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Diagnosis of Anti-Angiogenesis Peptide Binding to HUVECs Using FITC
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F Amini Sepehr1    , SM Asghari1 , M Shourian 2 |
1- Department of biology, Faculty of Sciences, University of Guilan, Rasht, Iran
2- Department of biology, Faculty of Sciences, University of Guilan, Rasht, Iran , shourian@guilan.ac.ir |
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Abstract: (2969 Views) |
Background & aim: Angiogenesis is the production of new blood vessels from existing vessels. Angiogenesis depends on the balance between its activating and inhibitory factors. Endostatin, a 20-kDa fragment of collagen XVIII, is a member of the angiogenesis inhibitory protein group that inhibits endothelial cell proliferation and migration and tubular-like structure. The use of colorimetric methods by fluorescent probes as a tracer is widespread, so the aim of this study was to determine and detect the binding of anti-angiogenic peptide to human cord endothelial cells using a fluorescence marker.
Methods: In the present experimental study, the 27 amino acid fragment of the endostatin protein amino acid domain was labeled with FITC as a fluorescence marker. Gel filtration chromatography with Sephadex G10 was used to separate the bound peptides and finally FTIR method was used to confirm the binding of FITC to the peptide. Human umbilical cord endothelial cells were treated with marker-bound peptides after culturing and studied by fluorescence microscopy to observe peptide binding to their receptor on the surface of these cells.
Results: findings by Fluorescence microscopy confirmed binding antiangiogenic peptide to its specific receptor. Various conditions such as concentration, time, and temperature were tested to achieve the proper conditions for marking peptides. Unlabeled FITCs separated by gel filtration Chromatography method with sephedex G10. Human umbilical endothelial cells (HUVECs) are harvested and treated with conjugate samples (FITC-peptide) from gel filtration chromatography. After the required time, the cells were fixed and the binding of labeled peptides to their receptors on HUVECs was analyzed by Fluorescence microscope.
Conclusion: The results of fluorescence microscopy confirmed the binding of this antiangiogenic peptide to its specific receptor on endothelial cells. Different experimental conditions such as peptide concentration and FITC and incubation time and temperature were studied to obtain the appropriate conditions for peptide labeling. Free FITCs were separated by conjugated FITC-peptide samples by gel filtration chromatography. Human umbilical cord endothelial cells (HUVEC) cultured with FITC-peptide conjugated samples were treated. The cells were then fixed and examined by fluorescence microscopy.
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| Keywords: Antiangiogenic peptide, Fluorescein isothiocyanat, Human umbilical endothelial cells (HUVECs) |
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Full-Text [PDF 3976 kb]
(692 Downloads)
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Type of Study: Research |
Subject:
Biochemistry
Received: 2019/04/19 | Accepted: 2019/09/2 | Published: 2019/09/21
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