Özet

体外体内模型来研究角膜内皮-间充质转换

Published: August 20, 2016
doi:

Özet

牛角膜内皮细胞的原代培养物用于研究角膜内皮 – 间充质转换的机制。此外,大鼠角膜内皮冷冻损伤模型被用来证明在体内角膜内皮-间充质转换。

Abstract

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema and diminished visual acuity. However, human CECs are prone to compromised proliferative potential. Furthermore, stimulation of cell growth is often complicated by gradual endothelial-mesenchymal transition (EnMT). Therefore, understanding the mechanism of EnMT is necessary for facilitating the regeneration of CECs with competent function. In this study, we prepared a primary culture of bovine CECs by peeling the CECs with Descemet’s membrane from the corneal button and demonstrated that bovine CECs exhibited the EnMT process, including phenotypic change, nuclear translocation of β-catenin, and EMT regulators snail and slug, in the in vitro culture. Furthermore, we used a rat corneal endothelium cryoinjury model to demonstrate the EnMT process in vivo. Collectively, the in vitro primary culture of bovine CECs and in vivo rat corneal endothelium cryoinjury models offers useful platforms for investigating the mechanism of EnMT.

Introduction

Corneal endothelial cells (CECs) play a vital role in maintaining corneal clarity and thus visual acuity by regulating the hydration status of the corneal stroma through active pumping1. Because of the limited proliferative potential of human CECs, the cell number decreases with age, and the repair of corneal endothelial wounds following injury is usually achieved through cell enlargement and migration, rather than cell mitosis2. When the CEC count decreases below a threshold of approximately 500 cells/mm2, the dehydration status of the corneal stroma cannot be maintained, leading to bullous keratopathy and vision impairment3,4.

The limited proliferative potential of human CECs has been attributed to several factors, including reduced expression of the epidermal growth factor and its receptor in aging cells5, antiproliferative TGFβ2 in the aqueous humor6, and contact inhibition2,7. Although some growth factors, such as basic fibroblast growth factor (bFGF), can increase proliferation in a cultured human corneal endothelium, the culture efficiency remains limited8,9. Furthermore, CECs may undergo a phenotypic change during ex vivo expansion, resembling epithelial-mesenchymal transition (EMT)10-13. Endothelial-mesenchymal transition (EnMT) is characterized by cell junction destabilization, apical-basal polarity loss, cytoskeletal rearrangement, alpha smooth muscle actin expression, and type I collagen secretion14. All of these characteristics may abrogate the normal function of CECs, hampering the use of ex vivo cultured CECs in tissue engineering. Moreover, EnMT has been associated with the pathogenesis of several corneal endothelial diseases, including Fuchs endothelial corneal dystrophy and retrocorneal membrane formation15,16. Therefore, understanding the mechanism of EnMT may aid in manipulating the EnMT process and facilitate the regeneration of CECs to enable competent function.

In this study, we described a method for isolating bovine CECs from the corneal button. In the primary culture in vitro, the EnMT process, including a phenotypic change, the nuclear translocation of β-catenin, and EMT regulators snail and slug, was observed. We further described a method for demonstrating EnMT in vivo by using a rat corneal endothelium cryoinjury model. Using these 2 models, we demonstrated that marimastat, a broad-spectrum matrix metalloproteinase (MMP) inhibitor, can suppress the EnMT process. The described protocols facilitate the detailed analysis of the EnMT mechanism and the development of strategies for manipulating the EnMT process for further clinical application.

Protocol

所有的程序遵循本研究中,在视觉与眼科声明协会研究给予了动物在眼科和视觉研究的用途和台大医院的机构动物护理和使用委员会批准。 1.隔离,原代培养制备与牛的CEC的免疫染色从本地屠宰场获得新鲜牛的眼睛。 消毒在3分钟10%w / v的聚乙烯吡咯烷酮 – 碘溶液中的眼睛。用磷酸盐缓冲盐水(PBS)溶液洗涤。 收获在无菌条件下解剖刀和剪刀角膜按钮。去皮后弹力与在…

Representative Results

牛的CEC的分离后,将细胞在体外培养, 图1给出了牛的CEC的相位对比图像。细胞在汇合的六边形表示细胞没有通过细胞分离过程中角膜基质成纤维细胞的污染。 图2描绘了使用针对ABC,蜗牛和蛞蝓的抗体在一个指定的时间点进行的免疫染色。除了 ​​在体外培养的表型变化,观察到的ABC和EMT调节的相应核易位。 图3示出马…

Discussion

的CEC是众所周知的倾向中的细胞增殖经受EnMT。制定战略用于抑制EnMT过程用于治疗目的时,EnMT机构的透彻理解是必要的。我们描述了2模型,研究EnMT,即在体 ​​外培养模型大鼠角膜内皮冷冻损伤模型牛CEC。我们的研究结果证明了这两款车型的EnMT过程。此外,马立马司他的EnMT抑制效应再现这两种模式,这表明这些2种型号共享相同的机制。

体外培养模型牛CEC提?…

Açıklamalar

The authors have nothing to disclose.

Acknowledgements

We thank the staff of the Second Core Lab, Department of Medical Research, National Taiwan University Hospital for their technical support.

Materials

trypsin ThermoFisher Scientific 12604-013
Dulbecco’s modified Eagle medium and Ham's F12 medium ThermoFisher Scientific 11330
fetal bovine serum ThermoFisher Scientific 26140-079
dimethyl sulfoxide Sigma D2650
human epidermal growth factor ThermoFisher Scientific PHG0311
insulin, transferrin, selenium  ThermoFisher Scientific 41400-045
cholera toxin Sigma C8052-1MG
gentamicin ThermoFisher Scientific 15750-060
amphotericin B ThermoFisher Scientific 15290-026
paraformaldehyde Electron Microscopy Sciences 111219
Triton X-100 Sigma T8787 
bovine serum albumin Sigma A7906
marimastat Sigma M2699-25MG
anti-active beta-catenin antibody Millpore 05-665
anti-snail antibody Santa cruz sc28199
anti-slug antibody Santa cruz sc15391
goat anti-mouse IgG (H+L) secondary antibody ThermoFisher Scientific A-11001 for staining of ABC of bovine CECs
goat anti-mouse IgG (H+L) secondary antibody ThermoFisher Scientific A-11003 for staining of ABC of rat corneal endothelium
goat anti-rabbit IgG (H+L) secondary antibody ThermoFisher Scientific A-11008 for staining of snail and slug of bovine CECs
antibody diluent Genemed Biotechnologies 10-0001
4',6-diamidino-2-phenylindole ThermoFisher Scientific D1306
mounting medium Vector Laboratories H-1000
laser scanning confocal microscope ZEISS LSM510
xylazine  Bayer N/A
tiletamine plus zolazepam Virbac N/A veterinary drug
proparacaine hydrochloride ophthalmic solution Alcon N/A veterinary drug
0.1% atropine Wu-Fu Laboratories Co., Ltd N/A clinical drug 
0.3% gentamicin sulfate Sinphar Group N/A clinical drug 
basic fibroblast growth factor ThermoFisher Scientific PHG0024 clinical drug 

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Bu Makaleden Alıntı Yapın
Ho, W., Su, C., Chang, J., Chang, S., Hu, F., Jou, T., Wang, I. In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition. J. Vis. Exp. (114), e54329, doi:10.3791/54329 (2016).

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