捺壓式細胞學及活體共軛焦顯微鏡在眼輪部缺損診斷上之比較研究

The role of impression cytology and in vivo confocal microscopy in the diagnosis of limbal insufficiency

許國軒(Kuo-Shen Hsu)

指導教授 ： 林中天

共同指導教授 ： 陳偉勵(Wei-Li Chen)

國立臺灣大學/獸醫專業學院/臨床動物醫學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.01800

摘要

實驗目的：以捺壓式細胞學(impression cytology, IC)檢查配合外眼照以及活體共軛焦顯微鏡檢查記錄觀察兔眼輪部幹細胞缺損模式下角膜上皮之癒合情況以及細胞形態之變化。
實驗方式：比較使用兩種大小孔徑(3μm，0.45μm)之Nitrocellulose filter paer以及不同之染色方式(PAS stain以及PAS 合併Papnicolau stain，double stain)在健康兔眼部採樣下較佳之採樣及染色呈現方式以決定後續使用方式；將實驗兔麻醉後移除眼輪部組織（包括部分角膜、結膜以及眼輪部上皮）以進行眼輪部幹細胞缺損模式之建立，同時在之後進行免疫組織切片確認模式之成功建立；術後以外眼照、捺壓式細胞學以及活體共軛焦顯微鏡每周一次記錄角膜癒合之病程以及細胞之變化。
實驗結果：在捺壓式細胞學實驗方式方面，使用0.45μm之小孔徑濾紙可以獲得較佳之染色呈現結果，而兩種不同染色方式皆可以獲得良好結果，但由於double stain較繁複費時因此後續決定使用PAS stain；術後進行之免疫組織染色顯示在術後14天即可以看到角膜中央有K4及K13之表現，表示出現角膜結膜化之情形而成功誘導出幹細胞缺損模式；在每周一次的觀察記錄中，術後外眼照顯示出顯著角膜水腫、新生血管以及不平整角膜上皮之情形，捺壓式細胞學染色下顯示初期結膜上皮有侵入角膜之現象並可以觀察到杯狀細胞，在IC之免疫螢光染色下也可以看到K4的表現，隨著上皮癒合過程演進，角膜上皮漸漸重新覆蓋並可以看到IC上K3之染色表現，在外眼照部分也漸恢復清澈之角膜狀態；在共軛焦顯微鏡下則可以看到一開始只有少部分正常角膜樣式之淺層上皮存在，但在第28天已可以觀察到大量正常角膜樣式之之淺層上皮細胞，但基底細胞則從第14天到第28天皆可以看到明顯細胞核且變為較大顆之細胞，顯示其持續處於活躍狀態。
結論：捺壓式細胞學配合免疫螢光染色的使用在進行眼表面的疾病包括眼輪部幹細胞缺損所引起之角膜上皮變化是一項快速、方便且非侵入式的診斷工具，活體共軛焦顯微鏡則可以更進一步且無侵入性地觀察細胞細微之改變，在臨床上可以提供相當重要的資訊以利於快速準確的診斷。

關鍵字

角膜 ； 結膜 ； 眼輪部幹細胞 ； 共軛焦顯微鏡 ； 捺壓式細胞學

並列摘要

Purpose： The aim of this study was to evaluate the ocular changes of the rabbit limbal deficiency model using external photography, impression cytology (IC), immunocytochemistry stain, and in vivo confocal microscopy.
Methods：Two pore sizes (3μm, 0.45μm) of nitrocellulose filter paper and different staining methods (PAS stain and PAS combined with Papanicolau stain, double stain) were compared to decide which one was superior in sampling and staining; Limbal epithelium of experimental rabbits including partial cornea, conjunctiva were surgically removed to create a limbal stem cell deficiency model under general anesthesia, and immunohistochemical stain was also performed at 14 days and 1 month post-operatively to evaluate the established animal model. External photographs, impression cytology and in vivo confocal microscopy were performed once a week to record the morphological changes during healing process.
Results：In the pore size test, smaller pore size (0.45μm) of filter paper presented better staining result; Both PAS stain and double stain provided good staining results, but we finally decide to use PAS stain due to its rapid, simple staining protocol. Immunohistochemical stain at 14 days and 1 month post-operatively showed the successful established rabbit model of limbal stem cell deficiency based on the expression of the K4 and K12 in the central part of the cornea, representing conjunctivalization of the cornea. In the experimental group, the external photography showed corneal edema, irregular ocular surface, and neovascularization. Conjunctival epithelial cells and goblet cells were observed by impression cytology with expression of K4. At healing of corneal epithelium, corneal epithelial cells gradually overlaid the corneal surface and transparent cornea could observed, and K3 could also be detected on IC exam. Initially there was few normal superficial corneal epithelial cells under in vivo microscopy, but lots of normal superficial corneal epithelial cells showed up at 28 days post-operatively. However, the basal corneal epithelial cells still remained in the active phase with obvious cell nucleus and large cell size.
Conclusions：It’s a rapid, convenient and non-invasive technique to diagnose ocular surface diseases by using impression cytology with immunofluorescein stain. In vivo confocal microscopy also provided details of cell morphologic changes. All of these techniques can provide rapid and useful information in clinical diagnosis.

並列關鍵字

cornea ； conjunctiva ； limbal stem cell ； impression cytology ； in vivo confocal microscopy

參考文獻

1. Adams AD. 1979. The morphology of human conjunctival mucus. Arch Ophthalmol 97:730-734.

2. Bolzan AA, Brunelli ATJ, Castro MB, Souza MA, Souza JL, Laus JL. 2005. Conjunctival impression cytology in dogs. Vet Ophthmol 8(6):401-405.

3. Chen WYW, Mui MM, Kao WWY, Liu CY, Tseng SCG. 1994. Conjunctival epithelial cells do not transdifferentiate in organotypic cultures: expression of K 12 is restricted to corneal epithelium. Curr Eye Res 13:765-768.

4. Chifflet S, Hernandez JA, Grasso S. 2005. A possible role for membrane depolarization in epithelial wound healing. Am J Physiol Cell Physiol 288:C1420-1430

5. Collin HB, Anderson JA, Richard NR, and Binder PS. 1995. In vitro model for corneal wound healing; organ-cultured human corneas. Curr Eye Res 14:331-339.