Date of Award

Spring 8-1-2004

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Life Sciences

First Advisor

H. Kathleen Dannelly

Second Advisor

Taihuang Peter Duong

Third Advisor

James Hughes

Abstract

The research project aims to determine the role of glycine receptor (GlyR) in the protection of plasma membrane of ischemic cells. Plasma membrane disruption is a progressive process during ischemia. Once plasma membrane becomes permeable to macromolecules, the cell injuries are irreversible. Ischemia can be modeled in the lab by using ATP-depletion medium. Previous results have shown that glycine (Gly) in extracellular medium prevents membrane permeabilization during ischemia. These studies suggest that the protection by glycine might be mediated by its binding to GlyR. But this hypothesis has never been confirmed. This study shows that glycine cannot protect HEK.293 cells during ATP-depletion as determined by the Lactate Dehydrogenase assay. The HEK.293 kidney cell line lacks the GlyR. To determine if the glycine can protect the HEK.293 cells during ATPdepletion when the GlyR is expressed in the HEK 293 cells, the plasmid pcDNA3.1(+) + GlyRalDNA was constructed and used to transfect HEK.293 cells and generate a permanent HEK.293 cell line which expressed GlyR. For a positive control MDCK cells were tested by RT-PCR and found to express endogenous GlyR by RT-PCR. The research illustrated that the role of glycine receptor in protecting cells during ischemia. The results of the research further our understanding on the mechanism of plasma membrane disruption during ischemia, and will help us find a way to protect cell when it lacks blood supply (ischemia), to stop these injuries ischemia at reversible stage.

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