Study on the effects of recombinant arginine deiminase and its modified form with a cell-penetrating peptide HBHAc-HE, in 2D and 3D cell cultures, respectively, of breast cancer cells

Autor: Po-Hsun Chang, 張博勛
Rok vydání: 2019
Druh dokumentu: 學位論文 ; thesis
Popis: 107
The differences between a monolayer cell culture and spheroids in human come from the heterogeneity of spheroids. Thus, current 2D cell culture is not enough to be an ideal model for predicting physiological conditions in human bodies. To make the difference between in vitro and in vivo assays smaller, the development of 3D in vitro cell culture which is similar to human body has drawn more and more attentions in recent years. This study built up 3D in vitro cell cultures of MCF-7 and MDA-MB-231 breast cancer cells and compared the differences of sensitivity to the protein drugs we prepared, ADI and HBHAc-HE-ADI, between 2D and 3D cell cultures. We would like to know the possibility of development and application of the drugs prepared in this study by a 3D in vitro cell culture which is more similar to spheroids in human body. On the part of drug preparation, we transformed the plasmid of ADI and HBHAc-HE-ADI into E.coli BL21. After confirming the sequences were correct, we cultivated the bacteria by 2X YT medium and added IPTG to make them express the protein in a large amount. The pellets were centrifuged and lysed to get the inclusion bodies. Then the proteins were soluted out and went through the steps of refolding, condensing, purification and dialysis. Finally, we got the ADI and HBHAc-HE-ADI. We confirmed the correctness of them by SDS-PAGE and western blots, and used Bradford protein assay to quantify the amount of proteins. With ADI activity assay, we knew the unit activity of ADI and HBHAc-HE-ADI were 259 and 177 mU/mg, respectively. On the part of 2D cell culture assays, we design different pH and oxygen concentrations in each group and compared the differences between MCF-7 and MDA-MB-231. The results showed that MDA-MB-231 was more sensitive to ADI and HBHAc-HE-ADI than MCF-7 either at different pH or oxygen concentrations, and no significant differences between the effects of HBHAc-HE-ADI and ADI on two cell cultures were found at different pH and oxygen concentrations. On the part of building up the 3D cell cultures, non-adherent culture was used and different methods of adding excellular matrix were compared in order to form up homogeneous MCF-7 and MDA-MB-231 spheroids. After measuring the diameter of spheroids formed by different seeding cells, the most appropriate seeding cell density was chosen and the metabolic activity of them at different days were measured. Finally, the dosing day was chosen after taking the morphology, diameter, and metabolic activity of spheroids into account. On the part of 3D cell culture assays, both MDA-MB-231 and MCF-7 spheroids showed resistance to ADI and HBHAc-HE-ADI that their cell viability had no significant differences between the doses ranged from 0.001 to 10 mU/mL. Also, the diameter of spheroids didn’t change during drug administration. These results showed that the sensitivity to drugs was different between 3D and 2D cell cultures. The 3D cell culture models built up in this study can be applied to the screening and development of other drugs, which would be a link between 2D cell culture assays and in vivo assays.
Databáze: Networked Digital Library of Theses & Dissertations
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