不同保存方法对羊膜生物学性能影响的比较研究

目的观察不同保存方法对羊膜生物学性能的影响。方法取10例健康剖宫产产妇胎盘,剥离羊膜,将羊膜平铺于无菌滤纸片上,上皮面朝上,剪成20 mm×20 mm小块,随机分为3组,分别采用Hank’s平衡盐/甘油溶液深低温（-80℃）保存、纯甘油4℃保存、冷冻干燥常温保存。分别于保存3、6、12个月取羊膜进行HE染色光镜观察、胶原蛋白酶Ⅳ降解速度测定和细胞因子含量放射免疫测定,并与新鲜羊膜进行对照。结果①新鲜羊膜及3种方法保存的羊膜均可见到羊膜的5层组织结构,自内向外依次为上皮细胞层、基底膜、致密层、纤维母细胞层、海绵层。新鲜羊膜的上皮细胞形态完好、排列较整齐紧密,其下可见厚而连续的基底膜,致密层、纤维母细胞层、海绵层结构紧密。冷冻干燥常温保存3个月,羊膜组织形态特征与新鲜羊膜基本相似,上皮细胞结构完整、排列紧密,连续的基底膜、致密层、纤维母细胞层、海绵层清晰可见;保存6个月,上皮细胞形态基本完好、排列整齐,但基底膜略显模糊,纤维母细胞层、海绵层相对疏松;保存12个月,上皮细胞形态基本完好但核上移,基底膜模糊,纤维母细胞层、海绵层疏松变薄,其内出现空泡。Hank’s平衡盐/甘油溶液深低温保存3个月,羊膜上皮细胞形态基本完好,基底膜略显模糊,致密层、纤维母细胞层、海绵层相对疏松;保存6个月,上皮细胞结构基本完整,连续排列但细胞间隙较为模糊,基底膜更显模糊,致密层、纤维母细胞层、海绵层进一步疏松;保存12个月,上皮细胞皱缩,上皮层连续性中断,基底膜及致密层基本完整,但各层结构模糊不清。纯甘油4℃保存3个月,羊膜上皮细胞皱缩,细胞间隙加大,基底膜及致密层基本完整,各层结构依稀可见;保存6个月,部分上皮细胞破裂,基底膜完整,致密层、纤维母细胞层、海绵层疏松;保存12个月,羊膜上皮细胞部分脱落,基质层厚度变薄,基底膜、致密层模糊不清,部分纤维母细胞层和海绵层缺失。②与新鲜羊膜相比,保存羊膜在胶原酶Ⅳ溶液中降解速度均有所加快。其中纯甘油4℃保存羊膜降解速度最快,冷冻干燥保存羊膜降解速度最慢。③冷冻干燥保存羊膜中被检测的8种细胞因子残存量均超过40%;Hank’s平衡盐/甘油溶液深低温保存和纯甘油4℃保存羊膜中8种细胞因子残存量均低于20%,明显低于冷冻干燥保存之羊膜细胞因子存留量（P〈0.05）。结论 3种方法中以冷冻干燥常温法保存羊膜效果最好,纯甘油4℃保存方法效果最差。冷冻干燥常温羊膜保存时限以不超过1年、Hank’s平衡盐/甘油溶液深低温保存不超过6个月、纯甘油4℃保存不超过3个月为宜。

Effects of different Preservation Methods on Biological Properties of Amniotic Membrane

Objective To investigate the effects of different preservation methods on biological properties of amniotic membrane（AM）.Methods AM obtained from 10 placentas of health women after caesarean section was minced into fragments（20 mm×20 mm） on sterile filter paper disks.The AM was preserved by 3 methods：Hank＇s balanced salt solution /glycerol at-80 ℃,pure glycerol at 4 ℃ and freeze-dried storage at room temperature.After preservation for 3 months,6 months and 12 months,the morphology of AM was observed by HE staining under light microscope and the degradation by type IV collagenase was analyzed.The levels of cytokines were determined by radioimmunoassay.All biological properties of preserved AM were compared with fresh AM.Results The structure layers,consisting of epithelial cells,basement membrane,compact layer,fibroblasts layer and strata spongiosum,were observed obviously in fresh AM and preserved AM.In fresh AM,epithelial cells were tightly arranged on a thick and continuous basement membrane.After freeze-dried preservation for 3 months,the morphologic characteristics of AM were similar to fresh AM.After freeze-dried preservation for 6 months,epithelial cells were largely intact,but basement membrane was slightly nebulous,and fibroblasts and strata spongiosum were somewhat loose.After freeze-dried preservation for 6 months,the nucleus of epithelial cells shifted to the top of cells,and nebulous basement membrane,loose and thin fibroblasts and strata spongiosum,and vacuolus were observed in AM.After cryopreservation in Hank＇s balanced salt solution/glycerol for 3 months,intact epithelial cells were observed in AM,but basement membrane was indistinct slightly,and fibroblasts and strata spongiosum were loose.After cryopreservation in Hank＇s balanced salt solution/glycerol for 6 months,basement membrane was more blurred,and compact layer,fibroblast and strata spongiosum were looser.After cryopreservation in Hank＇s balanced salt solution/glycerol for 12 months,epithelial cells shrank and structure layers were not clear.After preservation in glycerol for 3 months,epithelial cells shrank,intercellular space increased,and basement membrane and compact layer maintained structural integrity.After preservation in glycerol for 6 months,epithelial cells dissolved partly,and compact layer,fibroblasts and strata spongiosum were loose.After preservation in glycerol for 12 months,part of epithelial cells shed,and part of fibroblasts and strata spongiosum were lost.Compared with fresh AM,the velocity of degradation by collagenase IV increased in preserved AM.The maximum velocity was observed in AM preserved in glycerol and the minimum velocity in freeze-dried AM.The amount of cytokines in AM preserved in glycerol（20%）and AM preserved in Hank＇s balanced salt solution/glycerol（20%） was significantlylower than that in freeze-dried AM（40%）.Conclusion Freeze-dried storage is the most effective method for AM preservation among the 3 preservation methods.Freeze-dried storage,Hank＇s balanced salt solution/glycerol and pure glycerol for AM preservation should be used within 12 months,6 months and 3 months,respectively.