Özet

In vitro tr in vivo modellen voor de studie van het hoornvlies endotheel-mesenchymale transitie

Published: August 20, 2016
doi:

Özet

Een primaire kweek van runder corneale endotheelcellen werd gebruikt om het mechanisme van corneale endotheel-mesenchymale overgang te onderzoeken. Verder werd een rat hoornvliesendotheel cryoinjury model gebruikt om corneale endotheel-mesenchymale overgang in vivo te demonstreren.

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

Al de gevolgde procedures in deze studie overeenkomen met de Vereniging voor Onderzoek in Visie en Oogheelkunde Verklaring voor het gebruik van dieren in Oogheelkundige en Geluid Onderzoek en werden goedgekeurd door de Institutional Animal Care en gebruik Comite van de National Taiwan University Hospital. 1. Isolatie, Primary Cultuur Voorbereiding en Immunokleuring van Bovine CECs Verwerven van vers vlees van runderen ogen van een lokale slachthuis. Desinfecteren ogen in een 10% w / v povido…

Representative Results

Na de isolatie van runderen CECs, de cellen werden gekweekt in vitro. Figuur 1 toont de fasecontrastbeelden van runderen LME. De zeshoekige vorm van de cellen op confluentie aangegeven dat de cellen niet werden aangetast door corneale stromale fibroblasten gedurende celisolatie. Figuur 2 toont de immunokleuring die werd uitgevoerd met antilichamen tegen ABC, slak en slak op een aangegeven tijdstip. Naast fenotypische veranderingen in het in …

Discussion

ME-landen staan ​​bekend om hun neiging om EnMT ondergaan tijdens cel proliferatie. Strategieën te ontwikkelen voor het onderdrukken van de EnMT werkwijze voor therapeutische doeleinden, een grondig begrip van de EnMT mechanisme noodzakelijk. We beschreven 2 modellen om EnMT, namelijk het runder CEC in vitro cultuur model en rat hoornvliesendotheel cryoinjury model te onderzoeken. Onze resultaten toonden de EnMT proces in beide modellen. Bovendien is de EnMT-onderdrukkende effect van marimastat werd opgeno…

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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