液氮快速冻存法保存人羊膜抗冻剂筛选研究  

Cryoprotectant Screening to Preserve Human Amnion by Liquid Nitrogen Rapid Cryopreserving

作　　者：杨文贤[1] 刘亮[1] 朱富军[2] 吕礼芳[2] 詹球[2] 王磊[1,3] 辛海明[2] 童亚林[2] 

机构地区：[1]解放军181医院动物实验中心,广西桂林541002 [2]解放军181医院烧伤整形科,广西桂林541002 [3]广西师范大学生命科学学院

出　　处：《华南国防医学杂志》2013年第4期229-233,共5页Military Medical Journal of South China

基　　金：广西自然科学基金项目(0991290);广西科学研究与技术开发计划项目(1140003A-39);全军医学科学技术研究"十一五"计划课题(06MA121);全军医学科学技术研究"十二五"计划课题(CWS11J277)

摘　　要：目的观察不同抗冻剂预处理人羊膜后快速液氮保存的效果。方法剥取剖宫产胎膜中羊膜,分别用生理盐水(C)组、DMEM/甘油(D1)组、DMEM/甘油/二甲基亚砜(dimethyl sulfoxide,DMSO)(D2)组、DMEM/甘油/DM-SO/海藻糖(D3)组预处理并标记后直接投入液氮中保存。于冻存第1、3、6、12个月取出,行台盼蓝、苏木精-伊红(hema-toxylin-eosin,HE)染色,计算羊膜上皮细胞存活百分率(存活率)、上皮细胞结构(细胞结构),并与新鲜羊膜比较。结果C、D1组随着保存时间延长,存活率1>3>6>12个月(P<0.05),细胞结构破坏也逐步加重。D2组内存活率1>3个月(P<0.05),3>6>12个月(P>0.05),保存1、3个月的羊膜细胞结构接近新鲜羊膜,6、12个月有轻微改变。D3组内各时间点存活率有下降(P>0.05),细胞结构变化不明显。各组各时间点羊膜细胞存活率:新鲜羊膜>D3>D2>D1>C(P<0.05;D3组保存1个月与新鲜羊膜比较,P>0.05)。D2、D3组各时间点存活率>新鲜羊膜的70%。结论DMEM/甘油/DMSO/海藻糖抗冻剂预处理的羊膜,经快速液氮冻存1年后的效果最好。Objective To observe the effect of liquid nitrogen rapid cryopreserving of the human amnion （HAM） pretreated by different cryoprotectants. Methods The amnion was stripped from the fetal membrane inform the caesarean section, pretreated respectively by normal saline （group C）, DMEM/Glycerol （group D1）, DMEM/Glycerol/DMSO （group 132）,and DMEM/Glycerol/DMSO/Mycose （group D3）. The HAMs were wrapped up and marked, than input in the liquid nitrogen. After cryopreserving for 1,3,6 and 12 months （M）, the HAMs were taken out. Hematoxylin-eosin （HE） staining and typan blue staining were applied to observe the survival rate and the structure of amniotic epithelial cells,which were then compared with the fresh amnion. Results The survival rates of groups C and D1 were as follows： 1~3~6~12 M （P^0. 05） and the cell structure were gradually destroyed. The survival rate of group I）2 was 1〉3 M （P^0. 05）, 3〉6〉12 M（P^0. 05）, the structure of 1 and 3 M was similar to that of the fresh HAM and slightly changed at 6 and 12 M. The survival rate and the structure of group D3 decreased slightly （P^0. 05）. The survival rates at each time point were as follows：flesh HAM 2〉D3〉D2〉DI〉C （P%0. 05, except group 133 vs. fresh HAM at 1 M）. The sur- vival rate of groups I）2 and 193 at each time point were higher than 70 % of the fresh HAM. Conclusion The effect of liq- uid nitrogen rapid cryopreserving the HAM pretreated by the cryoprotectant DMEM/Glycerol/DMSO/Mycose after 1 year is the best.

关 键 词：人羊膜 羊膜上皮细胞存活率 抗冻剂 液氮快速冻存法 

分 类 号：R361.2[医药卫生—病理学]