Proteomics analyses of Fli-1 transgenic zebrafish
Date Issued
2011
Date
2011
Author(s)
Lyu, Syue-Lin
Abstract
The vascular endothelium is a thin layer of cells that lines the interior surface of blood vessels, forming an interface between circulating blood in the lumen and the rest of the vessel wall. Vascular endothelial cells play an important role in vascular development, blood coagulation, immune system and even in atherosclerosis and cancer. It has been reported that normal blood vessel formation is critical for embryonic development, whereas the aberrant blood vessel formation in the adult contributes to pathologies such as tumor growth. On the other hand, biomarker is a prevalent tool for early diagnosis of human diseases. Zebrafish fli-1 promoter is able to drive expression of EGFP in all blood vessels throughout embryogenesis. In this study, we intend to identify some novel biomarker of vascular endothelial cells by using Fli-1: EGFP transgenic zebrafish embryos. First, we collected and isolated cells of Fli-1: EGFP transgenic zebrafish embryo at 4hpf and 120hpf and then the EGFP positive vascular endothelial cells were sorted by Flow cytometry (FACSAria). The cells from 4hpf Fli-1 embryos were shortly co-cultured with feeder layer to increase the cell number (for expanding cell population) before sorting process. After resolving the total lysate from the sorted cells by SDS-PAGE, the separated proteins were subjected to trypsin in gel digestion followed by LC/MS/MS analysis. The data of proteomics analysis present the different protein patterns in these two stages. According to these data, we could identify some developmental stage-specific proteins and potential vascular endothelial cell biomarkers, Thrombospondin-4A and 4B. Furthermore, we clone the zebrafish TSP4A and TSP4B for the His-TSP4A and His-TSP4B fusion proteins and generate the antibodies against these antigens. By using immuno-staining, we could find out zebrafish Thrombospondin-4A respectively co-localize with VEGFR1、VEGFR2 and Tie-2. Thrombospondin-4A may be a biomarker for clinical diagnosis or prognosis in human disease management.
Subjects
zebrafish
vascular endothelial cells
biomarker
SDGs
Type
thesis
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