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Development of a Collagen Assay and testing pro- and antifibrotic compounds on fibroblasts in vitro

Bosma, Richard (2023) Development of a Collagen Assay and testing pro- and antifibrotic compounds on fibroblasts in vitro. Research Project, Pharmacy.

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Collagen is the most abundant protein found in mammals, making up to 30% of the total protein content. Disruption of collagen homeostasis can lead to diseases like liver fibrosis and cirrhosis. Due to the unique molecular structure of collagen, various assays have been developed to measure this protein. In this study, three assays will be compared to each other to determine the optimal assay for measuring collagen in NIH-3T3 fibroblasts. The three assays; Sirius Red assay, hydroxyproline assay and DPHAA assay each use a different characteristic to produce a signal from collagen. For these assays the positive control will be TGFβ. Additionally, the effect of IFNγ (a cytokine) and two natural compounds harmine and honokiol will be tested on the gene expression of fibrosis markers COL1A1, Fibronectin 1 (FN1) and α smooth muscle actin (αSMA). After which the IFNγ will be tested in combination with the three assays. The findings suggest that the Sirius Red assay is not suitable for collagen measurement in cell culture due to the fact that the dye also binds to the serum proteins. The hydroxyproline assay is not suitable because it needs large quantities of collagen to accurately measure the collagen content, while concentration of collagen in cell culture is significantly lower. The DPHAA shows promising results on measuring collagen in cell culture. Further optimization is needed however to increase the reliability of the assay.

Item Type: Thesis (Research Project)
Supervisor name: Poelstra, K. and Salvati, A.
Degree programme: Pharmacy
Thesis type: Research Project
Language: English
Date Deposited: 28 Apr 2023 11:43
Last Modified: 28 Apr 2023 11:43

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