[Binding characteristics between RGD-containing cyclic peptide and rat hepatic stellate cells: an in vitro study].
OBJECTIVE
To investigate the binding characteristics between an artificial Arg-Gly-Asp (RGD)-containing cyclic peptide [cyclo(CGRGDSPK)] and rat hepatic stellate cells (HSC).


METHODS
An artificial RGD-containing cyclic peptide was labeled with fluorescein isothiocyanate (FITC). HSCs were isolated by collagenase in situ liver recirculating and purified by density gradient centrifugation from normal rats. The cells were cultured for 5 days of primary culture (quiescent phenotype) or for 7 days of secondary culture (activated phenotype). To access the binding and uptake, HSCs were incubated with FITC-cRGD of different concentrations at 4 degree C or 37 degree C, and then the binding and uptake were investigated by flow cytometry. The location of FITC-cRGD in HSC was investigated by fluorescent microscopy. Kd and maximal binding sites per cell were calculated by radioligand binding assay (RBA) of receptors using 3H-cRGD. In the interim, FITC-cAGA was used as a peptide control devoid of any binding site.


RESULTS
The binding between FITC-cRGD and HSC was saturable, time- and dose-dependent and could compete with overdosed unlabeled cRGD. The fluorescence was mainly distributed in cytoplasma, especially near the nuclei. Kd was 7.05 x 10(-9) mol/L and Bmax per cell was nearly 6.79 x 10(5).


CONCLUSIONS
The results demonstrate that cRGD are specifically taken up by HSC through a receptor-mediated pathway. The information is useful for understanding the ligand-receptor interaction of HSC. FITC labeled cyclic RGD-peptides meet the standards of special ligands and FITC does not change the binding activation of cyclic RGD-peptides.
Du
Shi-lin
SL
Department of Gastroenterology, Zhongshan Hospital, Fudan University, Shanghai, China.
Wang
Ji-yao
JY
Lu
Wei-yue
WY
D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology*; D010456:Peptides, Cyclic / Q000494:pharmacology*; D011485:Protein Binding; D051381:Rats
D009842:Oligopeptides; D010456:Peptides, Cyclic; C047981:arginyl-glycyl-aspartic acid