羊膜保存方法及其对羊膜活性影响的研究

The effects on the vitality of amniotic membrane at different preservation methods

目的 :观察不同保存方法及保存时间对羊膜活性的影响。方法 :取健康剖宫产产妇胎盘 ,剥离其羊膜 ,分成 2 5× 2 5cm2 小块 ,随机分成四组 ,每组 8块 ,按四种不同方法保存。保存1、 3个月后分别取羊膜行光镜、电镜、酶组织化学〔乳酸脱氢酶 (LDH)、琥珀酸脱氢酶 (SDH)〕及免疫组织化学〔碱性成纤维细胞生长因子 (bFGF)〕观察 ,并与新鲜羊膜对比。结果 :①光镜下 ,甘油 4℃保存 1、 3个月羊膜上皮层结构破坏明显 ;DMEM :甘油 -70℃保存 1、 3个月羊膜组织结构基本完整 ;简化二、一步深低温保存 1、 3个月的羊膜上皮形态完好。②电镜下 ,甘油 4℃保存 1、 3个月上皮细胞破坏明显 ;DMEM :甘油 -70℃保存 1、 3个月羊膜上皮细胞线粒体肿胀 ,部分细胞核染色质边集 ;简化二、一步深低温保存 1、 3个月部分线粒体肿胀。细胞核变化不明显。③甘油 4℃、DMEM :甘油 -70℃保存法保存 1、 3个月羊膜上皮细胞SDH、LDH活性与对照组相比差别有极显著性 (P <0 0 0 1) ,保存 3个月与保存 1个月之间差别有极显著性 (P <0 0 0 1) ;简化二、一步深低温保存 1、 3个月的两种酶活性与对照组相比差异无显著性 (P >0 0 5 )。④bFGF分布于羊膜上皮和基质层。甘油 4℃和DMEM :甘油 -70℃保存1、 3个月bFGF在上皮层的表达与对?

Objective:To investigate the roles of different preservation methods of amniotic membrane (AM). Methods:Fresh AM was preserved by 4 ways,glycerol at -4℃,DMEM/glycerol at 70℃ and simplified two-step as well as simplified one-step cryopreservation method,every way was run for 1 and 3 months,respectively.The epithelial structure (light and electron microscopes) and the enzyme activity (SDH and LDH) of AM were analyzed.Immunohistochemical method combining with image analysis system was used to investigate the changes in the expression of basic fibroblast growth factor (bFGF). Results:Most of the epithelial cells in AM preserved in glycerol at 4℃ were destroyed.A few in DMEM/glycerol at -70℃ were lost,while AM preserved in rest two ways remained intact.SDH and LDH activities and the express of bFGF in the epithelial cells of AM preserved in two glycerol groups decreased significantly (P<0 001),while in the other two methods were as the same as that in the fresh control group (P>0 05). Conclusions:The vitality of AM preserved in glycerol at 4℃ and in DMEM/glycerol at -70℃ decreased significantly.Simplified two-step and simplified one-step cryopreservations maintained the vitality of AM.Simplified one-step cryopreservation was suitable for the long-term preservation of AM.