Saba AlmaeiÉcole d’optométrie

Université de Montréal

Expression of E- and N- cadherin in cultures of bovine corneal epithelium and endothelium

A tissue-engineered cornea based on untransformed corneal cells, should mimic the properties of the native cornea.

In addition to use as corneal replacement, a corneal equivalent could be a useful model for studying wound healing, physiology, toxicology, and pharmacology.

Background

- Cadherins are a vast super family of cell-cell adhesion proteins.

-calcium dependent cell membrane proteins and exist in cadherin-catenin

complex. (adherens junction proteins)

-with three subfamilies.

-roles: cell-cell contact, Tissue morphogenesis, inhibits cancer development

Cadherins super familly

http://www.hindawi.com/journals/vmi/2012/357187/fig1/

-Corneal epithelium is a polarized tissue with distinct plasma membrane on its apical and basolateral domains( dependent on adherens junction complexes).

-N-cadherin is essential for maintaining proper structure of corneal endothelial AJCs. Its ablation leads to increased endothelial permeability and corneal edema in mature eyes.

E- and N-cadherin and cornea

According to some experiments (Giasson et.al 2014) corneal equivalent by seeding fibroblasts into a collagen gel on which epithelial and endothelial cells were added on each side, cells from the epithelial and endothelial layers expressed adherens junction proteins, indicating the presence of cell–cell contacts.

Mol Vis. 2014; 20: 386–394.

Published online 2014 Mar 29.

background

The development of, E-cadherin and N-cadherin in corneal epithelial and endothelial cell culture ,starts in 2-4 days after seeding of the corneal epithelial and endothelial cells .

My hypothesis:

1-Separation and isolation of bovine corneal cells 2-subculture and feeding the cells 3- Passage of epithelium and endothelial cells 4-transfer the cells to 6-well 5-they were lifted to the liquid-air interface 6-TER measurement

7-Treatment of membrane and immunofluorescence 8-SDS-PAGE and western blots*

MATERIALS AND METHODS:

Isolation of corneal epithelium

Seeding the cells in membrane

Measurement of electrical resistance Electrical resistance measurements as a measurement of cell-cell

junctions, was used to determine the situation of adherence junctions of the cells.

Int Poster J Dent Oral Med 3 (2001), No. 2     (15.06.2001)

IF and WB and TER will be repeated at 2, 4,6 and days after seeding

Each experiment will be repeated 3 or 4 times Observation of IF and WB at these times: cadherins

formed? TER: Mean Resistance at each time will be plotted. Differences in mean resistance will be tested for

statistical significance with a repeated measure one way analysis of variance (time)(ANOVA).

Procedures

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K.Ohm

Cadherins and a-,b-catenins will be increasingly expressed with time after seeding of epi and endo cells by observation of IF and Western blots

N- and E-cadherins will be only expressed in endothelial and epithelial corneal cells, respectively

Cadherins and catenins are expressed after 3-4 days after seeding of corneal epithelial and endothelial cells in monoculture

Expected Conclusions (to be tested)

1. Nelson WJ, Adams CL, Chen YT, Smith SJ. Epithelial cell polarity from the outside looking in. News Physiol Sci. 2003;18:143–6. [PMC free article] [PubMed]

2-Vassil S. Vassilev, Michiko Mandai, Shigenobu Yonemura, Masatoshi Takeichi .Loss of N-Cadherin from the Endothelium Causes Stromal Edema and Epithelial Dysgenesis in the Mouse Cornea.Investigative Ophthalmology & Visual Science October 2012, Vol.53, 7183-7193. doi:10.1167/iovs.12-9949

3-Claud J.Giasson, Alexandre Deschambeault, Patrick Carrier and Luice Germain. Adherens junction proteins are expressed in collagen corneal equivalents produced in vitro with human cells. Mol Vis. 2014; 20: 386–394.

Published online 2014 Mar 29.

references

Endo