Date of Award

Spring 5-2022

Document Type

Honors Thesis

Degree Name

Bachelor of Science

Department

Biology

Advisor/Committee Chair

Amber Altrieth

Committee Member

Melinda Larsen

Abstract

In our blood vessels, cells called endothelial cells line the inside of veins and arteries. Due to the discovery that endothelial cells actually organogenesis during embryogenesis, our interest in these cells has increased to a new understanding of their functions in tissue repair and regeneration. These cells are no longer only considered passive modes of transport for bodily fluids and nutrients, but, with new research in the fibrosis of liver cells, are also considered to be key players in the repair of damaged organs and tissue. These cells release signals known as angiocrine factors that act in damaged areas of the body to induce regeneration, fibrosis, or inflammation. The angiocrine factors help to guide many steps of the process of self-renewal and differentiation of stem cells into functioning cells for the damaged tissue. These factors play a key role in the health and healing of organisms, but the question remains as to what factors are expressed from salivary gland endothelial cells, and how the expression of these factors changes due to injury. A good source of insight into this problem could be identifying the specific factors that contribute to the fibrotic phase in salivary gland injury and disease. First, we use a NonObese Diabetes (NOD) model mouse, which has phenotypes similar to Sjogren’s Syndrome, to examine the levels of expression of different genes in endothelial cells derived from NOD vs control mice. Second, to induce injury to the glands we performed a ductal ligation surgery, where a clip was applied to the salivary gland ducts, inducing a reversible fibrosis. We compared multiple protocols for releasing cells from fibrotic tissue to determine an optimized cell isolation protocol to use for single cell RNA sequencing. We found that utilizing the enzyme, Liberase, to dissociate cells in adult salivary glands, in combination with magnetic activated cell sorting to deplete EpCAM+ epithelial cells and red blood cells, produced the highest yield and cell viability with a yield of 4.175 x 106 cells and viability of 98.11% as compared to our other trials and our original collagenase/hyaluronidase cell dissociation protocol. We also released larger numbers of PDGFRβ+ fibroblasts and CD31+ endothelial cells with the optimized protocol. Compared to the mock sample, the ligated sample had a decrease in PDGFRβ+ cells. We then used the optimized cell dissociation protocol to release cells from a ligated salivary gland and a mock surgical gland and performed scRNASeq. Seurat-based clustering methods revealed a large number of endothelial cells released from both glands. This work paves the way for identification of differential gene expression by endothelial cells of the salivary gland following injury to inform future therapeutic strategies.

Available for download on Thursday, December 01, 2022

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