眼角膜炎及傷口修復之臨床與基礎研究

Abstract

角膜是眼表面非常重要且須維持透明的結構。角膜一旦受傷後，發炎及傷口修復的反應將會影響角膜維持其透光度的能力，視力可能因此而受損。任何角膜的傷口均可引起發炎的反應，而角膜的傷口可以是不小心所造成的，例如配戴隱形眼鏡所致;或蓄意人為所致，例如在角膜上進行雷射視力矯正手術等。瞭解這些傷口的修復過程以及為何會引起發炎的反應可使我們從中找出對病人最有利的預防方式及最有效的治療方法。本研究主要分兩個部份:第一部份是研究隱形眼鏡相關性微生物角膜炎啟動(initiation)的機制與綠膿桿菌第三類分泌系統 (Type three secretion system; T3SS)的關係;第二部份則結合了基礎和臨床的研究，主要針對雷射視力矯正手術後對角膜傷口修復與術後視力和其他臨床指標的影響。另外，也將探討血清備製劑在角膜傷口復原所扮演的角色。 論文中第一部份的研究與微生物角膜炎的致病機轉有關。配戴隱形眼鏡可能會因缺氧或摩擦等因素造成微小的上皮缺損，使伺機性的菌種，例如綠膿桿菌，有機會侵入並造成微生物角膜炎。這些與配戴隱形眼鏡有關的微生物角膜炎另稱為隱形眼鏡相關性微生物角膜炎(contact lens-associated microbial keratitis；CLMK)。根據許多研究的統計，發現綠膿桿菌是最常造成CLMK的菌種。因此，相當多的研究希望瞭解為何綠膿桿菌特別會造成CLMK，但由於所涉及的因素多且複雜，例如:隱形眼鏡、細菌的致病因子(virulence factors)和所引起的眼表面的變化及反應等，疾病真正的啟動機制目前仍然不明。在綠膿桿菌角膜炎的致病機轉中綠膿桿菌的T3SS是其重要的致病因子(virulence factor)之一。此分泌系統會製造及分泌會傷害角膜細胞的外毒素(exotoxin)而造成角膜的發炎及破壞。這系統主要是會製造一種分泌管道(secretion apparatus)將製造的外毒素從細菌的細胞質穿過細菌的細胞膜、細胞壁及最後穿越宿主細胞的細胞膜進入宿主細胞質內，造成宿主的破壞。具ExoS外毒素的菌株為侵入型的菌株(invasive phenotype)。具ExoU一種phospholipase外毒素的菌株為細胞毒性型菌株(cytotoxic phenotype)。本研究收集了近10年的綠膿桿菌菌株，發現綠膿桿菌角膜炎病人中細胞毒性菌株較多(P=0.002)。在進一步分析T3SS的基因型與隱型眼鏡附著的關係時，發現細胞毒性菌株與侵入型菌株對隱形眼鏡的附著量並無差異，但當比較T3SS正常株與PscC分泌管道蛋白突變株(PscC mutants)時，驚人的發現明顯有差異，顯現T3SS確實還是與細菌與隱形眼鏡附著有關。另外，研究發現隱形眼鏡的細菌附著也與隱形眼鏡的材質有關。較厭水性的矽水膠材質隱形眼鏡balafilcon較易有多量的細菌附著，而較親水材質矽水膠隱形眼鏡galyfilcon則明顯有較低量的細菌附著(P<0.05)。細菌的附著經SEM電子顯微鏡掃描後，發現多附著於材質的表面，但瞳孔變色水膠材質的隱形眼鏡(nelfilcon gray)會有細菌卡在孔洞內。我們研究的發現是第一個證實T3SS與綠膿桿菌隱形眼鏡的附著有關。 臨床上須倡導定期的更換隱形眼鏡的重要，避免細菌的滋生及感染。此結果為接下來的研究拓展了新的方向，並可使我們更加了解綠膿桿菌角膜炎的致病機轉，以利未來開發治療或預防感染的新方法。 本研究的第二部份主要與角膜傷口修復有關。首先，我們以活體共軛焦顯微鏡(in vivo confocal microscopy)觀察上皮細胞型態的特徵，並比較上皮雷射原位屈光角膜重塑術(epi-LASIK)中，不同角膜上皮瓣(epithelial flap) 處理的方式對於角膜傷口修復的影響。此前瞻性研究比較術後復位上皮瓣並保留上皮辦的on-flap組與不保留上皮辦的off-flap組的視力與上皮細胞恢復的時間。結果顯示，對於術後視力的影響並無差異。角膜上皮傷口完整癒合的時間以有使用mitomycin C(MMC)處理之off-flap組(4.27± 0.70天) 較on-flap組(5.84± 0.08天)快。 手術後一個月，以活體共軛焦顯微鏡觀察，發現復原至手術前基底細胞型態的的眼睛在on-flap組是87.5%，在off-flap組是 92.3%，這些眼睛均有在術中使用MMC；於未使用MMC處理之眼睛，恢復至正常術前的基底細胞型態的比例是on-flap 組86.3% ，off-flap 組是90.5%。在術後三個月不管有無使用MMC，off-flap組已完全恢復至正常型的上層細胞態但on-flap組僅有一半的眼睛恢復到正常型態。迴歸分析顯示角膜上皮瓣復位會明顯減緩上皮的生長與上層和基底上皮細胞之恢復(P<0.01)。由此研究的結論可知，上皮瓣處理的差別對於臨床上視力的的表現無顯著的差異，但以活體共軛焦顯微鏡的觀察可見off-flap者的角膜上皮細胞型態恢復較快。因此，原本認為保留上皮瓣對病人有益處的做法應被推翻。本研究的結論可作為未來臨床醫師處理角膜上皮瓣的依據。手術演進的過程與我們瞭解手術步驟對於傷口修復的影響確實有關。本研究的發現對於上皮修復的觀察是相當重要的，醫師對於手術的方式也因此而改變，使病人術後的恢復更快，預後更好。 我們以基礎的研究比較三種不同血清備製劑:週邊血液血清(peripheral blood serum:PBS) 、臍帶血清(cord blood serum：CBS)及新鮮冷凍血漿(fresh frozen plasma:FFP)對於上皮修復過程中，細胞移動或遷移、增生和分化的影響。 CBS與其他兩種備製劑相比含最高濃度的上皮細胞生長因子(epithelial growth factor: EGF)，轉化生長因子beta1(transforming growth factor β1: TGFβ1)和玻尿酸(hyaluronic acid:HA)(P<0.05)。在細胞增生實驗的結果可見CBS不論是10%或20%均比PBS和FFP更能促進細胞增生。細胞的遷移數目FFP比PBS (P<0.05) 和CBS差(P<0.05)。在本實驗中我們發現CBS在促進細胞增生方面的確比PBS和FFP好。在促進細胞分化的部分也是以CBS較好。總結而論，研究顯示CBS確實對上皮細胞的修復有幫助，特別是針對促進細胞增生與分化的方面。此發現使病人不必再忍受抽血的疼痛就能取得血清，並且也提供臨床醫師在治療困難癒合的角膜傷口時另一更方便的選擇。

The cornea is an avascular tissue that must remain clear for functional vision. Thus, any disease or trauma to the cornea that results in loss of clarity may severely hinder vision. Our investigation both from clinical and basic approach hopes to unravel the initiation mechanism involved in contact lens-associated microbial keratitis (CLMK) due to P. aeruginosa. We also hope to understand the differences in corneal wound healing process with laser vision correction (LVC) procedures and how different serum preparations may improve wound healing. Research results presented herin will determine the best treatment option and improve clinical outcome. Unintentional injury of the corneal epithelium during contact lens wear causes breakdown of the epithelial barrier allowing infection by opportunistic organisms such as P. aeruginosa. Extensive efforts have been done to understand the pathogenesis of CLMK in the hope of preventing this potentially sight threatening disease. Nevertheless, the actual initiation mechanism still remains elusive, mainly due to the complexity of the many factors involved: the contact lens, bacterial virulence factors, and ocular surface changes or response. An important virulence factor of P. aeruginosa is the Type III secretion system (T3SS). The T3SS produces potent exotoxins, the ExoS and ExoU, which is transported through the T3SS needle complex structure into host cells. Mutation in the needle complex structural protein PscC will prevent T3SS exotoxin secretion. In this investigation, we determine the distribution of invasive and cytotoxic genotypes among ocular isolates of P. aeruginosa and investigate the influence of the T3SS on adhesion to conventional, cosmetic, and silicone hydrogel contact lenses (CL). Among 87 of our total isolates, 64 strains were from microbial keratitis cases. Contact lens- related microbial keratitis (CLMK) isolates were mostly of cytotoxic genotype (expressing exoU) (P=0.002). No significant difference was found in the bacterial adhesion to all types of CL between the genotypes under T3SS inducing conditions. A trend for least bacterial adhesion of galyfilcon compared to the other CL was noted for both genotypes. Needle complex pscC mutants adhered less to all materials compared to wild type (P<0.05) indicating a role of the T3SS in contact lens adhesion. Arteficial tear fluid (ATF) incubated CL had significantly more bacterial adhesion (P<0.05). SEM showed most of the bacteria adhering on CL surfaces. In brief summary, this research provided for ther first time an indication of the involvement of the T3SS with other adhesins in CL adhesion. Different genotypes did not significantly differ in its adhesion to various CL. Contact lens material may play an important role in the adherence of both genotypes of P. aeruginosa. Therefore, this research is invaluable in extending our understanding of the initiation of CLMK and also provides new directions for on-going investigations. Intentional injury to the cornea usually involves planned surgical procedures to the cornea such as laser vision corrections or refractive surgeries. As the latter section of our research, we ask how various surgical procedures affect corneal wound healing. The effect of repositing the epithelial flap after epikeratome laser-assisted in situ keratomileusis (on-flap epi-LASIK) versus leaving it off (off-flap epi-LASIK) on epithelial wound healing is investigated.This prospective study comprised of 46 eyes in the on-flap group and 47 eyes in the off-flap group. Complete epithelialization was faster for mitomycin C (MMC) treated off-flap (4.27 ± 0.70 days) than on-flap (5.84 ± 0.08 days) eyes (P=0.01). Regression analysis revealed repositioned flap resulted in significant delay of epithelialization and apical/basal epithelial recovery (P<0.01). In summary, the results from this research showed that on-flap and off-flap epi-LASIK has comparable postoperatively clinical outcomes. Off-flap epi-LASIK had more rapid re-epithelialization and normalization of epithelial morphology compared to on-flap epi-LASIK by in vivo confocal microscopy. Therefore, from this study, it is suggested that leaving the epithelial flap off is a better procedure than the conventional on-flap technique. The second study compares the corneal epitheliotrophic capacity of different human blood-derived preparations, including cord blood serum (CBS), peripheral blood serum (PBS), and fresh frozen plasma (FFP) on bovine corneal epithelial cells. Of the three human blood derivatives evaluated, CBS had the highest concentrations of epithelial growth factor (EGF),transforming growth factor β1(TGFβ1), and hyaluronic acid (HA) (P<0.05). CBS demonstrated the highest ability to promote cellular proliferation, followed by PBS and FFP (P<0.05). CBS was also the best in promoting cellular differentiation, as shown by scanning electron microscopy and transepithelial resistance. CBS is generally superior to PBS in promoting corneal epithelial proliferation and differentiation. In conclusion, results from this study provided a new alternative to supplement epitheliotrophic factors for patients with poor corneal wound healing.