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Objective To investigate the effects of fresh and preserved amniotic membrane on polymorphonuclear neutrophils (PMNs) so as to understand the anti-inflammatory mechanism of amniotic membrane transplantation.Methods Conditioned medium was collected 48 hours after fresh or preserved amnions were cultured in DMEM and 5% CO 2 at 37℃. Then, polymorphonuclear cells were cultured in conditioned culture or DMEM. Fluorescent microscopy with 4’,6-diamidino-2-phenylindole (DAPI) staining and cytometry were performed 6, 9, 12, and 15 hours later. Results Apoptotic neutrophils were found in each group at different time points. The percentage of apoptotic cells at 6, 9, 12, and 15 hours after culture in the fresh and preserved amnion groups and the control group was 17.3%, 24.4%, 29.8%, 37.1%, and 16.2%, 20.1%, 23.7%, 27.7%, and 10.2%, 13.7%, 21.1%, 26.4%, respectively (t test, P 1<0.01, P 2<0.01 and P 3<0.01).Conclusion Amniotic membrane can accelerate apoptosis of polymorphonuclear neutrophils, reduce inflammation, and prevent ocular surface collagen from resolution, indicating that fresh amnion might have a stronger effect than preserved amnion.
Objective To investigate the effects of fresh and preserved amniotic membrane on polymorphonuclear neutrophils (PMNs) so as to understand the anti-inflammatory mechanism of amniotic membrane transplantation. Methods Conditioned medium was collected 48 hours after fresh or preserved amnions were cultured in DMEM and 5% CO 2 at 37 ° C. Then, the polymorphonuclear cells were cultured in conditioned culture or DMEM. Fluorescent microscopy with 4 ’, 6-diamidino-2-phenylindole (DAPI) staining and cytometry were performed 6, 9, 12, and 15 hours later. Results Apoptotic neutrophils were found in each group at different time points. The percentage of apoptotic cells at 6, 9, 12, and 15 hours after culture in the fresh and preserved amnion groups and the control group was 17.3%, 24.4%, 29.8% , 37.1% and 16.2%, 20.1%, 23.7%, 27.7% and 10.2%, 13.7%, 21.1% and 26.4% respectively (t test, P 1 <0.01, P 2 <0.01 and P 3 <0.01) .Conclusion Amniotic membrane can advance apoptosis of polymorphonuclear n eutrophils, reduce inflammation, and prevent ocular surface collagen from resolution, indicating that fresh amnion might have a stronger effect than preserved amnion.