抗血管生長因子藥物 bevacizumab 在隱形眼鏡引起之紐西蘭白兔角膜新生血管的療效探討

The therapeutic effects of bevacizumab on corneal neovascularization in a rabbit model of closed contact lens wear

陳彥名(Yan-Ming Chen)

指導教授 ： 胡芳蓉

國立臺灣大學/醫學院/臨床醫學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.03426

摘要

角膜為透明無血管的組織。在感染、炎症、缺氧、眼輪部缺損等疾病導致新生血管入侵角膜後，將導致炎症加劇及角膜透明度降低。角膜新生血管將增加角膜移植手術後的排斥率；也會在準分子雷射近視手術中造成出血以致影響手術結果。角膜新生血管的生成主因為角膜在發炎缺氧時，產生了特定的生長因子，吸引血管內皮細胞由結膜部位往中心角膜爬行；其中血管內皮生長因子VEGF (Vascular endothelium growth factor)主要控制了血管內皮細胞的增生、遷移、管壁形成和通透性的變化。
而在胡芳蓉教授和陳偉勵助理教授的實驗室中也發現:動物實驗中長期配戴隱形眼鏡而角膜長期缺氧的紐西蘭白兔角膜會產生新生血管，並且在這些角膜上會有 VEGF mRNA的增加。這些血管內皮生長因子(VEGF)的增加被認為和角膜缺氧有很大的關係。
而目前臨床上眼角膜新生血管的處理主要以類固醇等藥物來控制，但效果有限；而使用雷射的燒灼來治療眼角膜新生血管除了具侵入性外，效果也有限。而最新許多研究發現使用抗血管內皮生長因子anti-VEGF，Bevacizumab (Avastin) 此種抗癌藥物, 除了對老人性黃斑部病變新生血管、高度進視黃斑部病變新生血管、增殖性糖尿病視網膜病變等等視網膜新生血管病變有療效之外，對於抑制角膜新生血管的生成，anti-VEGF，Bevacizumab (Avastin)在動物實驗以及人體臨床試驗也有不錯的成效，然而給藥的時間點似乎會造成不同的治療效果。因此，本研究乃計畫嘗試使用(1) 在配戴隱形眼鏡的紐西蘭白兔身上早期(同時)給予Bevacizumab以及晚期(一個月後)再給予Bevacizumab結膜下注射(subconjunctival injection)來評估抑制角膜新生血管的效果以及(2) 比較戴一個月隱形眼鏡，當隱形眼鏡摘除後再給予Bevacizumab結膜下注射對於角膜新生血管消退是否有更好的效果 (3) 觀察配戴隱形眼鏡的紐西蘭白兔角膜新生血管上周圍細胞 (pericyte) 的變化，以及(4) 分析不同給藥組別的Bevacizumab這個藥物的角膜滲透性和停留的時間是否有差別。希望能藉此找出對臨床上許多因角膜長期發炎或缺氧而造成角膜新生血管的病人更安全有效的治療方法。
我們的實驗結果發現: (1) 在配戴隱形眼鏡的紐西蘭白兔身上早期給予Bevacizumab對於角膜新生血管是有抑制的作用,且有統計學上顯著的意義; 然而一個月後再給予結膜下藥物注射雖然對角膜新生血管也有些許抑制的作用,然而卻未達到統計學上顯著的意義 (2) 戴一個月隱形眼鏡，當隱形眼鏡摘除後再給予Bevacizumab結膜下注射對於角膜新生血管的消退雖然有比對照組稍快，然而卻也未達統計學上顯著的意義 (3) 在免疫螢光染色組織切片下可見白兔角膜新生血管上的確覆蓋有明顯的周圍細胞(pericyte)，這個現象推測可能和晚期給藥治療效果不彰有關 (4) Bevacizumab這個藥物在角膜的滲透性和停留的時間在正常角膜停留最久，至少可停留七天以上; 在早期給藥組和晚期給藥組則在第三天達到高峰且可滲透到角膜中心區域，到第七天則幾乎完全消失; 這種藥物快速消失的現象是否和藥物抑制角膜新生血管的作用有關則還需要未來更進一步深入研究和探討來釐清。
我們企圖找出對臨床上許多因角膜長期發炎或缺氧而造成角膜新生血管的病人更安全有效的治療方法。由我們動物實驗的研究結果顯示，使用Bevacizumab結膜下注射來治療因為配戴隱形眼鏡角膜缺氧所造成的角膜新生血管，越早給藥效果會比晚期再給藥效果來得好。另外，藉由免疫螢光組織染色的方法發現，白兔角膜新生血管上的確覆蓋有明顯的周圍細胞(pericyte); 此外，在治療組Bevacizumab這個藥物在角膜停留的時間都比正常角膜(對照組)來得短。雖然Bevacizumab這個藥物目前離真正大量臨床應用還有許多問題需要克服，但初步動物實驗的結果發現了許多寶貴的經驗，也替未來的應用提供了一條可能的出路。

關鍵字

角膜新生血管 ； 血管內皮生長因子 ； 抗血管內皮生長因子藥物 ； 血管周圍細胞

並列摘要

In normal cornea, the avascularity is important for maintaining corneal transparency. Corneal neovascularization induced by various pathological stimuli not only impairs visual accuracy, increases inflammation, increases the risk of graft rejection after corneal transplantation, but also impairs surgical outcome after LASIK. During the pathogenesis of corneal neovascularization, vascular endothelial growth factor (VEGF) plays a key role by controlling vascular endothelial cellular proliferation, migration, tube formation and change of transvascular permeability. In previous studies, prolonged wear of gas impermeable contact lens can induce corneal hypoxia and thus induce corneal neovascularization. In professor Hu and Dr. Chen’s lab, corneal neovascularization could be induced experimentally in New Zealand white rabbits by closed and prolonged wear of gas permeable contact lens. Interestingly, increased VEGF mRNA level was detected in these corneal samples. Clinically, corneal neovascularization is usually controlled by administration of steroid or argon laser photocoagulation. However, the result is not satisfactory. Steroid is well known to induce glaucoma and cataract formation. Argon laser photocoagulation has the potential to damage cornea. Recently, anti-VEGF antibody--- bevacizumab (Avastin) is reported to be safe and effective in treating various types of retinal vascular associated disorders. Besides, the effectiveness of bevacizumab on corneal neovascularization is also reported. Nonetheless, the time point of bevacizumab administration may result in different treatment effects. In this study, we aim to evaluate the possibility of using subconjunctival injection of bevacizumab to control corneal neovasculization induced by closed and prolonged contact lens wear in rabbit model and also to compare the treatment effects of different time points of bevacizumab administration in this animal model. We will also evaluate the formation of pericytes in the mature corneal neovasculization induced by closed and prolonged contact lens wear in rabbit model. Besides, we will also try to find out the intracorneal diffusion of bevacizumab (Avastin) in different treatment groups and also the control group. Finally, we will try to evaluate the effects of bevacizumab in treating corneal neovascularization induced by prolonged contact lens wear, and apply our study results to treat related patients.
Our results showed the proof of early but not late subconjunctival injection of bevacizumab in inhibiting corneal neovasculization induced by closed and prolonged contact lens wear in rabbit model. The mature vessels induced by closed and prolonged contact lens wear in rabbits were covered by pericytes. Comparing to normal corneas, the diffusion of bevacizumab in cornea is more transient in both early and late treatment groups. The preliminary results of the animal study provided us a new way and more information for further new methods in treating corneal neovascularization.

並列關鍵字

Corneal neovascularization ； Vascular endothelium growth factor(VEGF) ； Bevacizumab (Avastin) ； Pericyte

參考文獻

2.Epstein RJ, Stulting RD, Hendricks RL, et al. Corneal neovascularization: pathogenesis and inhibition. Cornea 1987; 6: 250-257.

3.Cursiefen C, Kuchle M, Naumann GO. Angiogenesis in corneal diseases: histopathologic evaluation of 254 human corneal buttons with neovascularization. Cornea 1998; 17:611–613.

4.Chang JH, Gabison EE, Kato T, et al. Corneal neovascularization. Curr Opin Ophthalmol 2001; 12: 242-249.

5.Ma DH, Chen JK, Zhang F, Lin KY, Yao JY, Yu JS. Regulation of corneal angiogenesis in limbal stem cell deficiency. Prog Retin Eye Res 2006;25:563-590.

8.Cursiefen C, Rummelt C, Kuchle M. Immunohistochemical localization of vascular endothelial growth factor, transforming growth factor alpha, and transforming growth factor beta1 in human corneas with neovascularization. Cornea 2000; 19:526–533.