Expression of E- and N-Cadherin in Bovine Corneal Cell Cultures
1. Expression of E- and N- cadherin in cultures of bovine
corneal epithelium and endothelium
2. 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.
3. - 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
http://www.hindawi.com/journals/vmi/2012/357187/fig1/
4. -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.
5. 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.
6. 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 .
7. 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*
8.
9.
10. 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)
11. 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).
13. 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
14. 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.
Editor's Notes
The observation of cultured cancer cells in vitro, showed that the first action in cancer cells is loss of
contact inhibition(CADHERINS) and the second step is being invasion and metastasis form.
-E-cadherins expressed in corneal equivalent (IF)
-3 weeks after seeding the cells
* sodium dodecyl sulfate polyacrylamide gel electrophoresis
Pen-strep solution(0.4%) ,
assay BCA(bicinchoninic acid assay ) is a biochemical assay for determining the total concentration of protein in a solution (0.5 μg/mL to 1.5 mg/mL). The total protein concentration is exhibited by a color change of the sample solution from green to purple in proportion to protein concentration, which can then be measured using colorimetric techniques.
Classical cadherins has the appropriate molecular weights of 120–130 kDa.
Blots exposed to ECL reagents for chemiluminescence.
Columns :TER KΩ
Rows : days
Mean and Standard deviation
Cornelal endothelial cell culture, in third passage.
