[The off-target effects of dynamin inhibitor Dyngo-4a during the internalization of dextran by bone marrow mesenchymal stem cells in rat].

Abstract

Objective To investigate the possible off-target effects of dynamin (DNM) inhibitor Dyngo-4a in dynamin-dependent endocytic pathways. Methods Bone marrow mesenchymal stem cells (BMSCs) obtained from SD rats were isolated and cultured, and identified by flow cytometry. The cells were divided into inhibitor control group, Dyngo-4a-treated group, negative control siRNA (si-NC) transfection group, DNM2 siRNA transfection (si-DNM2) group, si-DNM2 and Dyngo-4a co-treated group. Real time quantitative PCR and Western blot analysis were used to verify the silencing efficiencies of DNM2 gene and CCK-8 assay were used to detect the cell viability after Dyngo-4a treatment. Confocal microscopy was used to detect the number and mean fluorescence intensity (MFI) of transferrin-Dylight649-positive and dextran-TMR-positive vesicles. Results The mRNA and protein expression levels of DNM2 were down-regulated using small interfering RNA. The number of transferrin-Dylight649-positive vesicles significantly decreased in si-DNM2 group compared with si-NC group. For the number and MFI of dextran-TMR-positive vesicles, no significant change was observed between the si-DNM2 group and the si-NC group, but there was a significant reduction in the si-DNM2 and Dyngo-4a co-treated group compared with the si-DNM2 group. A significant decrease was also found in the Dyngo-4a-treated group compared with the inhibitor control group. Conclusion The off-target effects of dynamin inhibitor Dyngo-4a presents in the internalization of dextran by BMSCs.