Objective:To explore the effects of miR-21-5p exosomes derived from human umbilical cord mesenchymal stem cells on apoptosis of human granular cells.Methods:A granular cell apoptosis model was constructed by treating KGN cells with different concentrations (0 μmol/L, 30 μmol/L, 60 μmol/L, and 100 μmol/L) of phosphoramide nitrogen mustard for 48 h. The mRNA and protein levels of bax and bcl2 were detected using qPCR and Western blotting, respectively. The apoptosis rate was measured using flow cytometry to screen for the optimal concentration of phosphoramide nitrogen mustard for constructing an apoptosis model. Hsa-miR-21-5p overexpression plasmid was used for instantaneously transfecting human umbilical cord mesenchymal stem cells, and the expression level of hsa-miR-21-5p was detected by qPCR. The miR-21-5p exosomes were separated and identified by flow cytometry and electron microscope. Different concentrations (5 μg/mL, 10 μg/mL and 15 μg/mL) of miR-21 exosomes were added into successful KGN cell apoptosis model to overexpress miR-21. The mRNA and protein levels of bax and bcl2 were detected using qPCR and Western blotting, respectively. PKH-26 was used to trace the position of human granular cells. Results:The levels of bax mRNA and protein in KGN cells treated with 60 μmol/L phosphoramide nitrogen mustard were significantly higher than those in the 0 μmol/L phosphoramide nitrogen mustard group (all P<0.001), while the levels of bcl2 mRNA and protein were significantly higher than those in the 0 μmol/L phosphoramide nitrogen mustard group ( P=0.005, P<0.001). The apoptosis rate of KGN cells after 60 μmol/L phosphoramide nitrogen mustard intervention was (38.10±2.90)%, while the apoptosis rate of KGN cells after 30 μmol/L phosphoramide nitrogen mustard intervention was (16.75±2.55)%, they were all significantly higher than that of the 0 μmol/L phosphoramide nitrogen mustard intervention group ( P=0.020, P=0.006). Hsa-miR-21-5p was transiently transfected into human umbilical cord blood mesenchymal stem cells, and the expression of has-miR-21-5p was higher than that in control group detected by qPCR ( P<0.001). The positive rate of surface protein CD9, CD63 and CD81 was 14.9%, 16.4% and 31.4%. The exosome was observed as "tea tray" or "concave hemisphere" by electron microscope. The exosome labeled by PKH-26 entered the granular cells and exerted biological effects. There was no statistically significant difference in bax mRNA expression levels between the 5 μg/mL, 10 μg/mL, and 15 μg/mL empty plasmid exosomes groups and the 60 μmol/L phosphoramide nitrogen mustard group (all P>0.05). However, the expression levels of bax mRNA in the 5 μg/mL, 10 μg/mL, and 15 μg/mL miR-21-5-p exosomes groups were lower than those in the 60 μmol/L phosphoramide nitrogen mustard group ( P=0.008, P=0.003, P<0.001). However, there was no statistically significant difference in the expression of bcl2 mRNA among the groups (all P>0.05). From the perspective of protein levels, there was no statistically significant difference in BAX protein expression between the 5 μg/mL, 10 μg/mL, and 15 μg/mL empty exosomes groups and 60 μmol/L phosphoramide nitrogen mustard group (all P>0.05), while the 5 μg/mL, 10 μg/mL, and 15 μg/mL miR-21-5-p exosomes groups showed a decrease in BAX protein expression compared with the 60 μmol/L phosphoramide nitrogen mustard group, and the differences were statistically significant (all P<0.001). However, there was no statistically significant difference in the expression of BCL2 protein among the intervention groups (all P>0.05). Conclusion:Hsa-miR-21-5p exosomes derived from human umbilical cord blood mesenchymal stem cells can effectively exert the anti-apoptotic effect.

Objective:To explore the effects of miR-21-5p exosomes derived from human umbilical cord mesenchymal stem cells on apoptosis of human granular cells.Methods:A granular cell apoptosis model was constructed by treating KGN cells with different concentrations (0 μmol/L, 30 μmol/L, 60 μmol/L, and 100 μmol/L) of phosphoramide nitrogen mustard for 48 h. The mRNA and protein levels of bax and bcl2 were detected using qPCR and Western blotting, respectively. The apoptosis rate was measured using flow cytometry to screen for the optimal concentration of phosphoramide nitrogen mustard for constructing an apoptosis model. Hsa-miR-21-5p overexpression plasmid was used for instantaneously transfecting human umbilical cord mesenchymal stem cells, and the expression level of hsa-miR-21-5p was detected by qPCR. The miR-21-5p exosomes were separated and identified by flow cytometry and electron microscope. Different concentrations (5 μg/mL, 10 μg/mL and 15 μg/mL) of miR-21 exosomes were added into successful KGN cell apoptosis model to overexpress miR-21. The mRNA and protein levels of bax and bcl2 were detected using qPCR and Western blotting, respectively. PKH-26 was used to trace the position of human granular cells. Results:The levels of bax mRNA and protein in KGN cells treated with 60 μmol/L phosphoramide nitrogen mustard were significantly higher than those in the 0 μmol/L phosphoramide nitrogen mustard group (all P<0.001), while the levels of bcl2 mRNA and protein were significantly higher than those in the 0 μmol/L phosphoramide nitrogen mustard group ( P=0.005, P<0.001). The apoptosis rate of KGN cells after 60 μmol/L phosphoramide nitrogen mustard intervention was (38.10±2.90)%, while the apoptosis rate of KGN cells after 30 μmol/L phosphoramide nitrogen mustard intervention was (16.75±2.55)%, they were all significantly higher than that of the 0 μmol/L phosphoramide nitrogen mustard intervention group ( P=0.020, P=0.006). Hsa-miR-21-5p was transiently transfected into human umbilical cord blood mesenchymal stem cells, and the expression of has-miR-21-5p was higher than that in control group detected by qPCR ( P<0.001). The positive rate of surface protein CD9, CD63 and CD81 was 14.9%, 16.4% and 31.4%. The exosome was observed as "tea tray" or "concave hemisphere" by electron microscope. The exosome labeled by PKH-26 entered the granular cells and exerted biological effects. There was no statistically significant difference in bax mRNA expression levels between the 5 μg/mL, 10 μg/mL, and 15 μg/mL empty plasmid exosomes groups and the 60 μmol/L phosphoramide nitrogen mustard group (all P>0.05). However, the expression levels of bax mRNA in the 5 μg/mL, 10 μg/mL, and 15 μg/mL miR-21-5-p exosomes groups were lower than those in the 60 μmol/L phosphoramide nitrogen mustard group ( P=0.008, P=0.003, P<0.001). However, there was no statistically significant difference in the expression of bcl2 mRNA among the groups (all P>0.05). From the perspective of protein levels, there was no statistically significant difference in BAX protein expression between the 5 μg/mL, 10 μg/mL, and 15 μg/mL empty exosomes groups and 60 μmol/L phosphoramide nitrogen mustard group (all P>0.05), while the 5 μg/mL, 10 μg/mL, and 15 μg/mL miR-21-5-p exosomes groups showed a decrease in BAX protein expression compared with the 60 μmol/L phosphoramide nitrogen mustard group, and the differences were statistically significant (all P<0.001). However, there was no statistically significant difference in the expression of BCL2 protein among the intervention groups (all P>0.05). Conclusion:Hsa-miR-21-5p exosomes derived from human umbilical cord blood mesenchymal stem cells can effectively exert the anti-apoptotic effect.