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Home » Archive » 2013

TDK conference 2013

Modelling and regulating of paracellular transzport processes on IPEC-J2 cell modell
Csavojetz Anna - year 4
SzIU, Faculty of Veterinary Science, Department of Pharmacologi and Toxicology
Supervisor: Pásztiné Dr. Gere Erzsébet

Abstract:

The experiments were performed on IPEC-J2 cell line on which we were modelling the transport actions between the apical and the basolateral compartments of the small intestine emergent in vivo. We investigated the effect of matriptase and hypocalcaemia on the integrity of the cell line.

Integrity of the intestinal epithelium was reduced by inhibiting matriptase, which is a transmembrane serine protease expressed by cells of surface epithelial origin. We used a matriptase inhibitor, 4-(2-aminoetil)-benzolsulfonilfluorid to delay the differentiation of cells while we investigated the relationship between the activity of matriptase and the functioning of TJ komplexes, which regulate the paracellular transport actions. During the test, the value of the transepithelial electrical resistance (TER) decreased and then recovered, thus demonstrating the matriptase inhibitor reversible inhibition of the formation of TJ's. The matriptase thus may play an important role in maintaining the integrity of the epithelium and in case of inflammatory bowel diseases in restoring the homeostasis of the intestine.

Decrease of the extracellular Ca++ level and even more a partial disorder in the balance of the extracellular and intracellular Ca++ level can affect the resistance of the cell line, too. The latter can effect non physiology markings of the phosphorilation of the tight junctions and adherens junctions proteins. We investigated the extent of the cell layer damage in case of calcium depletion induced by apical EGTA and calcium free PBS treatment. The extent of the barrier damage is followed by 4 kDa flourescein isothiocyanate (FITC)-dextran paracellular marker penetration measuring with fluorimetric analysis and by periodic monitoring TER. After removal of extracellular Ca++ in the basolateral compartment increased level of FITC-dextran was measured therefore the paracellular permeability increased. Addition of Ca++ at 24 hours, the cell layer resistance is restored. The cells were treated with protective materials, 10 mM N-acetylcysteine (NAC), and 2 mM sodium butyrate (NB) for the purpose of restoring permeability. Ca++ withdrawal with concomitant NAC and NB had no effect on IPEC-J2 monolayer permeability.

The other task is to determine how TER decrease depends on the Ca++ level in the range of 0-1,05 mM. We also have to investigate the effect of the transport of Ca++ ions between the two compartments. We further investigate the role of Ca++ observing how extra- and intracellular Ca++ complexation or the inhibition of ER Ca++ ATP-ase take effect to the paracellular transport processes. Another task is the hypocalcemia caused intracellular oxidative stress measurement and monitoring of Ca++ homeostasis disruption effects of TJ complexes (claudin-2 distribution). Longterm study designed to develop an optimized biological test system, which allows important observations to be made to increase cell monolayer barrier function, or even by making the effective absorption.



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