准分子激光兔角膜切削术后应用抗氧化剂的实验研究
|
摘要
目的：探讨氧自由基对准分子激光屈光性角膜切削术(photorefractive keratectomy，PRK)后凋亡机制介导的角膜创伤愈合反应的作用，以及局部应用抗氧化剂维生素C(Vit C)，维生素E(Vit E)对术后角膜的影响。
方法：将28只兔分成3组，其中4只为正常对照；其余24只实验兔分成2组，每组12只，行双眼-5.0D PRK。一组术后左眼每日结膜下注射Vit C0.1g，右眼为对照；另一组术后左眼每日结膜下注射VitE25mg，右眼为对照。于术后1，3，7，14天测定角膜组织超氧化物歧酶(superoxide dismutase，SOD)，丙二醛(malondialdehyde，MDA)及谷胱甘肽过氧化物酶(glutathioneperoxide，GPx)的变化，同时术后定期制作病理切片，检测角膜基质细胞数量，采用脱氧核苷酸末端转移酶介导的缺口末端标记法(terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling，TUNEL)检测角膜细胞凋亡，光镜观察凋亡细胞形态，定量统计比较凋亡水平差别。
结果：(1) PRK术后1，3天角膜SOD，GPx活性小于对照组(P＜0.05)，MDA的水平高于对照组(P＜0.05)。局部应用抗氧化剂组其角膜SOD，GPx活性高于单纯激光组(P＜0.05)，MDA水平低于单纯激光组(P＜0.05)。(2) 角膜基质细胞凋亡在1-14天均与正常对照组有差别(P＜0.01)，在应用抗氧化剂组角膜基质细胞凋亡较单纯激光组减少(P＜0.05)。(3) 术后角膜基质细胞数增加，应用抗氧化剂组角膜基质细胞增生较单纯激光组减少(P＜0.05)。
结论：准分子激光屈光性角膜切削术后早期，角膜存在着脂质过氧化形式介导的自由基性的组纵损伤破坏，促进角膜细胞的凋亡；局部应用抗氧化剂
安徽医科大学硕士学位论文
vitC，VitE能最大限度的减轻PRK术后炎性反应，降低角膜过氧化损伤，阻止术
后细胞凋亡，减轻角膜基质反应性过度增生，降低术后屈光回退和haze形成。
Objective To investigate the effect of free oxygen radical in the rabbit cornea apoptosis-induced corneal wound healing respone after pholorefractive keratectomy and the influence of topical antioxidants Vitamin C, Vitamin E on cornea tissue having undergone PRK.
Methods 28 rabbits were divided 3 groups, 4 rabbits served as controls and the others served as experimental rabbits which were divided 2 groups, each consisting of 12 animals. They were underwent bilateral photorefractive keratectomy with 193nm argon fluoride excimer laser to correct 5 diopters of myopia. Following treatment, one group
the left eye of each rabbit was subconjunctival injection with Vitamin C 0.1 every day.
The right eye served as control. The other group the left eye of each rabbit was
subconjunctival injection with Vitamin E 25mg every day. The right eye served as control.Rabbits were killed at 1,3,7 and 14 days respectively, then measured the activity of corneal superoxide dismutase(SOD) with xanthine oxidase method and glutathioneperoxide(GPx) with modified glutathione, the level of corneal malondialdehyde(MDA) with barbituric acid reaction. At the same time, corneas were prepared for H.E.staining to observe histopathological changes and the quantities of corneal stroma cells after PRK. Corneal cell apoptosis was evaluated by terminal deoxyribonucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling (TUNEL) to detect DNA fragmentation.
Results (1) It was found that significant decrease in SOD, GPx activity (P<0.05) and increase in MDA level (P<0.05) compared with the control following 1,3 days after PRK. But the corneal SOD, GPx activity in effect of antioxidants group is significant higher than that of PRK group (P<0.05). The level of MDA in effect of antioxidants group is significant lower than that of PRK group (P<0.05). (2) Apoptosis was detected remarkably in anterior keratocytes from 1 day to 14 days (P<0.01). The level of keratocyte apoptosis in effect of antioxidants group less then PRK group (PO.05). (3) The number of keratocytes were increased after operation. The level of proliferation in effect of antioxidants group less then PRK group (P<0.05).
Conclusions The presence of free oxygen radical mediated tissue damage in the cornea with lipid peroxidation following excimer laser keratectomy. They may contribute to an increase apoptosis in cornea after PRK. The topical application of antioxidants Vitamin C or Vitamin E can decrease corneal oxygen radical tissue damage after excimer laser keratectomy, inhibiting keratocyte apoptosis, preventing the over-proliferation and
all of them may reduce postoperative corneal haze and regression.
方法：将28只兔分成3组，其中4只为正常对照；其余24只实验兔分成2组，每组12只，行双眼-5.0D PRK。一组术后左眼每日结膜下注射Vit C0.1g，右眼为对照；另一组术后左眼每日结膜下注射VitE25mg，右眼为对照。于术后1，3，7，14天测定角膜组织超氧化物歧酶(superoxide dismutase，SOD)，丙二醛(malondialdehyde，MDA)及谷胱甘肽过氧化物酶(glutathioneperoxide，GPx)的变化，同时术后定期制作病理切片，检测角膜基质细胞数量，采用脱氧核苷酸末端转移酶介导的缺口末端标记法(terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling，TUNEL)检测角膜细胞凋亡，光镜观察凋亡细胞形态，定量统计比较凋亡水平差别。
结果：(1) PRK术后1，3天角膜SOD，GPx活性小于对照组(P＜0.05)，MDA的水平高于对照组(P＜0.05)。局部应用抗氧化剂组其角膜SOD，GPx活性高于单纯激光组(P＜0.05)，MDA水平低于单纯激光组(P＜0.05)。(2) 角膜基质细胞凋亡在1-14天均与正常对照组有差别(P＜0.01)，在应用抗氧化剂组角膜基质细胞凋亡较单纯激光组减少(P＜0.05)。(3) 术后角膜基质细胞数增加，应用抗氧化剂组角膜基质细胞增生较单纯激光组减少(P＜0.05)。
结论：准分子激光屈光性角膜切削术后早期，角膜存在着脂质过氧化形式介导的自由基性的组纵损伤破坏，促进角膜细胞的凋亡；局部应用抗氧化剂
安徽医科大学硕士学位论文
vitC，VitE能最大限度的减轻PRK术后炎性反应，降低角膜过氧化损伤，阻止术
后细胞凋亡，减轻角膜基质反应性过度增生，降低术后屈光回退和haze形成。
Objective To investigate the effect of free oxygen radical in the rabbit cornea apoptosis-induced corneal wound healing respone after pholorefractive keratectomy and the influence of topical antioxidants Vitamin C, Vitamin E on cornea tissue having undergone PRK.
Methods 28 rabbits were divided 3 groups, 4 rabbits served as controls and the others served as experimental rabbits which were divided 2 groups, each consisting of 12 animals. They were underwent bilateral photorefractive keratectomy with 193nm argon fluoride excimer laser to correct 5 diopters of myopia. Following treatment, one group
the left eye of each rabbit was subconjunctival injection with Vitamin C 0.1 every day.
The right eye served as control. The other group the left eye of each rabbit was
subconjunctival injection with Vitamin E 25mg every day. The right eye served as control.Rabbits were killed at 1,3,7 and 14 days respectively, then measured the activity of corneal superoxide dismutase(SOD) with xanthine oxidase method and glutathioneperoxide(GPx) with modified glutathione, the level of corneal malondialdehyde(MDA) with barbituric acid reaction. At the same time, corneas were prepared for H.E.staining to observe histopathological changes and the quantities of corneal stroma cells after PRK. Corneal cell apoptosis was evaluated by terminal deoxyribonucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling (TUNEL) to detect DNA fragmentation.
Results (1) It was found that significant decrease in SOD, GPx activity (P<0.05) and increase in MDA level (P<0.05) compared with the control following 1,3 days after PRK. But the corneal SOD, GPx activity in effect of antioxidants group is significant higher than that of PRK group (P<0.05). The level of MDA in effect of antioxidants group is significant lower than that of PRK group (P<0.05). (2) Apoptosis was detected remarkably in anterior keratocytes from 1 day to 14 days (P<0.01). The level of keratocyte apoptosis in effect of antioxidants group less then PRK group (PO.05). (3) The number of keratocytes were increased after operation. The level of proliferation in effect of antioxidants group less then PRK group (P<0.05).
Conclusions The presence of free oxygen radical mediated tissue damage in the cornea with lipid peroxidation following excimer laser keratectomy. They may contribute to an increase apoptosis in cornea after PRK. The topical application of antioxidants Vitamin C or Vitamin E can decrease corneal oxygen radical tissue damage after excimer laser keratectomy, inhibiting keratocyte apoptosis, preventing the over-proliferation and
all of them may reduce postoperative corneal haze and regression.
引文
1．刘祖国，陈加祺．积极预防和减少屈光手术的并发症．中华眼科杂志2002；38(2)：65-66
2．刘美光，李镜海．近视手术方式的选择及联合手术．见：李镜海，肖瑛主编．近视手术治疗学(第一版)．北京：人民卫生出版社，2001：174
3. Wilson SE. Role ofapoptosis in wound healing in the cornea. Cornea, 2000 May; 19(3Suppl): S7-12
4. Wison SE, Kim WJ. Keratocyte apoptosis: implication on corneal wound healing tissue organization and disease, Invest Ophthahnol Vis Sci, 1998; 39(2): 220-226
5. Bilgihan K, Bilgihan A, Akata F, et al. Excimer laser corneal surgery and free oxygen radicals. Jpn J Ophthalmol, 1996; 40(2):154-157
6. Hayashi S, Ishimoto S, Wu GS, et al. Oxygen free radical damage in the cornea after excimer laser therapy. Br J Ophthahnol, 1997 Feb; 81 (2): 141-144
7. Pettit GH, Ediger MN, Hahn DW, et al. Electron paramagnetic resonance spectroscopy of free radicals in corneal tissue following excimer laser irradiation. Lasers Surg Med, 1996; 18(1): 367-372
8. O'Brien TP, Li Q, Ashraf MF, et al. Inflammatory Response in the early stages of wound healing after excimer laser keratectomy. Arch Ophthahnol, 1998; 116(11): 1470-1474
9. Wilson SE. Analysis of the keratocyte apoptosis, keratocyte proliferation, and myofibroblast transformation responses after photorefractive keratectomy and laser in situ keratomileusis. Trans Am Ophthahnol Soc, 2002; 100(2): 411-433
10. Mohan RR, Hutcheon AE, Choi R, et al. Apoptosis, necrosis, proliferation, and myofibroblast generation in the stroma following LASIK and PRK. Exp Eye Res 2003 Jan; 76(1): 71-87
11. Wilson SE, Mohan RR, Hong J W, et al. The wound healing response after laser in situ keratomileusis and photorefractive keratectomy. Arch Opthalmol, 2001; 119(6): 889-896
12. Ramirez-Florez S, Maurice DM. Inflammatory cells, refractive regression and haze after excimer laser PRK. J Refract Surg, 1996:12(3): 370-381.
13. Tuominen IS, Tervo TM, Teppo AM, et al. Human tear fluid PDGF-BB, TNF-alpha and TGF-betal vs corneal haze and regeneration of corneal epithelium and subbasal never plexus after PRK. Exp Eye Res, 2001; 72(6): 631-641
14．葛坚，郭彦．眼的发育生物学．见：葛坚主编．眼科学(第一版)．北京：人民卫生出版社，2002：4-5
15. Ormerod MG. Investigating the relationship between the cell cycle and apoptosis using flow cytometry. J Immunol Methods, 2002; 265(1-2): 73-80
16．李莹，庞国祥，金玉梅，等．共聚焦显微镜在屈光性角膜手术中的应用．眼科 2001；10(2)：91-94
17. Helena MC, Baerveldt F, Kim WJ, et al. Keratocyte apoptosis after corneal surgery. Invest Ophthahnol Vis Sci, 1998; 39(2): 276-283.
18. Wilson SE. Everett Kinsey Lecture. Keratocyte apoptosis in refractive surgery. CLAOJ, 1998; 24(3): 181-185
19. Weber BA, Gan L, Fagerholm PP. Short-term impact of corticosteroids on hyaluronan and epithelial hyperplasia in the rabbit cornea after photorefractive keratectomy. Cornea, 2001; 20(3): 321-324
20. Chen C, Michelini-Norris B, Stevens S, et al. Measurement of mRNAs for TGFss and extracellular matrix proteins in corneas of rats alter PPK. Invest Ophthalmol Vis Sci, 2000; 41(13): 4108-4116
21. Winkler MC, Reischl U, Lohmarm CP. Corneal haze after photorefractive keratectomy for myopia: role of collagen Ⅳ mRNA typing as a predictor of haze. J Cataract Refract Surg, 2002; 24(8): 1446-1451
22. Shimmura S, Masumizu T, Nakai Y, et al. Excimer laser-induced hydroxyl radical formation and keratocyte death in vitro, Invest Ophthalmol Vis Sci, 1999,40:1245-1249.
23．王雁，赵堪兴，Taylor HR，等．准分子激光术后超微结构变化与生物组织反应．眼科研究，2001；19(4)：293-296
24. Niizuma T, Ito S, Hayashi M, et al. Cooling the cornea to prevent side effects of photorefractive keratectomy. J Refract Corneal Surg, 1994; 10(2Suppl): S262-266
25. Kasetsuwan N, Wu FM, Hsioh F, et al. Effect of topical ascorbic acid on free radical tissue damage and inflammatory cell influx in the comea after excimer laser corneal surgery. Arch Ophthalmol, 1999 May; 117(5): 649-652
26. Costagliola C, Di Giovanni A, Rinaldi M, et al. Photorefractive keratectomy and cataract. Surg Ophthahnol, 1997; 42(Suppl 1): S133-140
27. Costagliola C, Balestrieri P, Fioretti F, et al. ArF 193nm excimer laser corneal surgery and photo-oxidation stress in aqueous humor and lens of rabbit: one-month following up. Curr Eye Res, 1996; 15(4): 355-361
28. Giasson CJ, Bleau G, Brunette I. Short-term oxidative status of lens and aqueous humor after excimer laser photorefractive keratectomy. J Refract Surg 1999; 15(6): 673-678
29. Wachtlin J, Blasig IE, Schrunder S, et al. PRK and LASIK-their potential risk of cataractogenesis:lipid peroxidation changes in the aqueous humor and crystalline lens of rabbits. Cornea, 2000, Jan; 19(1): 75-79
30. Siflinger-Birnboim A, Goligorsky MS, Delvecchio PJ, et al. Involvement of Ca~(2+) in the H_2O_2-induced increase in endothelial permeability. Am J Physiol, 1996; 270(6Pt1):L973-978.
31. Vetrugno M, Quaranta GM, Maino A, et al. A randomized, comparative study of fluorometholone 0.2％ and fluorometholone 0.1％ acetate after photorefractive keratectomy. Eur J Ophthahmol, 2000; 10(1): 39-45.
32. Brancato R, Schiavone N, Siano S, et al. Prevention of corneal keratocyte apoptosis after argon fluoride excimer laser irradiation with the free radical scavenger ubiquinone Q10. Eur J Ophthalmol, 2000; 10(1): 32-38
33. Wilson SE, Liang Q, Kim WJ, et al. Lacrimal gland HGF, KGF, and ECG mRNA levels increase after corneal epithelial wounding, Invest Ophthalmol Vis Sci, 1999; 40(10): 2185-2190
34. Wilson SE, Liu JJ, Mohan RR, et al. Stromal-epithelial interactions in the cornea. Prog Retin Eye Res, 1999; 18(3): 293-309
35．李纳，邹留河，董东生．抗氧化物质与角膜碱烧伤．眼科 2002；11(4)；243-246．
36. Cho KS, Lee EH, Choi JS, et al. Reactive oxygen species induced apoptosis and necrosis in bovine corneal endothelial cells, Invest Ophthalmol Vis Sci, 1999; 40(5): 911-919
37. Stoian I, Oros A, Moldoveanu E. Apoptosis and free radicals. Biochem Mol Med, 1996; 59(1): 93-97
38．李福洋，惠宏襄．氧自由基对基因表达的调节机理。生命科学，1999；11(S：S)：28-31
39. Allen RG, Balin AK. Oxidative influnce on development and differentiation. Free Radical Biology & Medicine, 1989; 6(6): 631-661
40．吴伟康．细胞凋亡与疾病．见：金惠铭主编．病理生理学(第五版)．北京：人民卫生出版社，2001：183-184
41. Behnding A, Svensson B, Marklund SL, et al. Superoxide dismutase isoenzymes in the human eye. Invest Ophthalmol Vis Sci, 1998; 39(3):471-475
42．美浓真，林洁．生物体内的活性氧清除系统：抗氧化剂．日本医学介绍，1994；15(7)：295-296
43. Yis O, Bilgihan A, Bilgihan K, et al. The effect ofexcimer iaser keratectomy on corneal glutathione peroxidase activities and aqueous humor selenium levels in rabbits. Graefies Arch Clin Exp Ophthalmol, 2002; 240(6): 499-502
44．李忌，郑荣梁．活性氧诱导细胞凋亡．细胞生物学杂志，1997；19(3)：115-119．
45. Brancato R, Fiore T, Papucci L, et al. Concimitant effect of topical ubiquinone Q10 and vitamin E to prevent keratocyte apoptosis after excimer laser photoablation in rabbits. J Refract Surg 2002; 18(2): 135-139
46. Bilgihan K, Ozdek S, Ozogul C, et al. Topical Vitamin E and hydrocortisone acetate treatment after photorefractive keratectomy. Eye 2000 Apr; 14(pt2):231-237.
47. Vetrugno M, Maino A, Cardia G, et al. A randomised, double masked, clinical trial of high dose vitamin A and vitamin E supplementation after photorefractive keratectomy. Br J Ophthahnol, 2001; 85(5): 537-539
48．赵保路．脂质过氧化和氧自由基．见：赵保路编著．氧自由基和天然抗氧化剂(第一版)．北京：科学出版社．1999：89
49. Bilgihan K, Bilgihan A, Adiguzel U, et al. Keratocyte apoptosis and corneal antioxidant enzyme activities after refractive corneal surgery. Eye, 2002; 16(1):63-68
2．刘美光，李镜海．近视手术方式的选择及联合手术．见：李镜海，肖瑛主编．近视手术治疗学(第一版)．北京：人民卫生出版社，2001：174
3. Wilson SE. Role ofapoptosis in wound healing in the cornea. Cornea, 2000 May; 19(3Suppl): S7-12
4. Wison SE, Kim WJ. Keratocyte apoptosis: implication on corneal wound healing tissue organization and disease, Invest Ophthahnol Vis Sci, 1998; 39(2): 220-226
5. Bilgihan K, Bilgihan A, Akata F, et al. Excimer laser corneal surgery and free oxygen radicals. Jpn J Ophthalmol, 1996; 40(2):154-157
6. Hayashi S, Ishimoto S, Wu GS, et al. Oxygen free radical damage in the cornea after excimer laser therapy. Br J Ophthahnol, 1997 Feb; 81 (2): 141-144
7. Pettit GH, Ediger MN, Hahn DW, et al. Electron paramagnetic resonance spectroscopy of free radicals in corneal tissue following excimer laser irradiation. Lasers Surg Med, 1996; 18(1): 367-372
8. O'Brien TP, Li Q, Ashraf MF, et al. Inflammatory Response in the early stages of wound healing after excimer laser keratectomy. Arch Ophthahnol, 1998; 116(11): 1470-1474
9. Wilson SE. Analysis of the keratocyte apoptosis, keratocyte proliferation, and myofibroblast transformation responses after photorefractive keratectomy and laser in situ keratomileusis. Trans Am Ophthahnol Soc, 2002; 100(2): 411-433
10. Mohan RR, Hutcheon AE, Choi R, et al. Apoptosis, necrosis, proliferation, and myofibroblast generation in the stroma following LASIK and PRK. Exp Eye Res 2003 Jan; 76(1): 71-87
11. Wilson SE, Mohan RR, Hong J W, et al. The wound healing response after laser in situ keratomileusis and photorefractive keratectomy. Arch Opthalmol, 2001; 119(6): 889-896
12. Ramirez-Florez S, Maurice DM. Inflammatory cells, refractive regression and haze after excimer laser PRK. J Refract Surg, 1996:12(3): 370-381.
13. Tuominen IS, Tervo TM, Teppo AM, et al. Human tear fluid PDGF-BB, TNF-alpha and TGF-betal vs corneal haze and regeneration of corneal epithelium and subbasal never plexus after PRK. Exp Eye Res, 2001; 72(6): 631-641
14．葛坚，郭彦．眼的发育生物学．见：葛坚主编．眼科学(第一版)．北京：人民卫生出版社，2002：4-5
15. Ormerod MG. Investigating the relationship between the cell cycle and apoptosis using flow cytometry. J Immunol Methods, 2002; 265(1-2): 73-80
16．李莹，庞国祥，金玉梅，等．共聚焦显微镜在屈光性角膜手术中的应用．眼科 2001；10(2)：91-94
17. Helena MC, Baerveldt F, Kim WJ, et al. Keratocyte apoptosis after corneal surgery. Invest Ophthahnol Vis Sci, 1998; 39(2): 276-283.
18. Wilson SE. Everett Kinsey Lecture. Keratocyte apoptosis in refractive surgery. CLAOJ, 1998; 24(3): 181-185
19. Weber BA, Gan L, Fagerholm PP. Short-term impact of corticosteroids on hyaluronan and epithelial hyperplasia in the rabbit cornea after photorefractive keratectomy. Cornea, 2001; 20(3): 321-324
20. Chen C, Michelini-Norris B, Stevens S, et al. Measurement of mRNAs for TGFss and extracellular matrix proteins in corneas of rats alter PPK. Invest Ophthalmol Vis Sci, 2000; 41(13): 4108-4116
21. Winkler MC, Reischl U, Lohmarm CP. Corneal haze after photorefractive keratectomy for myopia: role of collagen Ⅳ mRNA typing as a predictor of haze. J Cataract Refract Surg, 2002; 24(8): 1446-1451
22. Shimmura S, Masumizu T, Nakai Y, et al. Excimer laser-induced hydroxyl radical formation and keratocyte death in vitro, Invest Ophthalmol Vis Sci, 1999,40:1245-1249.
23．王雁，赵堪兴，Taylor HR，等．准分子激光术后超微结构变化与生物组织反应．眼科研究，2001；19(4)：293-296
24. Niizuma T, Ito S, Hayashi M, et al. Cooling the cornea to prevent side effects of photorefractive keratectomy. J Refract Corneal Surg, 1994; 10(2Suppl): S262-266
25. Kasetsuwan N, Wu FM, Hsioh F, et al. Effect of topical ascorbic acid on free radical tissue damage and inflammatory cell influx in the comea after excimer laser corneal surgery. Arch Ophthalmol, 1999 May; 117(5): 649-652
26. Costagliola C, Di Giovanni A, Rinaldi M, et al. Photorefractive keratectomy and cataract. Surg Ophthahnol, 1997; 42(Suppl 1): S133-140
27. Costagliola C, Balestrieri P, Fioretti F, et al. ArF 193nm excimer laser corneal surgery and photo-oxidation stress in aqueous humor and lens of rabbit: one-month following up. Curr Eye Res, 1996; 15(4): 355-361
28. Giasson CJ, Bleau G, Brunette I. Short-term oxidative status of lens and aqueous humor after excimer laser photorefractive keratectomy. J Refract Surg 1999; 15(6): 673-678
29. Wachtlin J, Blasig IE, Schrunder S, et al. PRK and LASIK-their potential risk of cataractogenesis:lipid peroxidation changes in the aqueous humor and crystalline lens of rabbits. Cornea, 2000, Jan; 19(1): 75-79
30. Siflinger-Birnboim A, Goligorsky MS, Delvecchio PJ, et al. Involvement of Ca~(2+) in the H_2O_2-induced increase in endothelial permeability. Am J Physiol, 1996; 270(6Pt1):L973-978.
31. Vetrugno M, Quaranta GM, Maino A, et al. A randomized, comparative study of fluorometholone 0.2％ and fluorometholone 0.1％ acetate after photorefractive keratectomy. Eur J Ophthahmol, 2000; 10(1): 39-45.
32. Brancato R, Schiavone N, Siano S, et al. Prevention of corneal keratocyte apoptosis after argon fluoride excimer laser irradiation with the free radical scavenger ubiquinone Q10. Eur J Ophthalmol, 2000; 10(1): 32-38
33. Wilson SE, Liang Q, Kim WJ, et al. Lacrimal gland HGF, KGF, and ECG mRNA levels increase after corneal epithelial wounding, Invest Ophthalmol Vis Sci, 1999; 40(10): 2185-2190
34. Wilson SE, Liu JJ, Mohan RR, et al. Stromal-epithelial interactions in the cornea. Prog Retin Eye Res, 1999; 18(3): 293-309
35．李纳，邹留河，董东生．抗氧化物质与角膜碱烧伤．眼科 2002；11(4)；243-246．
36. Cho KS, Lee EH, Choi JS, et al. Reactive oxygen species induced apoptosis and necrosis in bovine corneal endothelial cells, Invest Ophthalmol Vis Sci, 1999; 40(5): 911-919
37. Stoian I, Oros A, Moldoveanu E. Apoptosis and free radicals. Biochem Mol Med, 1996; 59(1): 93-97
38．李福洋，惠宏襄．氧自由基对基因表达的调节机理。生命科学，1999；11(S：S)：28-31
39. Allen RG, Balin AK. Oxidative influnce on development and differentiation. Free Radical Biology & Medicine, 1989; 6(6): 631-661
40．吴伟康．细胞凋亡与疾病．见：金惠铭主编．病理生理学(第五版)．北京：人民卫生出版社，2001：183-184
41. Behnding A, Svensson B, Marklund SL, et al. Superoxide dismutase isoenzymes in the human eye. Invest Ophthalmol Vis Sci, 1998; 39(3):471-475
42．美浓真，林洁．生物体内的活性氧清除系统：抗氧化剂．日本医学介绍，1994；15(7)：295-296
43. Yis O, Bilgihan A, Bilgihan K, et al. The effect ofexcimer iaser keratectomy on corneal glutathione peroxidase activities and aqueous humor selenium levels in rabbits. Graefies Arch Clin Exp Ophthalmol, 2002; 240(6): 499-502
44．李忌，郑荣梁．活性氧诱导细胞凋亡．细胞生物学杂志，1997；19(3)：115-119．
45. Brancato R, Fiore T, Papucci L, et al. Concimitant effect of topical ubiquinone Q10 and vitamin E to prevent keratocyte apoptosis after excimer laser photoablation in rabbits. J Refract Surg 2002; 18(2): 135-139
46. Bilgihan K, Ozdek S, Ozogul C, et al. Topical Vitamin E and hydrocortisone acetate treatment after photorefractive keratectomy. Eye 2000 Apr; 14(pt2):231-237.
47. Vetrugno M, Maino A, Cardia G, et al. A randomised, double masked, clinical trial of high dose vitamin A and vitamin E supplementation after photorefractive keratectomy. Br J Ophthahnol, 2001; 85(5): 537-539
48．赵保路．脂质过氧化和氧自由基．见：赵保路编著．氧自由基和天然抗氧化剂(第一版)．北京：科学出版社．1999：89
49. Bilgihan K, Bilgihan A, Adiguzel U, et al. Keratocyte apoptosis and corneal antioxidant enzyme activities after refractive corneal surgery. Eye, 2002; 16(1):63-68