(→Research idea and Goals)
(→Research Idea and Goals)
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==Research Idea and Goals==
==Research Idea and Goals==
Our goal is to determine whether we can enhance the proliferation of human corneal endothelial cells (HCECs) while maintaining their phenotype. We will use
Our goal is to determine whether we can enhance the proliferation of human corneal endothelial cells (HCECs) while maintaining their phenotype. We will use inhibitor and upregulation of nerve growth factor (NGF) to examine whether these proteins will enable HCECs to proliferate and avoid endothelial-mesenchymal transformation.
Revision as of 13:08, 7 May 2013
The aim of this project is to identify media additives that promote human corneal endothelial cell (HCEC) proliferation and preservation of phenotype during in vitro culturing.
The cornea is the transparent, outermost part of the eye. It consists of three distinct layers: an outer layer of epithelial cells; a thick stroma of collagen fibrils embedded with quiescent fibroblasts; and an inner epithelial layer, called the endothelium. The transparency of the cornea depends mainly on the endothelium, which can be damaged by surgery or disease, leading to a permanent loss of visual acuity. This deterioration is permanent because the endothelial cells do not inherently proliferate, and once they are damaged they are not replaced. Thus, research in the field has sought to identify growth factors that can stimulate HCEC proliferation. Progress has been hindered, however, by the transformation of endothelial cells to fibroblastic cells in culture, which abolishes the endothelial phenotype.
Research Idea and Goals
Our goal is to determine whether we can enhance the proliferation of human corneal endothelial cells (HCECs) while maintaining their phenotype. We will use ROCK inhibitor and upregulation of nerve growth factor (NGF) to examine whether these proteins will enable HCECs to proliferate and avoid endothelial-mesenchymal transformation.
(We will use Pipparelli, Aurélien, Yvan Arsenijevic, et al. "ROCK Inhibitor Enhances Adhesion and Wound Healing of Human Corneal Endothelial Cells." PLoS One. 8.4 (2013): n. page. Web. 5 May. 2013. <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3633902/> to design our methods because these researchers were asking a similar question about their inhibitor of interest -- specifically, whether it would allow for HCEC proliferation.)
Hong, J., Qian, T., NGF promotes cell cycle progression by regulating D-type cyclins via PI3K/Akt and MAPK/Erk activation in human corneal epithelial cells, Molecular Vision, Vol 18, Mar 30, 2012, Pages 758-764, <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3324366/>.
--> Nerve growth factor is a protein that promotes cell survival through the MAPKcascade. Without NGF the expression of pro-apoptotic proteins are increased, leading to cell death. NGF positively regulates HCEC cell cycle progression which can enhance cell proliferation. We would like to up-regulate NGF expression in order to allow the HCECs to proliferate during wound healing in vitro.
Okumura, Naoki, EunDuck Kay, et al. "Inhibition of TGF-β Signaling Enables Human Corneal Endothelial Cell Expansion In Vitro for Use in Regenerative Medicine." PLoS One. 8.2 (2013): n. page. Web. 5 May. 2013. <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3581499/>.
-->This paper demonstrated that the use of SB431542, a selective inhibitor of the TGF-β receptor, in culture media counteracted the development of fibroblastic phenotypes in HCECs. This suggests that SB431542 would be a useful additive to our HCEC growth media because it may allow the cells to maintain their phenotype as they proliferate.