Cytotoxicity of phthalic anhydride and methotrexate exposed HUVEC cells
Mäkinen, Anni (2025)
2025
Bioteknologian ja biolääketieteen tekniikan kandidaattiohjelma - Bachelor's Programme in Biotechnology and Biomedical Engineering
Lääketieteen ja terveysteknologian tiedekunta - Faculty of Medicine and Health Technology
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Hyväksymispäivämäärä
2025-02-11
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:tuni-202502112116
https://urn.fi/URN:NBN:fi:tuni-202502112116
Tiivistelmä
Pulmonary fibrosis is a progressive fibrotic disease that occurs in the lung tissue. In the fi-brotic lung tissue, excess number of fibroblasts and immune cells accumulate to the interstitial lung tissue. This disease does not have a working medication and thus will lead to the death of the patient without lung transplant. Due to these factors, finding a medication for pulmonary fi-brosis would be an important step. Creating a medication for disease requires understanding of its mechanisms. In order to find the mechanisms of pulmonary fibrosis in molecular level, expressed genes specific of a fibrotic cell need to be determined. For this purpose, genes ex-pressed in fibrotic cells need to be compared with expressed genes of a cell that has been ex-posed to poisonous substance that does not cause fibrosis. By this comparison, it is possible to separate the effect of poisonous chemical from specifically pulmonary fibrosis inducing chemical.
The aim of this bachelor’s thesis was to contribute to the creation of pulmonary fibrosis in vitro model to be able to create a medication for this disease. The goal of this experiment was to determine whether methotrexate and phthalic anhydride (PAH) could be used as reference chemicals in the in vitro model of pulmonary fibrosis. This was done by measuring the cyto-toxicity of these substances and calculating their IC50 and IC20 values.
The experiment was conducted with human umbilical vein endothelial cells (HUVEC). These cells model the structure of endothelial cells in the walls of the pulmonary veins. HU-VEC cells were cultivated in cell culture bottles for a week to ensure sufficient number of cells. After cultivation, cells were plated to 96-Well Microplates. Both potential reference substances had their own plate and then third plate was seeded to positive control substance sodium do-decyl sulfate. Exposure chemicals were added to their plates in 8 different concentrations with tracer compounds. After exposure, cell viability was measured from the plates with lumines-cence reader after 24,48 and 72 hours. One measurement round took 2 weeks to complete, and 3 rounds were performed to guarantee repeatability.
The results of this experiment were not according to the set objective. Within the used concentrations methotrexate did not decrease the viability of cells. Thus, it was not possible to determine the IC values for this substance. Phthalic anhydride showed inconsistent behavior through the experiment, and it was not possible to determine the IC values for this substance either. Based on the results of this study, higher concentrations for both substances could be used in future studies. Higher concentrations might be able to reduce the cell viability in the expected manner and then be suitable as reference chemicals for the in vitro model.
The aim of this bachelor’s thesis was to contribute to the creation of pulmonary fibrosis in vitro model to be able to create a medication for this disease. The goal of this experiment was to determine whether methotrexate and phthalic anhydride (PAH) could be used as reference chemicals in the in vitro model of pulmonary fibrosis. This was done by measuring the cyto-toxicity of these substances and calculating their IC50 and IC20 values.
The experiment was conducted with human umbilical vein endothelial cells (HUVEC). These cells model the structure of endothelial cells in the walls of the pulmonary veins. HU-VEC cells were cultivated in cell culture bottles for a week to ensure sufficient number of cells. After cultivation, cells were plated to 96-Well Microplates. Both potential reference substances had their own plate and then third plate was seeded to positive control substance sodium do-decyl sulfate. Exposure chemicals were added to their plates in 8 different concentrations with tracer compounds. After exposure, cell viability was measured from the plates with lumines-cence reader after 24,48 and 72 hours. One measurement round took 2 weeks to complete, and 3 rounds were performed to guarantee repeatability.
The results of this experiment were not according to the set objective. Within the used concentrations methotrexate did not decrease the viability of cells. Thus, it was not possible to determine the IC values for this substance. Phthalic anhydride showed inconsistent behavior through the experiment, and it was not possible to determine the IC values for this substance either. Based on the results of this study, higher concentrations for both substances could be used in future studies. Higher concentrations might be able to reduce the cell viability in the expected manner and then be suitable as reference chemicals for the in vitro model.
Kokoelmat
- Kandidaatintutkielmat [8996]