Available at: https://digitalcommons.calpoly.edu/theses/1938
Date of Award
MS in Biomedical Engineering
Biomedical and General Engineering
Kristen O'Halloran Cardinal
Developing a tissue-engineered Blood Vessel Mimic (BVM) to represent diabetic macrovascular disease could expedite design of new vascular devices specifically tailored to diabetic patients. In contribution toward this model, this thesis assessed Human Umbilical Vein Endothelial Cell (HUVEC) responses to high glucose conditions. Interleukin 6 (IL-6) and Cluster of Differentiation 36 (CD36) were selected to signify oxidative stress activity, a hallmark of diabetic macrovascular disease. Next, activity of potential reference genes B2M, HPRT1, and ACTB was assessed. All genes were found to exceed acceptable variability, so the E-ΔC T method of data analysis was selected. Next, cellular responses to high glucose treatment at 10.5 mM glucose and 25.5 mM glucose for 7 and 14 days were measured by qPCR. IL-6 mRNA expression increased significantly (p<0.001) following treatment with 25.5 mM glucose at both timepoints. Finally, fluorescent staining for Reactive Oxygen Species (ROS) production and cell viability was performed on HUVECs treated with 10.5- and 25.5-mM glucose for 24 and 48 hours. No differences in ROS production or cell viability were detected due to uncontrolled cell damage during the two-hour staining and imaging procedure. This thesis was limited by low reaction efficiency in qPCR reactions due to mistaken purchasing of primers with included probe-quencher reporters. Measurement of reaction efficiency facilitated valid analysis of data collected using these primers. Imaging experiments were unsuccessful due to a lack of incubation equipment designated for cells undergoing live staining and imaging. Alternative imaging assessments of oxidative stress activity were proposed to circumvent this problem.