[关键词]
[摘要]
目的 建立大鼠腔前卵泡的体外培养方法。方法 取性未成熟大鼠的卵巢,按照酶消化-机械结合法分离腔前卵泡,然后采用动态氧气法(初始孵育时氧气压力为4%,以后每隔24 h氧气压力增加1%,直至最后的氧气压力为11%)、向培养基中添加维生素C(VC)进行培养,观察获得的卵泡数量、形态,及对卵泡发育、激素生成、卵子发生的影响;并将体外卵母细胞的成熟情况与体内卵母细胞的生长情况进行比较,以判断体外培养方法是否成功。结果 酶消化-机械结合法及培养体系可获得大量基底膜完整的腔前卵泡;卵泡和卵母细胞直径显著增加,卵泡存活率91.14%、有腔卵泡形成率25.82%、卵丘细胞-卵母细胞复合体(COCs)排出率38.38%;体外培养卵泡分泌雌二醇(E2)和孕酮(P)与体内分泌特征一致。结论 本实验方法可以获得大量高质量的腔前卵泡,且对卵泡的长期培养发育无明显影响,与大鼠体内卵泡的发育一致。表明本实验成功建立大鼠腔前卵泡的体外培养方法。
[Key word]
[Abstract]
Objective To establish of an in vitro culture method for rat preantral follicles. Methods The ovary was obtained from premature rat and dissected according to isolation method of mechanical dissection with enzymatic digestion. Then they were cultured in the culture system which were dynamic oxygen delivery protocol (the oxygen tension in the incubator was initially set at 4%, and it was increased 1% every 24 h until a final oxygen tension of 11%) and the addition of VC to culture medium, then the quantity, morphology, and effects on follicular development, hormone production, egg occurance were assessed; In vitro maturation of oocytes were compared with in vivo growth of oocytes to determine the success of method. Results Mechanical dissection with enzymatic digestion and culture condition could obtain the large amount of preantral follicles, the average diameter of follicles was increased, the survival rate of follicles was 91.14%, 25.82% of the cultured follicles reached the pre-ovulatory stage, and 38.38% of COCs were released in this study. Conclusion This method could obtain the large amount of high quality preantral follicles, which also has no obvious effects on long development. The follicles development in vitro is consistent with that in vivo in rats. This study successfully establish the in vitro culture system for rat preantral follicle.
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[基金项目]
“重大新药创制”科技重大专项“十二五”计划(2012ZX09505001-001)