Explore The Fresh Amnion Perfect Save Method And Save Time To Promote Wound Healing

Objectives:This study randomly selected several healthy cesarean delivery of maternal (25～35 years old), prenatal maternal serological examination, eliminating the human immunodeficiency virus (HIV), hepatitis b virus (HBV), hepatitis c virus (HCV), cytomegalovirus, herpes virus, toxoplasma gondii, syphilis and other infectious diseases. To separate the maternal amniotic membrane(AM) under aseptic conditions and save them different months with pure glycerin, anhydrous alcohol and 50% DMEM medium after the sterilization processing, to study the best save method and save time by amniotic membrane transplantation experiments, HE staining slices and amniotic cells factor test when the biological function of amniotic membrane in reserve at the same time.Method:To save the amniotic membrane with three different ways 1-6 months respectively under different preservation conditions. From the day of amniotic starting preservation, to proceed experiment of amniotic membrane transplantation of mice mechanical trauma model by amniotic membrane of three kind of preserved way every months, comparing the effect of amniotic membrane preserved different way to promote wound healing of mice. In different months, taking three kind of saved amniotic membrane to do staining slice of HE, and observing the change of amniotic cells with the extension of save time under light microscope. To get three kinds of preserved amniotic membrane in equal amounts under aseptic conditions, dissolving amniotic cells and detecting the cytokine levels of epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (bFGF) and transforming growth factor-beta 1 (TGF-beta 1) of amniotic cells by ELISA method. Comparing the effect of three kinds of amniotic membrane preserved different month with cell factor levels of amniotic.Result:The wound healing time of mice which did amniotic membrane transplantation compared to did not amniotic membrane transplantation in mice significantly shorter (P<0.05). The wound healing time of mice which transplanted anhydrous alcohol amniotic membrane compared to the group of pure glycerin and 50% DMEM medium significantly shorter (P<0.05). The mice wound healing time of pure glycerin amniotic membrane transplant group has smaller difference with 50% DMEM medium amniotic membrane group (P>0.05). Three kind of preserved amniotic membrane, saving one month, cellular morphology basic integrity, intercellular increased. Saving two months, the part of epithelial cell nucleus osteoporosis, sponge layer structure is loose, the sponge layer of AM saved with pure glycerin and 50% DMEM vacuoles both occurred Cavitation sample. Saving three months, the part of epithelial cell is broken, and the nucleus depigmentation, dense layer changes litter, the structure of fibroblast and sponge layer is loose, sponge layer of three kinds of preserved amniotic membrane both occurred Cavitation sample, sponge layer of amniotic membrane preserved pure glycerin partially missing. Saving four months, intercellular space increased again, the nucleus depigmentation intensified, the sponge layer missing, the epithelial layer structure of AM preserved pure glycerin is not neat. Keep five months, epithelial cells appears a small amount of missing, part of cells necrosis, nuclei showed pyknosis, the structure of amniotic cells layer thin, part of fibroblasts layer pure of AM preserved glycerin and anhydrous alcohol is missing, the missing and necrosis of AM epithelial cell layer preserved 50% DMEM medium is serious. Kept up to six months, amniotic epithelial cell layer saved by pure glycerin is not continuous, part of fibroblast layer is missing, the structure of amniotic epithelial cell layer of AM preserved anhydrous alcohol is loose, fibroblast layer falls off large area,50% DMEM medium amniotic epithelial cells occurred pyknosis and necrosis, substrate layer appears air ball sample, basement membrane is dim. This experiment detects the level of cytokines of three kinds of preserved amniotic membrane in different months by ELISA method. We found that the levels of four kind cytokines decrease with the saving time extended of AM. The levels of fibroblast growth factor (bFGF), transforming growth factor beta 1 (TGF beta 1) and Epidermal growth factor (EGF) all decline significantly after amniotic membrane preserved in May (P<0.05), vascular endothelial growth factor (VEGF) content decline significantly during AM kept in February (P<0.05).Conclusion:The effect of amniotic membrane to promote wound healing, the integrity of the cell morphology and cell factor levels all reduce as the preservation time extension. The effect of amniotic membrane preserved by anhydrous alcohol promotes wound healing and the integrity of amniotic membrane structure better than amniotic membrane preserved by pure glycerin and 50% DMEM medium. It is obvious that the cytokines content of three kinds of preserved amniotic membrane declines in the overall level after five months.