羊膜对多形核白细胞调亡的影响

The Effect of Amniotic Membrane on Polymorphonuclear Cells

目的 :探讨羊膜对多形核白细胞凋亡的影响 ,同时比较新鲜羊膜和保存羊膜对其影响的差异 ,以初步揭示羊膜移植减轻炎症等作用的有关机制。方法 :先将新鲜羊膜和保存羊膜进行组织块培养 2天后收集条件培养基 ,然后将其加入己分离好的多形核白细胞中继续进行培养 (DMEM培养基作对照组 ) ,培养后 6、 9、 12、 15小时分别应用Dapi荧光染色和流式细胞仪的方法进行凋亡的研究。结果 :每组在各时间点均可见到凋亡细胞 ,凋亡细胞的数量 (流式细胞仪 )随时间的延长而逐渐增多。新鲜羊膜组、保存羊膜组和对照组在 6、 9、 12、 15小时多形核白细胞的凋亡百分比分别为 17 3%、 2 4 4%、 2 9 8%、 37 1% ,16 2 %、 2 0 1%、2 3 7%、 2 7 7%和 10 2 %、 13 7%、 2 1 1%、 2 6 4%。统计学上三组间的差异具有显著性 (组间差值t检验 ,P1<0 0 1,P2 <0 0 1,P3<0 0 1)。结论 :羊膜可以促进多形核白细胞的凋亡 ,进而影响其功能的变化 ,从而减轻炎症和阻止眼表面基质的溶解。新鲜羊膜比保存羊膜更能促进其凋亡 。

Objective:To investigate the effect of fresh and preserved amniotic membrane on polymorphonuclear neutrophils in order to uncerstand the associated anti inflammatory mechanism of amniotic membrane transplantation. Methods:Conditioned medium was collected 48h later when fresh or preserved amnion were cultured in DMEM at 5% CO 2,37℃.Then polymorphonuclear cells were cultured in conditioned culture or DMEM.Fluoreceint microscopy with Dapi staining and cytometry were performed 6h,9h,12h,and 15h later. Results:Apoptotic neutrophils were found in every group at different check point.The percentage of apoptotic cells at 6h,9h,12h,and 15h in fresh amnion group,preserved amnion group and 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 P 3<0 01). Conclusions:Amniotic membrane can fasten apoptosis of polymorphonuclear neutrophils to reduce inflammation and prevent ocular surface collagen from lysis.It seemed that fresh amnion may act stronger than preserved amnion.