目的 建立大鼠腔前卵泡的體外培養(yǎng)方法。方法 取性未成熟大鼠的卵巢,按照酶消化-機(jī)械結(jié)合法分離腔前卵泡,然后采用動(dòng)態(tài)氧氣法(初始孵育時(shí)氧氣壓力為4%,以后每隔24 h氧氣壓力增加1%,直至最后的氧氣壓力為11%)、向培養(yǎng)基中添加維生素C(VC)進(jìn)行培養(yǎng),觀察獲得的卵泡數(shù)量、形態(tài),及對卵泡發(fā)育、激素生成、卵子發(fā)生的影響;并將體外卵母細(xì)胞的成熟情況與體內(nèi)卵母細(xì)胞的生長情況進(jìn)行比較,以判斷體外培養(yǎng)方法是否成功。結(jié)果 酶消化-機(jī)械結(jié)合法及培養(yǎng)體系可獲得大量基底膜完整的腔前卵泡;卵泡和卵母細(xì)胞直徑顯著增加,卵泡存活率91.14%、有腔卵泡形成率25.82%、卵丘細(xì)胞-卵母細(xì)胞復(fù)合體(COCs)排出率38.38%;體外培養(yǎng)卵泡分泌雌二醇(E2)和孕酮(P)與體內(nèi)分泌特征一致。結(jié)論 本實(shí)驗(yàn)方法可以獲得大量高質(zhì)量的腔前卵泡,且對卵泡的長期培養(yǎng)發(fā)育無明顯影響,與大鼠體內(nèi)卵泡的發(fā)育一致。表明本實(shí)驗(yàn)成功建立大鼠腔前卵泡的體外培養(yǎng)方法。

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.

“重大新藥創(chuàng)制”科技重大專項(xiàng)“十二五”計(jì)劃(2012ZX09505001-001)