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研究生： 賴俊杰
Lai, Chun-Chieh
論文名稱： Toll樣受體3在紫外線引起的眼翳形成扮演一個致病的角色
The pathogenic role of Toll-like receptor 3 in the ultraviolet light-induced pterygium formation
指導教授： 謝奇璋
Shieh, Chi-Chang
曾順輝
Tseng, Sung-Huei
許聖民
Hsu, Sheng-Min
學位類別： 博士
Doctor
系所名稱： 醫學院 - 臨床醫學研究所
Institute of Clinical Medicine
論文出版年： 2021
畢業學年度： 110
語文別： 英文
論文頁數： 75
中文關鍵詞： 眼翳 、眼翳表皮細胞 、Toll樣受體3 、中波紫外線(UVB) 、核因子活化B細胞κ輕鏈增強子 、腫瘤蛋白p63 、去氧核糖核酸
外文關鍵詞： NF-κB, pterygium, pterygial epithelial cells, p63, RNA, Toll-like receptor 3, ultraviolet B
相關次數： 點閱：90  下載：10 
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    • Toll樣受體3(TLR3)藉由偵測損害相關分子模式，可以檢測到中波紫外線(UVB)損傷皮膚細胞而導致死亡所釋放出來的self-RNA，進而造成類似曬傷的皮膚急性炎症。眼翳（或名:翼狀贅肉）也被認為是和紫外線輻射相關的疾病，眼表面細胞因長期曝照於紫外線，導致細胞活化增生，造成類似腫瘤形成的變化，這可解釋眼翳在經常太陽光照射的病人身上有高患病率。由於眼翳是紫外線相關不受控制的細胞增殖，我們推測TLR3相關UVB損傷途徑可能也與眼翳的發病機制有關。在這裡，我們研究TLR3在眼翳以及眼翳培養細胞的表現。用於本研究的檢體取自同一患者的眼翳和同側眼正常的結膜，並研究TLR3，p63和NF-κB的表現。另外我們也培養眼翳上皮細胞，並使細胞暴露於UVB輻射下，以確定UVB對TLR3表現和NF-κB活化的影響。相較於正常結膜，我們發現在眼翳組織中，細胞質TLR3的表現與NF-κB轉移至細胞核中及p63的表現皆具有較強的現象。另外，TLR3的表現在眼翳的分層鱗狀上皮中具有梯度分佈，在最淺層上皮細胞中染色最強，而最深層基底細胞層的染色較弱。這樣暗示了紫外線照射與TLR3表現之間的關係，因為紫外線照射通常是造成表層細胞暴露最多也最強。在qPCR實驗中，也發現眼翳和結膜組織有不同的TLR3基因表現量。UVB照射後的眼翳培養細胞，TLR3蛋白表現和NF-κB的活化高於未受照射的細胞。免疫螢光研究發現，UVB輻射可誘導眼翳培養細胞的NF-κB入核。相反地，在培養細胞接受TLR3基因的抑制之後，這些眼翳細胞就不會因為UVB輻射而導致NF-κB的活化，表示這樣的一個反應是仰賴於TLR3這個因子的表現與否。本研究首次發現眼翳和同側眼正常結膜組織之間的TLR3和p63表現有差異，表明TLR3和p63可能在眼翳的發病機轉中扮演重要角色。眼翳培養細胞中TLR3的表現增加以及UVB輻射誘導的NF-κB入核，也表明了UVB輻射、TLR3的表現和NF-κB的活化可能是導致眼翳形成的一個致病過程。

    Toll-like receptor 3 (TLR3), as a damage-associated molecular pattern sensor, can detect self noncoding RNA, which could be released from necrotic cells induced by ultraviolet B (UVB) radiation. The formation of pterygium is believed to be a tumorigenesis-like process in association with UVB exposure. In this study, we aimed to investigate the expression pattern of TLR3 in pterygial tissues and cultured pterygial epithelial cells (PECs).
    In this study, human pterygial specimens and ipsilateral pterygium-free conjunctival specimens from the same patients were used. The expression of TLR3 and nuclear localization of NF-κB were investigated in these tissues. Besides, PECs were irradiated with UVB to determine the effect of UVB radiation on the expression of TLR3 and the activation of NF-κB.
    In our experiments, an immunofluorescence study showed stronger TLR3 expression in superficial epithelial cells in the pterygium when compared with the normal conjunctival epithelium. The expression of TLR3 decreased in intensity from the superficial epithelium toward the basal cell layers, implying a correlation between TLR3 expression and UVB exposure. A qPCR study showed differential expression patterns of TLR3 between pterygial tissues and conjunctival tissues. PECs after exposure to UVB radiation had significantly higher levels of TLR3 proteins and phospho-NF-κB than those of the PECs without UVB exposure. The immunofluorescence studies showed that UVB radiation induced the nuclear translocation of NF-κB in the PECs. In PECs with the targeted TLR3 gene silencing, the expression of phospho-NF-κB was not induced to increase by UVB irradiation. In conclusion, our results indicate that UVB damage, TLR3 expression, and NF-κB activation may be a critical sequence that leads to the pterygium formation.

    Contents Chapter 1. Introduction 1 1.1. Tumorigenesis in pterygium 1 1.2. Ultraviolet B (UVB) in pterygium 6 1.3. Toll-like receptor 3 (TLR3) in pterygium 9 1.4. Treatments for pterygium and managements of complications 12 Chapter 2. The application of pushed-type monocanalicular intubation (MCI) in patients with obstruction of lacrimal drainage system at NCKUH 16 2.1. Balloon dacryocystoplasty combined with pushed-type MCI as the primary surgical management for congenital nasolacrimal duct obstruction 16 2.2. Balloon dacryocystoplasty with pushed-type MCI as a primary surgical management for primary acquired nasolacrimal duct obstruction 18 Chapter 3. The pathogenic role of Toll-like receptor 3 in the ultraviolet light-induced pterygium formation 22 3.1. Objective of this study 22 3.2. Materials and methods 23 3.3. Results 31 3.3.1. TLR3 was highly expressed in the tissue of pterygium 31 3.3.2. The nuclear localization of NF-κB in pterygium was more significant than ipsilateral pterygium-free conjunctiva 33 3.3.3. UVB irradiation induced upregulation of TLR3 and nuclear translocation of NF-κB in pterygial epithelial cells (PECs) 33 3.3.4. The TLR3 ligand, poly(I:C), promoted cell proliferation of PECs 34 3.3.5. UVB-induced phosphorylation of NF-κB in PECs was dependent on the TLR3-signaling pathway 34 3.3.6. RNA may play a key role in UVB-induced TLR3-TRIF-NF-κB signaling pathway in PECs 35 3.3.7. UVB irradiation induced production of IL-6 in PECs 36 3.3.8. The cell lysates from UVB-irradiated PECs induced production of IL-6 in PECs 36 3.3.9. Loop a and Loop b of synthetic U1 RNA induced production of IL-6 in PECs 36 3.3.10. U1 RNA may play a key role in the TLR3-TRIF signaling pathway in PECs 37 3.3.11. UVB irradiation induced inflammatory reaction and increased the expression of TLR3 in the eye of mice 37 Chapter 4. Discussions 39 Chapter 5. Conclusion 47 Chapter 6. Figures and figure legends 48 References 70

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