两种2-细胞鼠胚体外培养方法比较

目的应用鼠胚质控中的小鼠胚胎体外培养模型,探讨两种胚胎培养方式（四孔皿与微滴法）在单胚观察时间上的差异以及对2-细胞鼠胚体外发育潜能的影响。方法取6-8周龄的昆明白雌性小鼠。采用HMG10IU促排卵,48 h后注射HCG 10IU促卵泡成熟,取形态正常的2-细胞鼠胚。每5-10个胚胎培养在含500μL培养基的四孔皿中（A组）,或单个胚胎接种在含50μL的培养微滴中（B组）。培养后,每隔24 h在倒置显微镜下观察一次,计算单胚观察时间,并检测24 h时的≥4细胞胚形成率、48 h的融合胚形成率7、2 h的囊胚与扩张囊胚形成率、96 h囊胚孵化率。结果两种培养方式于同一试验条件下分别试验5次,A组培养83个胚胎,B组培养69个2-细胞鼠胚。在每一个观察点上,微滴培养的单胚观察时间远超过四孔皿培养（P〈0.001）。但两组各时间点的胚胎发育率相似,无显著差异（P〉0.05）。结论尽管微滴单胚培养方式的胚胎暴露培养箱外时间长,但与四孔皿多胚培养方式比较,两者间2-细胞鼠胚的体外发育潜能相似。

Prospective Trial of Two Cultural Systems on the Developmental Capacity of Mouse 2-cell Embryos in Vitro

Objective To evaluate two cultural systems including either well or micro-drop systems in vitro for comparing the observation time of single embryo and mouse 2-cell embryos developmental competence. Methods Female mice in 6 - 8 weeks were treated for superovulation with 10IU HMG, combined with 10IU HCG with the interval of 48 h. 5- 10 of 2-cell mouse embryos with normal morphology, were cultured in each weU of a 4-well dish containing 500 μL of culture medium（ group A）, or single embryo was placed in a mineral oil coated 50 μL of medium volume （group B ）. At 24 hour intervals, the embryo＇s developmental stages were observed under 400 x magnification in an inverted microscope. It was calculated for the observation of single embryo. The cleavage rate of ≥ 4 cells on 24 hours, morula rate on 48 hours, blastocyst and expanded blastocyst rate on 72 and hatched blastocysts rate on 96 hours were calculated, respectively. Results 5 repeated tests were performed for each of experimental group. Group A and group B included 83 and 69 embryos observed respectively. Observing the development of single embryo in micro-drops results in more exposure time per embryo than that in 4-well dishes at any point in time （ P 〈 0.001 ）. However, the incidences of cleavage rate of ≥4 cells, morula rate, blastocyst rate, expanded blastocyst rate and hatched blastocysts rate were detected in micro-drops, there was no significant difference in the rates of 2-cell embryos development among 4-well dish and micro-drop systems （P 〉 0.05）. Conclusion These results indicate that the micro-environment produced by micro-drop system produced similar developmental competence of 2-cell mouse embryos in vitro to the well system, although it increases more exposure time for observation.