Impact of exosomes from IGFBP7-transfected human adipose-derived mesenchymal stem cells on apoptosis of malignant melanoma A375 cells
Objective To analyze the uptake capacity of the malignant melanoma cell line A375 towards exosomes derived from insulin-like growth factor binding protein 7(IGFBP7)transfected human adipose-derived mesenchymal stem cells(ADSCs)and the subsequent effects on apoptosis,providing new insights for gene therapy in malignant melanoma.Methods Primary adipose-derived stem cells were prepared by adherent culture of tissue blocks in vitro and passaged to obtain the second-generation.Cell surface markers were identified by cell climbing immunofluorescence.IGFBP7-GFP overexpression vectors were transfected into ADSCs to create ADSCsIGFBP7,followed by measuring the mRNA level of IGFBP7 by fluorescent quantitative PCR.Exosomes were isolated and extracted,and their ultrastructure was observed under electron microscopy.The protein expressions of CD63 and CD81 proteins were detected using Western blot.ADSCsIGFBP7 exosomes were applied to A375 cells,with the uptake capacity of A375 cells for ADSCsIGFBP7 exosomes being observed under a fluorescence microscope.Apoptosis of A375 cells was detected using flow cytometry.Results Immunofluorescence results showed positive expression of CD44 and negative expression of CD34 in the isolated cells,indicating that the isolated and cultured cells displayed a specific phenotype of ADSCs.IGFBP7 mRNA expression in the transfection group was higher compared to the control group(t=11.50,P<0.001),suggesting successful transfection of IGFBP7-GFP overexpression vector into ADSCs to construct ADSCsIGFBP7cells.Transmission electron microscopy revealed that the exosomes had distinct heterogeneity,showing the morphology of exosomes of ADSCsIGFBP7 appeared as spherical structures of varying sizes with a darker color in the center and low-density substances at the edges.Western blot results indicated positive expression of exosome marker proteins CD63 and CD81,which confirmed the successful extraction of exosomes.After the exosomes of ADSCsIGFBP7 were co-cultured with A375 cells for 6 hours and the exosomes were labeled with PKH26,it was observed that A375 cells exhibited an increased uptake of ADSCIGFBP7 exosomes after 6 hours compared with the control group.The apoptosis rate of A375 cells in the A375 cell group and A375+ADSCIGFBP7 exosome group were(0.743±0.050)%and(4.990±0.196)%,respectively.The difference was statistically significant compared to the control group(t=20.93,P<0.001),with the apoptosis rate of A375+ADSCs IGFBP7 exosome cells being higher.Conclusions ADSCsIGFBP7 exosomes can be taken up by A375 cells and promote their apoptosis.
万方数据
维普
