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角膜間質層角質細胞的自殺行為與角膜移植手術的關係(II)

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角膜間質層角質細胞的自殺行為與角膜移植手術的關係(II)

英文計劃名稱

The association of keratocytic apoptosis with corneal transplantation

(II)

計劃編號

NSC89-2314-B-002-499

執行期限

89 年 8 月 1 月至 90 年 7 月 31 日

主持人資料

主持人:王一中 共同主持人:胡芳蓉 計劃參與人員:陳偉勵 協同主持人:高惠陽 執行機構:臺大醫院眼科部

關鍵詞

角膜,自殺反應,角膜間質細胞,角膜表皮細胞,全層角膜移植

Key Words

cornea, keratocyte, apoptosis, penetrating keratoplasty, corneal epithelial cell

中文摘要

角膜細胞自殺反應為近幾年來國內外文獻的熱門研究主題。無論在準分子雷射 近視手術或角膜移植手術當中,角膜細胞的反應都將可能影響術後的傷口復原或視力 進步狀況。本計劃乃利用紐西蘭白兔的角膜作為研究材料。旨在探究不同的角膜貯存 方式,或角膜運送過程中的晃動現象是否引發角質細胞自殺反應;本實驗在去年的研 究結果初步得知,角膜保存時間愈久,處理過程中經過搖晃等等將導致較嚴重角膜表 皮細胞或角膜間質細胞的自殺行為。今年則將角膜間質細胞的狀況再度量化,已確定

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去年之結果為正確無誤。

Abstr act:

Several investigators have shown recently that anterior stromal keratocytes undergo programmed cell death (apoptosis ) in response to corneal epithelial injury, and it is

believed that this phenomenon plays an important role in wound healing after many kinds of corneal surgery such as photorefractive keratectomy (PRK). However, It is not known whether degree of apoptosis may account for variations of wound healing observed after penetrating keratoplasty (PKP). The purpose of this study is to investigate whether keratocyte apoptosis takes place during storage and process period of donor cornea. New Zealand white rabbits are used as the animal models. The preliminary result of last year showed that the way of preservation and transportation will affect keratocyte apoptosis in corneal buttons. In this year, we further quantify the previous result, and confirm the previous result.

計劃緣由及目的

The finding of ker atocyte apoptosis

In the past, the stromal keratocytes of adult cornea have been characterized as quiescent cells populating the stroma. Recent researches and the introduction of laser refractive procedures have made this notion more important.

Keratocyte apoptosis extend to a depth of 50to 200 micrometer, depending on the

magnitude of the epithelial injury and the species studied. Typical in situ staining with the terminal deoxyribonucletidyl transferase-mediated d-uridine 5’-triphosphate-dihoxigenin nick-end labeling (TUNEL) assay is first noted at approximately 1 hour after injury, but it has been noted to peak at approximately 4 hours later .The later prominence of TUNEL staining likely is caused by continued internucleosomal deoxyribonucleic acid (DNA) fragment after the earliest cell morphologic changes are noted. Control of this response has the potential to regulate wound healing in the cornea in response of corneal surgeries, such as photorefractive keratectomy (PRK), laser-assisted in situ keratomileusis (LASIK) or penetrating keratoplasty (PKP).

Ker atocyte apoptosis as an initiator of the wound-healing r esponse in the

cor nea

Cellular morphologic changes consistent with apoptosis are observable in keratocytes immediately after an injury to the corneal epithelium. The disappearance of the keratocytes is noted after the scraping of the epithelium in preparation for excimer laser photorefractive keratectomy (PRK), after a microkeratome injury to the epithelium in laser-assisted in situ keratomileusis (LASIK) , and is in association with manipulations that occur during other corneal surgical procedures. The keratocytes that die are thought to be replaced over a period of time of a few days by proliferation and migration of remaining keratocytes from

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components associated with stromal healing and regression of the effect of PRK).The activated keraticytes also express increased hepatocyte growth factor and other growth factors, which may stimulate proliferation and inhibit terminal differetiation of corneal epithelial cells.

Effect of stor age condition on cor neal epithelial pr eser vation

The success of penetrating keratoplasty depends on many perioperative factors, including preoperative, operative and postoperative management of recipients and donors. However, the epithelial condition of the donor buttons has long been ignored. It may not be a major problem in western countries with the new corneal preservative media; however, it remained a major concern in many other areas of the world. The period prior to arrival at the donor button’s destination is long, and there is unavoidable disturbance during transportation. Is there any effect on the epithelium of corneal buttons from long-term vibration? Does the macroscopic or microscopic epithelial defects trigger keratocytic apoptosis of corneal buttons like cornea in vivo? Is there any possibility to inhibit this phenomenon during the storage period of donor button? We want to resolve this questions in this study.

Delayed post-oper ative epithelial healing after penetr ating ker atoplasty

Penetrating keratoplasty has been recognized as a very effective surgical method to treat lots of corneal problems. But unfortunately, there still exists some problems to be solved. For example, persistent epithelail defect after operation may cause lots of postoperative problems such as corneal melting, calcium deposotion and infection, and bother many ophthalmologists.

Since keratocyte apoptosis was found to take place after corneal epithelial damage in vivo, and this is supposed to play some role in post-traumatic or post-operative wound healing, it is interesting to investigate the effect of keratocyte apoptosis of donor buttons on postoperative wound healing, and we further focus it on postoperative poor epithelization. What is the effect of this reaction on postoperative results? Which steps from donor cornea harvesting to transplantation may trigger keratocyte apopatosis? Can we block this

phenomenon and improve the surgical results? All these questions remained to be solved.

結果與討論

Gr oup 1. Keratocytic apoptosis directly after removing the corneal epithelium of the central cornea of 8 mm diameter

TUNEL-positive ker atocytes (% )

Under debr ided epithelium Cor nea

Under intact epithelium

Anter ior Str oma Middle Str oma Poster ior str oma Pr eliminar y r esult of last year

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1 5 % 78% 24% 3%

2 10% 64% 12% 6%

3 6% 50% 16% 5%

4 8% 68% 8% 2%

Result of this year

5 9% 56% 24% 9% 6 12% 68% 30% 11% 7 4% 74% 12% 18% 8 6% 52% 29% 7% 9 15% 50% 24% 14% 10 7% 68% 36% 18% 11 9% 45% 13% 12% 12 12% 78% 24% 11%

Gr oup 2 Donor cornea storaged in moist chamber at 4 °C.

TUNEL-positive cells (% ) Cor nea Stor age

Time

Temper atur e Epithelial condition

Str oma Epithelium Endothelium

1 12 hr 4 °C. Intact 2% 4% 0% 2 12 hr 4 °C. Intact 4% 2% 4% 3 24 hr 4 °C. Intact 0% 6% 6% 4 24 hr 4 °C. Intact 3% 7% 2% 5 36 hr 4 °C. Mildly exfoliated 9% 8% 8% 6 36 hr 4 °C. Mildly Exfoliated 15% 59 5% 7 48 hr 4 °C. Totally exfoliated 46% Totally exfoliated 9% 8 48 hr 4 °C. Totally exfoliated 25% Totally exfoliated 12%

Gr oup 3. Donar cornea storaged in Optisol GS at 4 °C.

or nea Stor age Time Temper atu r e Epithelial condition TUNEL-positive cells (% )

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1 12 hr 4 °C Intact <5% <5% <5% 2 12 hr 4 °C Intact 6% 5% 6% 3 24 hr 4 °C Intact 13% 8% <5% 4 24 hr 4 °C Intact <5% 6% 7% 5 36 hr 4 °C Intact 12% 8% 5% 6 36 hr 4 °C Partial exfoliated 12% 5% 6% 7 48 hr 4 °C Intact 15% 5% 7% 8 48 hr 4 °C Partial exfoliated 22% 10% 12%

Gr oup 4 Donar cornea put into Optisol GS at 4 °C. Shaking at a speed of 5 rpm (0.001475g) for 10 hours.

TUNEL-positive cells (% ) Cor nea Stor age

Time

Temper atur e

Epithelial condition

Str oma Epithelium Endothelium

1 12 hr 4 °C Intact <5% 6% 9% 2 12 hr 4 °C Intact <5% <5% 7% 3 24 hr 4 °C Intact <5% 5% <5% 4 24 hr 4 °C Intact 7% <5% 6% 5 36 hr 4 °C Intact 8% 10% 5% 6 36 hr 4 °C Partially exfoliated 17% 19% 12% 7 48 hr 4 °C Partially exfoliated 18% 21% 17% 8 48 hr 4 °C Partially exfoliated 23% 21% 18%

Figur e 4 Cornea 7 in Group III

After storage at Optisol GS at 4°C and shaked at 10 Hz for 48 hrs, the corneal epithelium was partially desquamated. TUNEL stain revealed the positive reaction at whole layers of keratocytes.

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計劃結果自評

由本計劃結果得知,角膜間質細胞的自殺行為確與不同的角膜保存及處理過程有 關。但本計劃結果的缺點是 TUNEL stain 的結果往往無法非常確定(因染色結果常常 太淡);因此已用 annexin V and PI 來確認 apoptosis 的結果。另外,隨著保存時的增長 角膜間質細胞確實有較嚴重的自殺行為出現,此表現隱約呈現一種現線性關係。以細 胞培養方式,或許可以在定量上提供更好的角度。

參考文獻

(1) Wilson SE,Walker JW, Chwang EL, et al. Hepatocyte growth factor (HGF) ,

keratinocyte growth factor (KGF) , their receptors, FGF Receptor-2, and the cells of the cornea. Invest Ophthalmol Vis Sci. 1993 ;34: 2544-2561.

(2) Wilson SE, He Y-G, Weng J, et al .Effect of epidermal growth factor, hepatocyte growth factor , and keraticocyte growth factor, on proliferation, motility , and differentiation of human corneal epithelial cells. Exp Eye Res. 1994;59:665-678.

(3) Howell SJ, Doane KJ.Type VI collagen increases cell survival and prevents anti-beta a intergrin–mediated apoptosis. Experiemntal Cell Research. 1998;241(1): 230-41. (4) Mohan RR, Liang Q, Kim WJ, et al. Apoptosis in the cornea: further characterization

of Fas/Fas ligand system. Experimental Eye Research . 1997;65(4):575-89.

(5) Nakayasu K. Stromal changes following removal of epithelium in rat cornea. Jpn J Ophthalmol. 1988;21: 113-125

(6) Crosson CE. Celluar changes following epithelial abrasion. In: Beuerman RW, Crosson CE, Kaufman HE, eds. Healing Processes in the cornea. Houston, TX: Gulf Publishing; 1989: 3-14.

(7) Campos M, Szerenyi K, Lee M, McDonnell JM, Lopez PF, McDonell PJ. Keratocyte loss after corneal de-epithelialization in primates and rabbits. Arch Ophthalmol. 1994;112:254-260.

(8) Wyllie AH, Kerr JFR, Currie AR. Cell death: the significance of apoptosis. Int Rev Cytol. 1980;68:251-306.

(9) Wilson SE, Pedroza L, Beuerman R, et al. Herpes simples virus type-1 infection of corneal epithelial cells induced apoptosis of the underlying keratocytes. Exp Eye Res.1997;64:775-779.

(10) Gavrieli Y, Sherman Y, Ben-Sasson SA. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation . J Cell Biol.1992;119:493-501. (11) Michel-Salamin LA, Tosi-Couture E, Gautier A, et al. Electron microscopy of the

chromosomes of dinoflagellate Porocentrum micans: confirmation of Bouligand’s liquid crystal hypothesis. J Ultrastruct Mol Strut Res. 1986;97:10-30.

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Invest. 1994;71:219-225.

(13) Mori C, Nakamura N, Okamoto Y, et al.Cytochemical identification of programmed cell death in the fusing fetal mouse palate by sspecific labelling of DNA fragmentation. Anat Embryol.1994;190:21-28.

(14) Wilson SE, He Y-G, Weng J, et al. Epithelial injury induces keratocyte apoptosis : hypothesized role for interleikin-1 system in the modulation of corneal tissue

organization and wound healing . Exp Eye Res. 1996;62:325-338.

(15) Wilson SE, Li Q., Weng J, et al. The Fas/Fas ligand system and otther modulators of apoptosis in the corbnea. Invest Ophthalmol Vis Sci. 1996;37:1582-1592.

(12) Li Q, Weng J, Mohan RR, et al. Hepatocyte growth factor (HGF) and HGF receptos in the lacrimal gland , tears , and cornea. Invest Ophthalonol Vis Sci. 1996;37:727-739. (16) Stuart PM. Griffin TS, Usui N, et al. CD95 ligand (FasL) –induced apoptosis is

necessary for corneal allograft survival. Journal of clin Invest. 1007:99 (3):396-402. (17) Helena MC, Kim WJ,Wilson SE,et al. Keratocyte apoptosis after corneal surgery.

Invest Ophthalomol Vis Sci. 1998;39:276-283.

(18) Wilson SE. Programmed cell death , wound healing, and laser refractive surgery. J Refract Surg. 1997:13: 171-175.

(19) Gao J, Gelber-Schwalb TA, Addeo JV, Stern ME. Apoptosis in the rabbit cornea after photorefractive keratectomy. Cornea.1997;16:200-208.

數據

Figur e 4 Cornea 7 in Group III

參考文獻

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