arthritis, atherosclerosis, cancer, or even sepsis while it’s over activated. Identification of TLRs antagonists is therefore considered as a promising direction in the regulation of TLRs associated inflammatory disorder. Aptamers are single-stranded RNA or DNA molecules isolated through an in vitro selection process. In this study, I use a selection strategy that combines immunopreciptate (IP) SELEX, cell-based SELEX and endocytic SELEX. This modification facilitates the screening of high-affinity aptamers against human TLR2, which can further be engulfed by TLR2-expressing cells. After 16 rounds of selection, the aptamers against TLR2 with the ability to trigger endocytosis were identified. My data suggested that the isolated aptamers were effectively engulfed through TLR2-dependent pathway. These aptamers were further analyzed and characterized to find out if they could antagonize TLR2 receptor by using NF-κB reporter assays. Several isolated aptamers exhibited strong inhibition of LTA-induced TLR2 response. Moreover, I had conjugated the isolated aptamer with p65 siRNA to further knockdown the NF-κB signal induced by TLR2 independent pathway.