不同抗冻剂预处理对直接快速液氮冻存人羊膜活性的影响

EFFECT OF PRETREATMENT WITH DIFFERENT CRYOPROTECTANTS ON BIOACTIVITY OF HUMAN AMNIOTIC MEMBRANE BY DIRECT PLUNGING INTO LIQUID NITROGEN

目的:筛选一种抗冻剂配方,为建立深低温高活性保存的羊膜库提供依据。方法:选取12只健康剖宫产产妇羊膜,根据预处理抗冻剂的不同随机分为4组:A组(空白对照组),B组(DMEM/甘油组),C组(DMEM/甘油/DMSO组),D组(DMEM/甘油/DMSO/海藻糖组)。冷冻保存后第1,3个月取羊膜行HE与台盼蓝染色,分别观察羊膜结构,计算羊膜上皮细胞死亡率,并与新鲜羊膜(E组)比较。结果:E组:上皮细胞呈柱状、排列整齐紧密,细胞膜清晰,细胞核圆润蓝染,基底膜连续,细胞死亡率为(6.95±1.95)%。A组:保存1,3个月羊膜上皮层结构完整性均破坏、核固缩呈深蓝染,基底膜渐变为模糊不清。细胞死亡率分别为(39.96±5.84)%,(81.50±4.50)%,两组比较差异有统计学意义(t=13.796,P<0.05)。B组:保存1,3个月细胞变形,核固缩加剧,基底膜渐变为不连续。细胞死亡率分别为(21.61±6.65)%,(36.76±10.72)%,两组比较差异有统计学意义(t=2.940,P<0.05)。C组:保存1个月后类似新鲜羊膜,保存3个月后羊膜上皮细胞扁平,细胞核深染固缩,基底膜较完整。细胞死亡率分别为(14.08±3.21)%,(29.68±8.19)%,两组比较差异无统计学意义(t=4.344,P>0.05)。D组保存1,3个月形态结构接近新鲜羊膜,死亡率分别为(8.51±1.75)%,(12.70±5.70)%,两组比较差异无统计学意义(t=1.722,P>0.05)。保存1个月的A、B、C组死亡率与D、E组比较均有统计学差异(P<0.05),D、E组间比较差异无统计学意义(P>0.05);保存3个月的A、B、C、D、E各组间比较均有统计学差异(P<0.05),但D组存活率仍然达到87.3%。结论:DMEM/甘油/DMSO/海藻糖可作为新型抗冻剂用于深低温高活性保存羊膜。

Objective： To observe the effect of different eryoprotectants on bioactivity of human amniotic membrane by cryopreservation. Methods： Fresh amniotic membranes from 12 healthy parturient by caesarean section were randomly selected and divided into 4 groups according to different cryoprotectants： groups A, B, C and D. All amniotic membranes were preserved by direct plunging into liquid nitrogen as soon as being pretreated with different cryoprotectants except group A. Hematoxylin-eosin staining, trypan blue direct staining were done. Results： Group E. Amniotic epithelial cells appeared prismatical, lined up in order and compact, cell membrane clear, cell nucleolus round and blue-stain, basilar membrane continuous, the cell mortality was （6. 95±1.95）%. In group A： After saved 1, 3 months, the structure of integrity of amniotic epithelial layer was destroyed, and appeared karyopyknosis and deep blue-stain, basilar membranefrom appreciably complete became unclear. Cell mortality rates were （39.96 ± 5.84）%, （81.50±4.50）%, the difference between them had statistically significance （ t = 13. 796, P 〈0.05）. Group BAfter saved 1, 3 months, cytomorphosis, karyopyknosis aggravated, basilar membrane became discontinued. Cell mortality rates were （21.61 4- 6.65）%, （36. 76±10.72）%, the difference between them hadstatistically significance （ t =-2. 940, P %0.05）. Group C. After saved 1 month, amniotic epithelial layer structure similar to fresh amnion, after saved 3 months, the amniotic epithelial layer was thin and flat, cell nucleolus anachromasis and pycnosis, basilar membrane appeared complete. Cell mortality rates were （14.08±3.21）%, （29.68± 8.19）%, the difference between them had statistically significance （t = 4. 344, P 〈0.05）. Group DAfter saved 1 month and 3 months, the cell structure appeared no significant changes, morphological structure was closed to the fresh amniotic membrane, mortality rates were （8.51± 1.75） %, （12.70±5.70）%, the difference between them had statistically significance （ t = 1. 722, P 0.05）. The mortality rates in groups A, B and C had significant difference as compared with groups D and E after saved 1 month （ P〈0.05）, it had no significant difference between groups D and E （ P〉0.05）. There were significant differences between groups A, B, C, D and E after saved 3 months （ P〈0.05）, and the survival ratio in group D was 87.3%. Conclusion The compounded cryoprotectants of DMEM/ glycerin/DMSO/trehalose can well preserve the bioactivity of amniotic membrane for direct liquid nitrogen preservation.