Objective: To investigate the binding of the chemosynthetic analogue of N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) FAM-Aca-SDKP to hepatic stellate cell-T6 (HSC-T6) cells and basic physical characteristics.
Methods: The Ac-SDKP analogue short-peptide FAM-Aca-SDKP carrying green fluorescence was synthesized chemically. Quantitative real-time PCR was used to evaluate its effect on the secretion of HSC collagen and verify the consistency in the biological effect between FAM-Aca-SDKP and Ac-SDKP. A fluorescence microscope was used to observe the binding between FAM-Aca-SDKP and HSC-T6, and flow cytometry was used to evaluate the time-concentration effect of the binding between FAM-Aca-SDKP and HSC-T6. The t-test or rank sum test was used for the statistical analysis of different types of data.
Results: After HSC-T6 was incubated with Ac-SDKP or FAM-Aca-SDKP for 24 hours, the expression of type I collagen in HSC-T6 was increased, when the action time was 0.5 hour, Ac-SDKP and FAM-Aca-SDKP caused a 30%-50% reduction in the expression of type I collagen. After HSC-T6 was incubated with FAM-Aca-SDKP, strong green fluorescence was observed on cell surface under a fluorescence microscope, and after Ac-SDKP was added, Ac-SDKP significantly reduced the fluorescence intensity on cell surface due to competitive inhibition. Flow cytometry showed that when the concentration of FAM-Aca-SDKP was 0-50μmol/L, the rate of fluorescence-positive cells rapidly increased from 0 to 12%; when the concentration was 50-100μmol/L, the rate of fluorescence-positive cells only increased from 12% to 14%; co-incubation with Ac-SDKP significantly reduced the rate of fluorescence-positive cells. The number of positive cells reached the peak at the 45-minute point of the incubation and then decreased gradually.
Conclusions: FAM-Aca-SDKP can bind to the surface of HSC-T6 cells, and this process has ligand-receptor binding characteristics such as competitive inhibition, saturability, and time-concentration effect.