小鼠体外受精、胚胎培养及胚胎快速冷冻的研究

目的　为扩大胚胎来源并获取特定胚龄胚胎 ,建立小鼠冷冻胚胎库。方法　运用超数排卵、体外受精与胚胎培养及胚胎冷冻技术系统研究了小鼠受精卵的体内发育与运行规律。卵母细胞的体外成熟与受精、单细胞胚胎培养及胚胎快速冷冻。结果　 (1)注射hCG后 12～ 2 0h受精卵发育至原核期 ,4 2～ 4 8h为 2 细胞期 ,4 8～ 6 0h为 4 细胞期 ,6 0～ 6 8h为 8 细胞期 ,以上各期受精卵均处于输卵管中 ;75～ 78h为桑椹胚 ,78～ 80h为致密桑椹胚 ,90～ 92h为早期囊胚 ,92～ 96h为囊胚 ,以上各期均处于子宫角中。 (2 )培养液中添加促性腺激素 (FSH与hCG) ,能显著提高卵母细胞的体外受精率 ,添加FCS和激素组的体外受精率又显著高于单独添加激素组 ,FCS还能显著提高胚胎发育。 (3)在培养液中添加EDTA ,能有效克服小鼠胚胎的 2 细胞阻断 ,其 2 细胞胚的发育率达 10 0 % ,8 细胞胚发育率达 5 5 %以上 ;牛、羊上皮细胞培养液上清也能有效克服 2 细胞阻断。添加乳酸钠和丙酮酸钠可使 2细胞与 8细胞期胚的发育率显著提高。 (4)以D PBS +甘油 +蔗糖为冷冻液 ,以D PBS +蔗糖为稀释液 ,对小鼠胚胎进行快速冷冻 ,桑椹胚的存活率为 6 9 3% ,早期囊胚的存活率为 6 0 4 %。结论　研究为将生物技术应用于小鼠 ,扩大卵子和胚胎来源

Study on in vitro Fertilization, Embryonic Culture and Embryonic Rapid-Freezing in Mice

Objective\ To collect age\|specific embryos, increase the resources of embryos, and establish the bank of frozen embryos in mice. Methods\ The in vivo development and movement of zygotes, the in vitro maturation and fertilization of oocytes , the in vitro culture and rapid\|freezing of embryos were studied. Results\ (1) Zygotes developing to pronuclei occurred at 12-20h after injection of hCG;2\|cell, 4\|cell, and 8\|cell embryos formed at 42-48 h, 48-60 h, and 60-68 h, respectively. The embryos of the above stages were in the oviducts. Morula, early blastocysts, and blastocysts, which were in the uterus horns, formed at 75-70 h, 90-92 h, and 92-96 h, respectively. (2)Adding FSH and hCG to the culture medium could increase significantly the percentage of in vito fertilization of oocytes. Adding FCS and FSH and hCG simultaneously to the culture medium was better than adding hormone alone. FCS could increase significantly the rate of the embryo development. (3)Adding EDTA to the culture medium could effectively overcome the 2\|cell block in mice. The rates of embryos developed to 2\|cell stage and to 8\|cell stage were 100% and over 55%, respectively. Adding sodium lactate and pyruvate sodium to the culture medium could stimulate significantly the embryonic development. (4) After frozen in the freezing solution (D\|PBS+glycerol+sucrose), and thawed in DPBS +sucrose solution, the survival rate of morula and early blastocysts were 69.3% and 60.4%, respectively. Conclusion\ The present results laid the preliminary basis for the establishment of embryo bank in mice.