上輪部角結膜炎之病理機轉及臨床治療之研究

Abstract

上輪部角結膜炎(superior limbic keratoconjunctivitis, SLK)是一種眼表疾病(ocular surface disorder)，其臨床表徵主要為單側或雙側上部球結膜鬆弛以及上部瞼結膜和球結膜的發炎表現。此病症雖有明顯的炎症表現，但其致病機轉至今仍然未明。為進一步探討其致病機轉，我們收集包括SLK患者和正常受試者的上部球結膜和淚液樣本。 首先，我們將基質金屬蛋白酶（matrix metalloproteinase, MMP）-1和-3列為研究的主要焦點，因為它們在結膜鬆弛症(conjunctivochalasis)中有過度表達的情形，而結膜鬆弛症是另一種也合併有球結膜鬆弛病徵的眼表疾病。我們發現，在SLK患者的結膜上皮下基質中MMP-1和-3免疫染色的程度與對照組有顯著差異。同樣地，我們以SLK患者手術中所取下之結膜樣本進行培養，其纖維母細胞也被發現與正常受試者相比具有明顯的MMP-1和-3過度表現。其次，為了證實肥大細胞穩定劑在SLK治療中臨床應用效果的基礎理論，我們研究兩個會促使肥大細胞遷移和活化的因子在SLK的表現，分別是幹細胞因子（stem cell factor, SCF）和胸腺基質淋巴細胞生成素（thymic stromal lymphopoietin, TSLP）。我們發現SLK患者結膜上皮細胞中SCF和TSLP免疫染色的強度都高於對照組，並且，TSLP的免疫染色分級與結膜上皮下基質中的肥大細胞數量有顯著的正相關。此外，由於淚液中細胞因子（cytokine）的表現已被認為炎症性眼表疾病的其中一種客觀且可量化的量度因子，我們於是進行一系列研究來探討SLK患者是否有淚液之細胞因子不平衡的現象。結果發現，單核細胞趨化蛋白（monocyte chemoattractant protein, MCP）-1的表現在SLK患者治療前的淚液中有明顯的增加，且MCP-1的濃度與SLK臨床分級呈現正相關的情形，隨後在SLK的藥物或手術治療後則可見MCP-1表現出很明顯的降低情形。 關於SLK臨床轉譯方面的研究，我們還進行了相關的臨床診斷和治療方式的研究。 SLK診斷的幾個關鍵特徵包括上部角膜輪狀區域呈現點狀螢光染色。儘管使用螢光劑作為標記染料來評估眼表的緊密連接通透性已被廣泛接受，但角膜螢光染色的機轉仍未達成共識。經由動物實驗（紐西蘭白兔之乾眼症模型）和體外細胞實驗的證實，我們發現螢光劑得以進入角膜上皮細胞中，而且其中一個進入細胞的方法是透過單羧酸鹽轉運蛋白（monocarboxylate transporter, MCT）家族所主導的。未來對於MCT與角膜上皮細胞的相關研究可能會有助於許多眼表疾病的鑑別診斷及病理機轉的了解。關於治療方面，雖然眼部局部藥物使用通常是SLK的第一線治療選擇，但結膜切除手術治療迄今為止則是有效控制該疾病的最佳方式。為了探討及提高手術治療效果的可能性，我們分析並改良手術方式，主要對於藥物治療無反應之SLK患者施以上部球結膜切除術合併Tenon囊切除。在所有的手術眼中，病人眼部臨床症狀和徵象在手術後三個月都有顯著減少的現象。結膜切除術後40眼中僅有3眼有邊緣復發的情形（7.5％），復發之眼睛都於再次手術後完全緩解。研究結果顯示以上部球結膜切除術合併Tenon囊切除作為SLK患者的常規手術治療的確具有很大的前景。 此博士研究主要著重於SLK的分子和臨床兩方面的研究。我們發現結膜上皮基質和上皮細胞中分別有MMP-1，MMP-3，SCF和TSLP的過度表現。另外，在手術標本中注意到有顯著數目的肥大細胞。在淚液樣品收集中，也發現藥物及手術治療前後之MCP-1的表現有顯著差異。然而，在對藥物治療無反應的患者中，我們進行了結膜切除合併Tenon囊切除術，這對緩解患者的眼部症狀和表徵則非常有效。 在未來，儘管眼表面的機械性摩擦被認為是SLK的主要原因之一，但這一概念尚未能被清楚的直接證明。因此，我們希望能開發一種工具，使我們能夠明確地確定機械摩擦力對SLK發病機轉的作用。若能順利建立了所謂的“模擬摩擦力裝置”，直接測試機械摩擦力對於角膜及結膜細胞發生的影響。我們相信這樣的努力將會加深我們對SLK的了解，最終讓受SLK患者受益。

Superior limbic keratoconjunctivitis (SLK) is an ocular surface disorder characterized by unilateral or bilateral redundancy of the superior bulbar conjunctiva with inflammation of the superior palpebral and bulbar conjunctiva. While SLK has been known as an inflammatory disease, the pathogenesis at molecular level remains largely unexplored. To this end, we first established a tissue collection including superior bulbar conjunctiva and tear samples from SLK patients and normal subjects. Matrix metalloproteinase (MMP)-1 and -3 were the main focus of our study due to their overexpression in conjunctivochalasis, a pathological process associated with redundant inferior bulbar conjunctiva. We found that MMP-1 and -3 immunostaining were more prominent in the subepithelial stroma of the SLK patients than that in controls. Consistently, the primary cultivated conjunctival fibroblasts obtained from SLK patients were also found with apparent overexpression of MMP-1 and -3, comparing with those from normal subjects. Furthermore, in order to provide a molecular basis for the clinical application of mast cell stabilizer in the treatment of SLK, we also investigated the roles of stem cell factor (SCF) and thymic stromal lymphopoietin (TSLP), two factors involving mast cell migration and activation, in SLK. We found that the intensity of SCF and TSLP immunostaining were higher in the conjunctival epithelium of SLK patients than control subjects, and that the TSLP grading is significantly correlated with the number of mast cells. In addition, because tear cytokine profile has been represented as an objective and quantifiable measure of inflammatory ocular surface disorder, we also intended to investigate whether tear cytokine is indeed imbalance in SLK patients. We found that the level of monocyte chemoattractant protein (MCP)-1 is augmented in SLK patients prior to treatment and is subsequently decreased following the medical or surgical treatment of SLK. To enhance the translational significance of our study on SLK, we also conducted the associated studies with respect to its clinical diagnosis as well as treatment. Positive fluorescein staining over superior limbal area is among several key characteristics for SLK diagnosis. Despite the use of fluorescein as a marker dye for the evaluation of tight junctional permeability is widely accepted, the interpretation of corneal fluorescein staining is still not in consensus. Based on our in vitro as well as in vivo experiments employing the rabbit model, we found that, rather than a passive permeabilization, fluorescein ingress in corneal epithelial cell is in fact mediated by the monocarboxylate transporter (MCT) family. Future investigation of MCT-mediated transport on human corneal epithelial cells may potentially benefit differential diagnosis and contribute better understandings of ocular surface disorders. Regarding the treatment, although topical medication is usually the first-line treatment choice for SLK, surgical intervention is thus far the most effective modality to manage this disease. To explore the possibility of improving the effectiveness of surgical treatment, we conduct a study analyzing the outcome of the modified surgical modality, of which the superior bulbar conjunctival resection combined with Tenon’s capsule excision, on SLK patients who were unresponsive to medical treatment. In all operated eyes, the clinical symptoms and signs subsided significantly three months after operation. Only three out of 40 eyes (7.5%) had recurrence from the margin of conjunctival resection, and this was relieved after reoperation. This suggests that the modified modality has great promise as the routine surgical treatment for SLK patients. In conclusion, this Ph.D. study primarily focuses on SLK molecularly and clinically. We showed higher levels of MMP-1, MMP-3, SCF, and TSLP in conjunctival subepithelial stroma and epithelial cell, respectively. Also, a prominent number of mast cell was noted in the surgical specimens. In the tear sample collection, we found a significant differential expression of tear MCP-1 and IL-6 before and after medical/surgical treatment. In patients unresponsive to medical treatment, we performed conjunctival resection combined with Tenon’s capsule excision, which is very effective to relieve patients’ symptoms and signs. In the future, although mechanical friction force over the ocular surface is considered the primary cause of SLK, this concept has yet clearly and directly demonstrated. In this regard, we set to develop a device that allows us to determine the role of mechanical friction force on the pathogenesis of SLK unambiguously. In cooperation with National Chin-Hwa University bioengineering department, we established so-called “the modulated friction force device” to directly test the effect of mechanical friction force on the genesis of SLK. We believe our endeavor will improve our understanding on SLK, ultimately benefiting patients afflicted by this disease.