不同冻存方法对羊膜超微结构和上皮细胞活性的影响

摘要

背景：羊膜冻存方法众多，对羊膜超微结构和生物活性的影响不一，目前尚无有效的冻存方法。　　目的：比较不同冻存方法对羊膜超微结构和活性影响的研究，探寻更为理想的冻存方法。　　方法：将新鲜羊膜采用深低温和玻璃化冻存法保存，分别于冻存后3，6个月复苏羊膜，以新鲜羊膜组织为对照组，比较羊膜的超微结构差异、羊膜上皮细胞离体氧分压和乳酸脱氢酶活性。　　结果与结论：不同冻存方法保存的羊膜超微结构有明显改变，但玻璃化冻存对其超微结构的影响相对较小；与新鲜羊膜相比较，深低温冻存组3，6个月羊膜的乳酸脱氢酶灰度值和氧分压明显降低(P <0.05)；玻璃化冻存组6个月后的羊膜乳酸脱氢酶灰度值和氧分压明显降低(P <0.05)，而玻璃化冻存组3个月后的上述检测结果与新鲜羊膜相比差异无显著性意义(P >0.05)。结果证实，羊膜的玻璃化冻存技术优于深低温冻存技术，不仅维持了羊膜的超微结构，而且保持了羊膜上皮细胞的功能和活性。%BACKGROUND: There are currently many cryopreservation methods for the aminotic membrane, which have varying effects on the ultrastructure and biological activity of amniotic membrane, but on no one is effective. OBJECTIVE: To compare the effects of different cryopreservation methods on the ultrastructure and viability of aminotic membrane and to seek the ideal cryopreservation method. METHODS: Aminotic membrane separated from the fresh placenta was preserved respectively with deep-frozen cryopreservation and vitrification, and everyway was run for 3 and 6 months. Fresh aminotic membrane was used as control. The ultrastructure of aminotic membrane was observed by transmission electron microscopy, and the viability of aminotic membrane was assessed by microcomputer analysis system for biological oxygen consumption, and immunohistochemical staining combined with image analysis system was used for lactate dehydrogenase activity. RESULTS AND CONCLUSION:After 3 and 6 months of crypreservation, the damage to the ultrastructure of aminotic membrane by vitreous cryopreservation was slighter than that of amniotic membrane cryopreserved at-80℃. Compared with the fresh aminotic membrane, the gray value of lactate dehydrogenase and partial pressure of oxygen were significantly decreased in the cryopreserved aminotic membrane by deep-frozen cryopreservation at 3 and 6 months (P < 0.05) and by vitreous cryopreservation at 6 months (P < 0.05), but there was no statisticaly significant difference in the change rate of oxygen partial pressure and the gray value of lactate dehydrogenase between the fresh aminotic membrane and the cryopreserved aminotic membrane by vitreous cryopreservation at 3 months. The present study led to the conclusion that vitreous cryopreservation protocol alows to not only maintain the integrity of AM, but also to preserve the viability of the cels. So the vitreous cryopreservation is superior to the deep-frozen cryopreservation for cryopreservation of aminotic membrane.