摘要
目的：研究两种冷冻保护剂乙烯已二醇(Ethylene Glycol,EG)和甘油(Clycero,GC)在不同浓度和暴露时间的条件下对桑葚胚及囊胚的毒性影响；比较桑葚胚、早期囊胚和囊胚经玻璃化法(Vitrification)、EG慢速冷冻和GC慢速冷冻三种方法冻融后胚胎继续发育的潜能；初步确定玻璃化法冷冻种植前晚发育时期胚胎的最佳阶段。
方法：1．桑葚胚、早期囊胚和囊胚分别暴露于1.5M EG磷酸盐缓冲液、1.5M GC磷酸盐缓冲液20min，6.0M EG磷酸盐缓冲液5min，以暴露于不含冷冻保护剂的磷酸盐缓冲液为对照，观察在胚胎体外培养系统M16培养液继续发育的情况。2．桑葚胚、早期囊胚和囊胚三个发育阶段的胚胎均用玻璃化法、EG慢速冷冻和GC慢速冷冻法冻存，比较三种方法冻融后胚胎继续发育的情况。3．比较三个阶段胚胎经玻璃化法冻存后冻融胚胎继续发育的情况。
结果：1在1.5M EG磷酸盐缓冲液、1.5M GC磷酸盐缓冲液中暴露20min，6.0M EG磷酸盐缓冲液暴露5min后，在体外培养中桑葚胚、早期囊胚和囊胚继续发育与对照组相比没有显著性差异，对胚胎没有明显毒性影响。2桑葚胚经玻璃化法、EG慢速冷冻法和GC慢速冷冻法三种方法冻存后，冻融胚胎囊胚形成率分别为：85.7％、71.1％、48.7％，囊胚孵出率分别为：59.5％、42.2％、23.1％，玻璃化法和EG慢速冷冻法高于GC慢速冷冻法(P＜0.01，P＜O.05)，玻璃化法与EG慢速冷冻法相比没有显著性差异(P＞0.05)。三种方法冻存早
天津医科大学硕士学位论文
期襄胚融解后扩张襄胚形成率分别为65.钱、26.既、41%，班胚孵出率51.钱、
l外、28.既，冻存班胚融解后囊胚重新扩张率为43.既、21 .1%、22.5%，.
胚孵出率为29.2%、10.5%、1既。在早期襄胚和澳胚的冻存中，玻璃化法冻
融后胚胎继续发育率高于EG慢速冷冻法和GC慢速冷冻法(P<0 .01，P<0 .05)
EG慢速冷冻法与Gc慢速冷冻法相比没有显著性差异(P>0 .05)。
结论:1.SMEG、1.SMGC溶液及6.0施G在本研究采用的冷冻条件下对胚
胎没有毒性影响。对干桑甚胚，玻璃化法和EG慢速冷冻法优于GC慢速冷冻
法;早期囊胚和囊胚玻璃化法冷冻效果优于EG和Gc慢速冷冻法。在慢速冷
冻中，EG较GC更适于桑套胚。桑甚胚可能是玻璃化法冻存种植前晚发育时
期胚胎的最佳阶段。
Objective:To study the toxic effects of the different concentration of the two cyroprotectants and the different exposure time on the mouse morulae and blastocysts; To compare the developmental potential of mouse morulae, early blastocysts and blastocysts cyropreserved by vitrification , slow- freezing with ethylene glycol or glycerol; To find out the stage which can withstand cyropreservation best in the late development stage of preimplanted mouse embryo.
Methed: 1.The embryotoxicity of cyroprotectants was tested by exposing the morulae and blastocysts to FB1 medium which contained 1.5M EG or 1.5M GC for 20min, 6.0M EG FB1 medium for 5min at the room tempreture, compared with FB1 medium contained no cyroprotectant as the control group. The developmental rate after cutured in the M16 medium were examed. 2 morulae, early blastocysts and blastocysts were
cryopreserved by vitrification , slow- freezing with EG or GC ,compared the developmental rates after thawing.3 compared the developmental rates when these three stage embryos cryopreserved by vitrification.
Results: When morulae, early blastocysts and blastocysts exposed to 1.5M EG or 1.5M GC FB1 medium for
20min, 6.0M EG FB1 medium for 5min at the room tempreture , the rates of embryos develop to the next stage during the culture have no statistically significant compared with the control group.2 Both blastocyst rates and hatching rates have no significant different when vitrification compared with EG slow-freezing after the morulae thawing(85.7%vs71.1%, 59. 5%vs42. 2%),but both of
them higher than GC protocol ( P<0. 01 , P<0.05 ) .By
vitrification , slow-freezing with EG or GC, the expanded blastocystrates of thawed early blastocysts respectively are 65. 7%, 26. 2%, 41%, hatching rates are 51. 4%, 19%, 28. 2%; the re-expanded blastocyst rates are 43.8%, 21. 1%, 22. 5%, and hatching rates are 29. 2%, 10. 5%, 10%. At the early blastocyst and blastocyst stage , the developmental rates by vitrification are higher than by two slow-freezing, but no differents between EG and GC protocol.
Conclusion :1.5M EG, 1.5M GC and 6. OM EG appears low toxic effect on mouse morula and blastocyst stage in the freezing protocol. Vitrification make a better results than slow-freezing at early blastocyst and blastocyst stage,
EG slow-freezing protocol as good as vitrification for morulae. In slow-freezing , EG is more adaptable to morulae than GC,but has no siginificant in early blastocyst and blastocyst stage. And that the morula is likely the most feasible stage for embryo vitrification.
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