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中华眼科医学杂志(电子版)

中华眼科医学杂志(电子版) ›› 2019, Vol. 09 ›› Issue (05) : 298 -304. doi: 10.3877/cma.j.issn.2095-2007.2019.05.006

论著

建立在体角膜内皮细胞损伤后再生动物模型的实验研究
苏冠羽1, 韦振宇1, 王乐滢1, 梁庆丰1,†()   
  1. 1. 100005　首都医科大学附属北京同仁医院　北京同仁眼科中心　北京市眼科研究所　眼科学与视觉科学北京市重点实验室
  • 收稿日期:2019-08-16 出版日期:2019-10-28
  • 通信作者: 梁庆丰
  • 基金资助:
    国家自然科学基金面上项目(81470607); 北京市卫生系统高层次卫生技术人才培养基金(2014-3-016); 2017年北京市百千万人才工程培养项目(2017A10)

Experimental study on regeneration of corneal endothelial cells in vivo

Guanyu Su1, Zhenyu Wei1, Leying Wang1, Qingfeng Liang1,†()   

  1. 1. Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing Institute of Ophthalmology, Beijing Key Laboratory of Ophthalmology and Visual Sciences, Beijing, 100005, China
  • Received:2019-08-16 Published:2019-10-28
  • Corresponding author: Qingfeng Liang
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引用本文：

苏冠羽, 韦振宇, 王乐滢, 梁庆丰. 建立在体角膜内皮细胞损伤后再生动物模型的实验研究[J/OL]. 中华眼科医学杂志(电子版), 2019, 09(05): 298-304.

Guanyu Su, Zhenyu Wei, Leying Wang, Qingfeng Liang. Experimental study on regeneration of corneal endothelial cells in vivo[J/OL]. Chinese Journal of Ophthalmologic Medicine(Electronic Edition), 2019, 09(05): 298-304.

目的

探讨建立在体角膜内皮损伤动物模型的方法及其损伤后再生的潜能。

方法

选取2周龄的正常六角龙鱼30只。采用数字表法随机分为正常观察组、NaOH损伤组及机械损伤组，各10只。采用活体共聚焦显微镜检查、组织病理学检查、茜素红-曲利苯蓝内皮染色和氯化金角膜神经染色等方法观察。对正常观察组六角龙鱼，观察其眼球和角膜正常结构；通过NaoH溶液和器械两种方法损伤角膜内皮层，建立角膜内皮细胞损伤模型，观察其角膜内皮细胞再生的情况。对4个时间点角膜内皮细胞的密度进行正态性检验和方差齐性检验，使用重复测量方差分析对不同组别、不同时间点角膜内皮细胞密度的均值进行比较，事前进行Mauchly球形检验，如果检验结果显著，采用多元方差分析，否则选用校正自由的F检验。

结果

六角龙鱼角膜厚度约为(75.75±7.51)μm，角膜上皮细胞层较厚，约占角膜厚度的一半。经NaoH溶液和器械两种方法损伤其角膜内皮细胞后，六角龙鱼角膜内皮细胞密度明显降低。NaoH损伤组和机械损伤组在不同时间点，六角龙鱼角膜内皮细胞密度的比较，具有统计学意义(F＝31.38，51.77；P<0.05)。而各个时间点间的差异，两组均无统计学意义(F＝1.37，2.67，0.70，4.14；P>0.05)。在损伤后3 d时，NaoH损伤组六角龙鱼角膜内皮细胞的密度为(128±14)个/mm2，机械损伤组为(113±11)个/mm2。与损伤前比较，其角膜内皮细胞密度的差异均有统计学意义(t＝19.39，8.78；P<0.05)；在损伤后7 d时，NaoH损伤组六角龙鱼角膜内皮细胞的密度为(157±20)个/mm2，机械损伤组为(169±19)个/mm2。与损伤后3 d时比较，其角膜内皮细胞密度均有所恢复，差异均有统计学意义(t＝3.75，8.07；P<0.05)；在损伤后14 d时，可见NaoH损伤组六角龙鱼角膜内皮细胞的密度为(198±17)个/mm2，机械损伤组为(223±17)个/mm2。与损伤后3 d时和7 d时比较，均有明显恢复，差异均有统计学意义(t＝10.05，8.07；P<0.05)和(t＝4.94，6.70；P<0.05)。随着时间延长，两组六角龙鱼角膜内皮细胞的密度均逐渐恢复。在伤后14 d时，其角膜内皮细胞形态、大小和密度均基本恢复正常状态。

结论

使用NaoH溶液和器械两种方法均可成功建立六角龙鱼角膜内皮细胞损伤的动物模型。初步观察结果表明，其角膜内皮细胞具有一定的再生潜能。此方法可为角膜内皮细胞损伤后再生研究提供一种新的动物模型。

关键词: 动物模型, 角膜, 组织病理, 内皮损伤
Objective

This study was aim to explore the method of establishing animal model of corneal endothelial injury and the potential of endothelial regeneration after injury.

Methods

30 normal axolotls of 2 weeks old were selected. They were divided into normal observation group, NaoH-injured group and mechanical-injured group with 10 axolotls each. In vivo confocal microscopy, histopathological examination, alizarin red-trichlorobenzene blue endothelial staining and gold chloride corneal nerve staining were used to observe. In the normal observation group, the normal structure of the eye and cornea was observed. Corneal endothelial cells were damaged by NaoH solution and mechanics, and the endothelial cell injury model was established to observe the regeneration of corneal endothelial cells. The corneal endothelial cell density at four time points was tested by normality test and homogeneity test of variance. The mean corneal endothelial cell density at different time points in different groups was compared by repeated measurement variance analysis. Mauchly spherical test was performed beforehand. According to the test results, if significant, the results of multivariate analysis of variance are adopted; otherwise, the correction free F test is selected.

Results

The corneal thickness of axolotls was about (75.75±7.51) μm, and the corneal epithelial cell layer was about half of the corneal thickness. After the corneal endothelial cells were damaged by NaoH solution and mechanic, the density of corneal endothelial cells decreased significantly. The density of corneal endothelial cells in NaoH-injured group and mechanical-injured group at different time points was significantly different (F=31.38, 51.77; P<0.05). There was no significant difference between NaoH-injured group and mechanical-injured group at each time point (t= 1.37, 2.67, 0.70, 4.14; P>0.05). At 3 days after injury, the density of corneal endothelial cells in NaoH-injured group and mechanical-injured group were (128±14)/mm2 and (113±11)/mm2. And the density of corneal endothelial cells in NaoH-injured group was (157±20)/mm2 and that in mechanical-injured group was (169±19)/mm2 at 7 days after injury (t=19.39, 8.78; P<0.05). Compared with 3 days after injury, the density of corneal endothelial cells recovered with statistical significance (t=3.75, 8.07; P<0.05). At 14 days after injury, the density of corneal endothelial cells in NaoH injury group was (198±17)/mm2, and that in mechanical injury group was (223±17)/mm2. Compared with 3 days and 7 days after injury, there were significant differences (t=10.05, 8.07; P<0.05) and (t=4.94, 6.70; P<0.05). Over time, the density of corneal endothelial cells in both groups was gradually recovered. At 14 days after injury, the morphology, size and density of corneal endothelial cells almost turned back to normal.

Conclusions

Both NaoH solution and mechanic can successfully establish the animal model of corneal endothelial cell injury in axolotls. The preliminary results showed that the endothelial cells had a certain regeneration potential. This method could provide a new animal model for the study of corneal endothelial cell regeneration after injury.

Key words: Animal model, Cornea, Histopathologic, Endothelial injury
图2 六角龙鱼正常角膜活体共聚焦显微镜下显微结构图像　图2A显示活体角膜上皮层(×800)；图2B显示活体角膜上皮下神经(×800)；图2C显示活体基质层角膜神经(×800)；图2D显示活体角膜内皮细胞(×800)；图2E显示茜素红-曲利苯蓝染色下的角膜内皮细胞(×400)；图2F显示神经氯化金染色下的角膜浅基质层神经(×400)
图4 六角龙鱼角膜内皮损伤后不同时间点活体共聚焦显微镜下的显微图像　图4A、4B、4C、4D显示NaoH损伤组角膜内皮经损伤后不同时间点活体共聚焦显微镜下观察内皮细胞形态及密度变化(×800);图4E、4F、4G、4H显示机械损伤组角膜内皮经损伤后活体共聚焦显微镜观察内皮细胞形态及密度变化(×800)
图5 两个角膜内皮损伤组六角龙鱼角膜内皮细胞密度损伤后随时间变化的情况
表1 正常观察组与损伤前后NaoH损伤组及机械损伤组六角龙鱼角膜内皮细胞密度的比较(±s，个/mm2)
分组 数量(只) 损伤前 损伤后3 d 损伤后7 d 损伤后14 d
正常观察组 10 271±39      
NaoH损伤组 10 283±36 128±14a 157±20ab 198±17 abc
机械损伤组 10 259±16 113±11 a 169±19ab 223±17 abc
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