联合乙二醇和二甲亚砜玻璃化冷冻人卵裂早期胚胎的研究

发布时间：2018-05-28 01:22

本文选题：玻璃化冷冻 + 玻璃化溶液　；参考：《昆明医学院》2007年硕士论文

【摘要】： 目的：以普通冷冻麦管为冷冻载体，联合冷冻保护剂乙二醇(ethylene glycol，EG)和二甲亚砜(dimethyl suloxide，DMSO)玻璃化冷冻人早期卵裂期胚胎，比较它们在不同浓度组合下的冷冻效果，以期获得最佳浓度组合，为临床提供一种有效的玻璃化溶液。方法：以人类体外受精发育至第三天的废弃卵裂早期胚胎为实验对象，以常用的冷冻麦管为载体，冷冻保护剂EG和DMSO分别取20％和25％两个浓度水平，在这两个浓度水平EG和DMSO交叉配伍组成不同的玻璃化溶液，分别对胚胎进行玻璃化冻存，并与程序化慢速冷冻法进行对比。胚胎冻存时间平均为1月，，复苏后，通过倒置显微镜下形态学观察，比较胚胎的存活率和继续发育能力，另外，复苏后存活胚胎经罗丹明123(Rhodamine 123)染色，激光扫描共聚焦显微镜摄片分析，测定胚胎线粒体跨膜电位。结果：EG和DMSO在20％和25％两个浓度水平交叉配伍组成的四种玻璃化溶液E20D20、E20D25、E25D20、E25D25都能有效地冻存人早期卵裂期胚胎，胚胎复苏后存活率依次为51.0％、72.7％、62.0％、46.3％，均比程序化慢速冷冻组的存活率39.7％高，其中最高的是20％EG和25％DMSO配伍组，与程序化冷冻组相比差异有统计学意义(P＜0.05)，而其余各组与程序化冷冻组相比差异无统计学意义(P＞0.05)。复苏后囊胚形成率依次为11.8％、18.2％、18.0％、9.3％，也均高于程序化冷冻组的囊胚形成率8.6％，其中E20D25组最高，但是各组间的差异均无统计学意义(P＞0.05)。复苏后存活胚胎线粒体膜电位的比较，E20D25组膜电位值最高，与未冷冻对照组相比差异无统计学差异，与其它各组相比，除E25D25组外差异均有统计学意义。程序化冷冻组膜电位低于未冷冻对照组，差异有统计学意义(P=0.001＜0.05)。结论：1．以EG和DMSO为主体的玻璃化溶液能有效地冷冻人早期卵裂期胚胎。2．以常用的冷冻麦管为载体，20％EG和25％DMSO配伍组成的玻璃化溶液能获得较好的冷冻效果，可作为临床应用时一种可供选择的玻璃化溶液。3玻璃化冷冻技术比程序化慢速冷冻法能获得更好的冷冻效果，能够有效保存人早期卵裂期胚胎。
[Abstract]:Aim: to vitrify human early cleavage embryos with common cryopreservation wheat tube as cryopreservation carrier, combined with ethylene glycol glycoline and dimethyl sulfoxide dimethyl suloxide-DMSO. and compare their freezing effects under different concentration combinations. In order to obtain the best concentration combination, to provide an effective vitrification solution for clinical. Methods: the abandoned early cleavage embryos from the third day of human IVF development were used as experimental objects. The cryopreservation agents EG and DMSO were taken at 20% and 25% concentrations, respectively, using common cryopreserved wheat tube as the carrier, and the concentration of EG and DMSO were 20% and 25%, respectively. At these two concentrations EG and DMSO crosscompatibility composed of different vitrification solution, the embryos were vitrified and frozen, and compared with programmed slow freezing method. The average cryopreservation time was 1 month. After resuscitation, the survival rate and developmental ability of the embryos were compared by morphological observation under inverted microscope. In addition, the surviving embryos were stained with Rhodamine 123(Rhodamine 123 after resuscitation. The transmembrane potential of embryo mitochondria was measured by laser scanning confocal microscope. Results the four vitrified solutions E20D20, E20D25, E20D25, E25D20, E25D20, E25D25 and E25D25 could effectively freeze human early cleavage embryos at 20% and 25% levels, respectively. The survival rate after embryo resuscitation was 51.0% and 72.7%, respectively, which was higher than that of programmed slow freezing group (39.7%). The highest one was 20%EG and 25%DMSO (P < 0.05), but there was no significant difference between the other groups and programmed freezing group (P > 0.05). The blastocyst formation rate after resuscitation was 11.8and 18.2and 18.00.It was also higher than the blastocyst formation rate of programmed freezing group (8.6%). The highest blastocyst formation rate was found in E20D25 group, but there was no significant difference between each group (P > 0.05). Comparison of mitochondrial membrane potential in surviving embryos after resuscitation group E20D25 had the highest membrane potential value, but there was no significant difference compared with other groups except E25D25 group. The membrane potential in programmed freezing group was significantly lower than that in unfrozen control group (P < 0.05). Conclusion 1. Vitrification solution with EG and DMSO as main body can effectively freeze human early cleavage embryos. The glass solution composed of common frozen wheat tube and EG and 25%DMSO can obtain better freezing effect. It can be used as an alternative vitrification technique in clinical application, which can obtain better cryopreservation effect than programmed slow freezing method, and can effectively preserve human early cleavage embryos.
【学位授予单位】：昆明医学院
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R321-33

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