目的 研究抗苗勒激素（AMH）和生長分化因子-9（GDF-9）對環(huán)磷酰胺（Cy）降低卵巢儲備力機(jī)制中的作用。方法 成年雌性SD大鼠25只，取雙側(cè)卵巢組織，并切制成500 μm厚的卵巢片，將卵巢片分為7組：空白組、Cy組、Cy＋卵泡刺激素（FSH）組、Cy＋AMH組、Cy＋GDF-9組、Cy＋FSH＋AMH組、Cy＋FSH＋GDF-9組，放入含DMEM-F12培養(yǎng)液的培養(yǎng)皿中分別培養(yǎng)2 d或4 d。除空白組外，其余各組于培養(yǎng)液中分別添加Cy 100 ng/mL、FSH 10 mIU/mL、AMH 100 ng/mL、GDF-9 100 ng/mL。利用雙抗體夾心酶聯(lián)免疫吸附技術(shù)（ELISA）檢測雌二醇（E₂）、抑制素B（INHB）的量；光鏡下計(jì)數(shù)始基卵泡（PMF）及閉鎖卵泡；運(yùn)用DNA缺口原位末端標(biāo)記法（TUNEL）檢測細(xì)胞凋亡情況。結(jié)果 ①與空白組相比，Cy組PMF數(shù)目明顯降低，閉鎖卵泡明顯增多；Cy＋AMH組及Cy＋FSH＋AMH組PMF數(shù)目明顯多于除空白組之外的其他各組。各組培養(yǎng)4 d較培養(yǎng)2 d始基卵泡數(shù)目明顯降低，閉鎖卵泡數(shù)目明顯增多。②與空白組相比，Cy組培養(yǎng)2 d和4 d時E₂及INHB水平明顯降低；與Cy組相比，培養(yǎng)2 d和4 d時Cy＋FSH組、Cy＋GDF-9組及Cy＋FSH＋GDF-9組E₂、INHB明顯升高。與Cy組相比，培養(yǎng)2 d和4 d時Cy＋AMH組及Cy＋FSH＋AMH組E₂明顯升高。培養(yǎng)4 d時Cy＋AMH組、Cy＋FSH＋AMH組INHB水平明顯升高。③Cy組與空白組比較，凋亡細(xì)胞明顯增多；Cy＋FSH組、Cy＋GDF-9組與Cy組相比，凋亡細(xì)胞的數(shù)量并沒有顯著減少。Cy＋AMH組、Cy＋FSH＋AMH組與Cy組相比凋亡細(xì)胞的數(shù)量顯著減少。結(jié)論 Cy可加速PMF的募集與耗竭，從而使卵巢儲備力降低乃至衰竭。FSH及GDF-9可加速PMF的募集與發(fā)育，使更多的PMF進(jìn)入生長卵泡階段而受到Cy細(xì)胞毒的損害，以致PMF庫存逐漸耗竭；AMH可抑制PMF的募集與發(fā)育，從而減緩Cy對PMF的加速募集與耗竭，起到保護(hù)卵巢儲備力的作用。

Objective To research the effects of anti-mullerian hormone (AMH) and growth differentiation factor-9 (GDF-9) on cyclophosphamide (Cy) reducing ovarian reserve. Methods Twenty-five adult female SD rats were used to remove the bilateral ovarian tissues and cut them into 500 μm slices. The slices were divided into control, Cy, Cy + follicle-stimulating hormone (FSH), Cy + AMH, Cy + GDF-9, Cy + FSH + AMH, and Cy + FSH + GDF-9 groups, and put into petri dishes including DMEM-F12 for 2 or 4 d. Except the control group, the other groups were added with Cy 100 ng/mL, FSH 10 mIU/mL, GDF-9 100 ng/mL, and AMH 100 ng/mL. The contents of estradiol (E₂) and inhibitant B (INHB) were detected by ELISA, the numbers of primordial follicles (PMF) and atresic follicles were counted under light microscope, and the rat ovarian cell apoptosis was detected by TUNEL. Results ①Compared with the control group, the numbers of PMF were significantly reduced and the numbers of atresic follicles were increased in Cy group. In Cy + AMH and Cy + FSH + AMH groups, the numbers of PMF were obviously more than those in the other groups except control group. The numbers of PMF were significantly reduced and the atresic follicles were significantly increased in 4 d culture compared with 2 d culture, ② Compared with the control group, the E₂ and INHB contents of rats in Cy group were significantly Reduced; Those in GDF-9 groups were significantly increased, and the E₂ content of rats in Cy + AMH and Cy + FSH + AMH groups was significantly increased. At the day 4, the INHB contents of rats in Cy + AMH and Cy + FSH + AMH groups were significantly increased. ③ Compared with the control group, the apoptotic cells in Cy group were significantly increased. Compared with Cy group, the numbers of apoptotic cells in Cy + FSH and Cy + GDF-9 groups were not significantly reduced, and the numbers of apoptotic cells in Cy + AMH and Cy + FSH + AMH groups were significantly reduced. Conclusion Cy could accelerate the recruitment and depletion of the PMF, so that the ovarian reserve is reduced and even failure. FSH and GDF-9 could accelerate the recruitment and the development of the PMF, leading to more PMF into the growing follicle stage and suffering from Cy cytotoxic damage, so the PMF stock is gradually depleted. AMH could restrain the PMF recruitment and development, thus slow down the acceleration of the PMF recruitment and depletion by Cy, and protect the ovarian reserve.

國家自然科學(xué)基金資助項(xiàng)目（30801479）