小鼠1-细胞胚胎体外培养条件的研究

小鼠1-细胞胚胎体外培养条件的研究*

【摘要】目的检验培养基(Whitten、CZB、SOM和KSOM)、培养器皿(进口、国产新旧平皿)与配制培养基的水质对小鼠胚胎体外发育的影响。方法采用微滴培养法培养昆明种小白鼠1-细胞胚胎，以胚胎发育到囊胚的比例为判断培养效果的标准。结果Whitten、CZB、SOM和KSOM培养基中2-细胞发育率分别为100%、100%、100%和98.6%，而囊胚发育率却分别为0%、78.3%、4.8%和9.5%；用三蒸水、五蒸水和去离子水配制的CZB培养基，培养囊胚发育率分别为40.8%、54.1%和52.4%；国产和进口新平皿的囊胚发育率分别为68.0%和72.2%；国产和进口旧平皿的培养囊胚发育率为27.2%和4 6.1%。结论昆明小白鼠早期胚胎存在体外发育阻断现象；CZB培养基培养昆明小白鼠早期胚胎效果最好；用三蒸水、五蒸水和去离子水配制的CZB 培养基培养结果无显著差别；进口和国产新平皿的培养效果相近，但再次使用时培养效果显著下降。

【关键词】1-细胞胚胎培养系统小鼠

STUDIES ON THE CONDITIONS FOR THE CULTURE OF

ONE-CELL MOUSE EMBRYOS

Liu Zhonghua, Tan Jinghe△, He Guixin

(Department of Biotechnology, North-East Agricultural University, Harbin)

【Abstract】Objective To study the effects of culture media, types of culture dish and types of water used for preparation of media on the in vitr o development of one-cell embryos in the mice of Kunming breed. Metho

d One-cell mous

e embryos collected from the oviduct o

f the superovulated mice were cultured in microdrops of medium and the percentage of embryos developed to blastocyst stage was used as evaluation criteria. Results (1)Wh en one-cell mouse embryos were cultured in the Whitten's, CZB, SOM and KSOM media, the cleavage percentages were 100%,100%,100% and 98.6%, respectively, and the blastocyst percentages were 0%, 78.3%, 4.8% and 9. 5%, respectively. (2)The blastocyst percentages obtained from the CZB medi a prepared from the triple-distilled, fivefold-distilled and deionized water wer e 40.8%, 54.1% and 52.4%, respectively. (3)The blastocyst percentages prod uced from the Chinese-made and the imported culture dishes were 68.0% an d 72.2%, respectively, but those obtained when the dishes were used for the second time were decreased significantly (bein

g only 27.2% and 46.1%, res pectively). Conclusions There existed a development block during the in vitro development of the mouse embryos of the Kunming breed. Among the me dia tested CZB was the best in supporting the development of one-cell mou se embryos to blastocyst stage. No significant difference was found among t he CZB media prepared from the triple-distilled, fivefold-distilled and deioni zed water in blastocyst percentages. Bot

h the Chinese-made and the importe

d cultur

e dishes produced good results o

f embryo development when used f or the first time but the blastocyst percentages decreased significantly when the culture dishes were washed and used for the second time.

【Key words】One-cell embryo; Culture system; Mouse

胚胎体外培养是胚胎工程研究的重要基础条件。因此，建立稳定、可靠的哺乳动物胚胎体外培养系统已成为当前这一领域的重要研究课题之一。在体外培养哺乳动物早期胚胎时，几乎都会不同程度的发生体外发育阻断(in vi tro block of develo-pment)现象［1］。采用体细胞与胚胎共同培养和改进培养基成分，以形成可克服体外发育阻断的化学成分明确培养基，是当前人们试图解决这一难题的两个主要方向，而化学成分明确培养基(chemically defined m edium)培养胚胎，这种方法以其操作简便，有利于发育阻断机理研究而尤为人们重视。CZB，SOM和KSOM培养基就是最近出现的几种效果较好的成分明确培养基［2，3］。除培养基外，影响胚胎培养效果的因素还很多，其中培养器皿与配制培养基的水质是两个不容忽视的因素。William研究发现不同厂家，不同批次的培养器皿以及不同的清洗、灭菌和包装方法可明显影响小鼠胚胎体外发育效果［4］。因此，本次实验采用微滴培养法对昆明小白鼠1-细胞胚胎进行培养并依据胚胎发育到囊胚的比例为判断标准，对培养基、培养器皿及配制培养基的水质进行了研究，以期为建立完善的哺乳动物早期胚胎培养系统提供一些参考。

材料和方法

控光饲养(14h光照，10h黑暗)的8～12周龄昆明白雌鼠腹腔注射孕马血清促性腺激素(PMSG)10 IU/只，间隔48h腹腔注射人绒毛膜促性腺激素(hCG) 10 IU/只，进行超排处理。注hCG后与公鼠合笼，次日见阴道栓者于注射hC G后22～24h断颈椎处死，于输卵管中冲取1-细胞胚胎，用300 IU/ml的透明质酸酶去除卵丘细胞后用于实验。

CZB培养基，SOM培养基，KSOM培养基，Whitten培养基以及M2按文献［2，3］所载配方配制。

去离子水采用SYBROY BARNSTEAD(美国产)纯水器制取；三蒸水用自动双重纯水蒸馏器(上海玻璃仪器一厂生产)蒸馏制得；五蒸水为三蒸水再次经过自动双重纯水蒸馏器蒸馏制得。3种纯水经电导仪测量其电导值分别为2.1μΩ-1、2.6μΩ-1和1.8μΩ-1。

进口平皿为美国COSTAR公司产，规格为35×10 mm；国产平皿购自安普公司，规格也为35×10 mm。培养用过的新平皿回收，用脱脂棉在碱液中擦洗干净，清水冲洗，干燥，4%盐酸浸泡24h，清水冲净，蒸馏水冲洗至少3遍，纯水冲洗2遍后烘干。用前紫外线灭菌30min。

用微量移液器吸取培养液在平皿中制成50μl的培养滴，覆盖石蜡油置C O2培养箱中平衡2h后，将待培养胚胎移入微滴，于5%CO2，37℃饱和湿度的培养箱中培养，每滴中培养胚胎10个左右。

实验所得数据均采用百分数t检验进行统计学处理。