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目的 :探索适合临床使用的可维持新鲜羊膜活性的方法 ,确定其有效的维持时间 ,以便为临床使用制定新鲜羊膜的标准贮存方法。同时分析影响其有效贮存时间的相关因素。方法 :共取人胎盘 6个 ,分别剥离羊膜、胎膜 (羊膜和绒毛膜 )置于生理盐水、PBS液、DMEM液中 4℃贮存和 3 7℃下的组织培养贮存 ,贮存后 6h、 12h、 18h、 2 4h、 48h、 72h和 1周分别进行台盼蓝染色检测羊膜上皮的活性细胞数量和比例 ,同时进行电镜和光镜检查其相应时间点的羊膜组织变化。结果 :单纯羊膜在生理盐水、PBS、DMEM液 (4℃ )和组织培养中的有效贮存时间明显不同 (P1<0 0 1,P2 <0 0 1,P3 <0 0 1)。在PBS液 4℃贮存条件下 ,胎膜的有效贮存时间 (3 0 %左右死亡 )明显比羊膜为短 (P <0 0 1)。DMEM液 4℃下 2 4h时两者差别不大 (P >0 0 1) ,但胎膜在DMEM液中 48h时的上皮存活百分比明显比羊膜少 (P <0 0 1)。单纯的离体新鲜羊膜在生理盐水 4℃环境下贮存 6小时、在PBS液 4℃下贮存 12小时、DMEM液中 2 4小时和组织培养贮存法 1周仍可维持 70 %以上的羊膜上皮细胞存活 ,并且不会引起其基质胶原的变化。结论 :不同的贮存液和绒毛膜是否同时存在会不同程度地影响新鲜羊膜的贮存时间。单纯的离体新鲜羊膜在生理盐水 4℃环境下贮存
OBJECTIVE: To explore ways to maintain fresh amniotic activity suitable for clinical use and to determine the effective maintenance time for standard amniotic storage for clinical use. At the same time analyze the factors that affect the effective storage time. Methods: A total of 6 human placenta were removed and amniotic membrane was separated. The fetal membranes (amniotic membrane and chorion) were stored in saline, PBS solution, DMEM solution at 4 ℃ and tissue culture at 37 ℃ for 6h, 12h , 18h, 24h, 48h, 72h and 1 week respectively. The numbers and proportions of active cells in amniotic epithelium were detected by trypan blue staining. The changes of amniotic membrane were observed by electron microscope and light microscope. Results: The storage time of simple amniotic membrane in physiological saline, PBS, DMEM solution (4 ℃) and tissue culture were significantly different (P1 <0.01, P2 <0.01, P3 <0.01). The effective storage time of the membranes (about 30% of the deaths) was significantly shorter than that of the amniotic membranes (P <0.01) under the storage conditions of PBS liquid at 4 ° C. There was no significant difference between the two groups (P> 0 01) at 4 ℃ for 4 days in DMEM solution. However, the percentage of epithelium survival in DMEM solution was significantly lower than that in amniotic membrane at 48h (P <0.01). Pure isolated fresh amniotic membrane was stored at 4 ℃ in physiological saline for 6 hours, stored in PBS for 12 hours at 4 ℃, 24 hours in DMEM medium and 1 week in tissue culture and storage for more than 70% of amniotic epithelial cells Survive, and do not cause changes in its matrix collagen. Conclusion: Whether different storage solution and chorion coexist will affect the storage time of fresh amniotic membrane to some extent. Pure fresh amniotic membrane in vitro stored in saline at 4 ℃