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Notch signalling and its role in corneal epithelium survival and differentiation

Ma, Aihua. 2006. Notch signalling and its role in corneal epithelium survival and differentiation. PhD Thesis, Cardiff University.

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To elucidate mechanism of corneal epithelial cell differentiation, proliferation and stratification is crucial for maintaining epithelial cell homeostasis, manipulating corneal wound healing and developing therapeutic strategy for treatment ocular surface diseases. The Notch signalling system regulates cell fate decisions and cell function. In human, there are four Notch receptors, Notch 1 to 4, and five ligands, including Deltal, 3, 4, Jaggedl, 2. Activation of Notch upon ligand binding is accompanied by proteolytic processing regulated by gamma-secretase. This study aimed to determine whether the components of the Notch are expressed in the human corneal epithelial cells and the role of Notch signalling in corneal epithelial homeostasis, stratification and wound healing. METHODS Immunohistochemistry was employed for the localisation of the Notch receptors and their ligands in fresh human cornea and embryonic chicken cornea. Gene expression of Notch receptors and their ligands was determined using reverse transcriptase- polymerase chain reaction (RT-PCR) in cultured human corneal epithelial cells and keratocytes. Western Blotting analysis, immunocytochemistry in the presence or absence gamma-secretase inhibitor and Jaggedl were used to correlate Notch with Ki67 (a marker of cell proliferation) and cytokeratin 3 (a marker of cell differentiation) expressions for a functional study of proliferation and differentiation in corneal epithelial cells. The co-culture model with amniotic membrane and an organ culture model of intact rat cornea were used to investigate the function of Notch in corneal epithelial cell stratification. Also, an organ culture of wounded cornea was used to study the role of Notch in corneal epithelial and stromal wound healing. RESULTS Immunohistochemical results showed that Notch 1 and Notch2 expressed throughout the corneal epithelium in superficial and suprabasal layers. Delta 1 and Jaggedl appeared to be expressed throughout all cell layers of the corneal epithelium. The expression of activated Notch 1, Notch2 and Ki67 was decreased and cytokeratin 3 was increased after the Notch pathway was blocked by a gamma-secretase inhibitor. In contrast, activation of Notch pathway by Jaggedl induced the increase of the expression of activated Notch 1, Notch2 and Ki67 and decreased the expression of cytokeratin 3. The corneal stratification was inhibited by activation of Notch. In wound healing study, Notch inhibited the wound repair at late stage. In addition, the expression pattern of Notch was exhibited in developmental embryonic chicken cornea. CONCLUSIONS Notch suppresses differentiation and stratification in corneal epithelium. Activation of Notch results the retardation of corneal wound repair. Notch signalling system plays a pivotal role in maintenance of corneal epithelial cell homeostasis and wound healing.

Item Type: Thesis (PhD)
Status: Unpublished
Schools: Optometry and Vision Sciences
Subjects: R Medicine > R Medicine (General)
ISBN: 9781303204289
Funders: National Eye Research Centre, UK, Overseas Research Studentship Award
Date of First Compliant Deposit: 30 March 2016
Last Modified: 12 Feb 2016 23:15

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