類風濕性關節炎 (rheumatoid arthritis，簡稱RA) 是一種自體免疫功能失調造成之慢性發炎疾病，可導致關節發炎甚至失去功能。特徵包括免疫細胞浸潤於關節腔、滑膜細胞活化及增生、血管新生、發炎性趨化激素及細胞激素的釋放，進而引起分解性酵素分泌導致軟、硬骨受到破壞。類鐸受體 (toll like receptor，簡稱TLR) 為一群可透過辨識病原相關分子型態 (pathogen associated molecular patterns，簡稱PAMPs) 而誘導核轉錄因子κB (nuclear factor kappa B，簡稱NF-κB)及干擾素調控因子 (interferon-regulatory factor，簡稱IRF) 訊息傳遞的膜蛋白， 過去文獻指出可辨識單股核醣核酸 (single strandribonucleic acid，簡稱ssRNA) 的類鐸受體七號 (TLR7) 在RA 病人之關節組織有高量表現的情況。因此在本實驗中，我們首先偵測在膠原蛋白誘發之大鼠關節炎 (collagen-induced arthritis，簡稱CIA)發病過程中TLR7 表現，發現在關節炎發病初期至中期，大鼠關節萃取液中TLR7表現量較正常大鼠表現量高，因此我們進一步構築帶有TLR7核酸干擾因子 (RNA interference，簡稱RNAi) 之慢病毒，命名為Lt.shTLR7，進一步評估Lt.shTLR7 在動物體及細胞實驗中抑制TLR7的效果。我們發現在Lt.shTLR7 治療之CIA 大鼠，其關節炎之臨床症狀、X 光影像 (radiography) 與組織病理學 (histology) 上之病理特徵都有改善的情況。此外，Lt.shTLR7 治療之大鼠可減緩血管新生、T細胞浸潤數目及細胞激素介白素六號 (interleukin-6，簡稱IL-6) 的釋
放。我們進一步利用TLR7 受質imiquimod (INN) 刺激滑膜細胞後發
現只有滑膜纖維母細胞 (synovial fibroblast，簡稱SF) 可隨INN 的劑量增高而促使血管內皮生長因子 (vascular endothelial growth factor，簡稱VEGF) 釋放情況增加，且參與RA 血管新生的過程。最後我們也發現CIA 關節滑液中刺激SF TLR7 訊息可受到核醣核酸酶及內質網成熟抑制劑的抑制，這也意味著關節滑液中確實存在TLR7 的內生性RNA 受質。總結來說，本實驗旨在對RA 的致病及臨床應用研究上提供一個不同於以往的觀點，利用慢病毒載體攜帶TLR7 的RNA干擾因子提供長效性的基因沉默作用以達到治療關節炎的目的。

Rheumatoid arthritis (RA) is a chronic autoimmune disorder that causes loss of function in the joints. It is characterized by infiltration of immune cells into the joints, activation of synovial cells, and production of inflammatory cytokines together with degradative enzymes that progressively leads to the cartilage and bone destruction. Toll like receptors (TLRs) are a family of membrane proteins that trigger the nuclear factor-κB-dependent signaling pathway. Recent studies have shown that TLR7, the receptor of single strand RNA (ssRNA), is highly expressed in the RA joints. In this study, we firstly demonstrated that TLR7 expression was unregulated in the ankle joint tissue extracts during disease progression in a rat model of collagen-induced arthritis (Firestein et al.). Furthermore, we constructed short-hairpin RNA of rat TLR7 gene to a lentivirial vector, designated Lt.shTLR7, and evaluated the efficiency of Lt.shTLR7 to knock-down TLR7 gene expression both in vitro and in vivo. Intra-articular administration of Lt.shTLR7 reduced articular circumference and index, radiological score, and histological score in rats with CIA. Moreover, Lt.shTLR7-treated rats showed reduced angiogenesis and T cells infiltration. We further used TLR7 ligand imiquimod to stimulate synoviocytes and found that imiquimod could induce synovial fibroblsts (SFs) release vascular endothelial growth factor (VEGF), that is involved in the process of angiogenesis in the pathogenesis of RA. Finally, we demonstrated that pretreatment of CIA rat joint fluid with RNase and chloroquine could abolish TLR7 signaling in SFs, suggesting that ssRNA ligand was present in arthritic joint fluid. Taken together, our studies provided different insights into the pathogenesis and potential therapy of RA by utilization of lentiviral vector expressing TLR7 shRNA for the long-term gene silencing.

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