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两种冷冻方法对人大块卵巢组织卵泡活性的影响
Effects of different freezing methods on the follicular viability of large pieces of human ovarian tissues
目的 探讨两种冷冻方法冻融人类大块卵巢组织的冷冻效果及对卵泡活性的影响。方法 收集15例人卵巢组织，每例修剪成3块卵巢组织
（约15 mm×15 mm×2mm），分别随机分配到新鲜对照组（A组）、玻璃化冷冻组（B组）、两步法冷冻组（C组）,组织学分析3组卵泡形态学变
化，并进行卵巢组织体外培养后测定培养液上清中雌激素、孕激素浓度，免疫组织化学染色测定培养后的卵巢组织细胞核增殖相关抗原Ki67 及
B细胞淋巴瘤/白血病-2基因（Bcl-2）表达水平，评估卵泡增殖及凋亡活性。结果 A组正常形态卵泡比率（91.9%）显著高于B组（71.3%）和C组
（82.9%），差异有统计学意义(P<0.05)，C组高于B组，差异有统计学意义(P<0.05)。C组的始基卵泡和初级卵泡的正常形态率分别为86.8%、
54.4%，均分别高于B组73.8%、44.4%，差异有统计学意义(P<0.05)。同一时期3组激素测定结果差异不显著(P>0.05)。C组Ki67的阳性率为73%，
分别显著高于A组50%和B组55%(P<0.05)，A组低于B组(P<0.05)。Bcl-2阳性率，A组为57%和C组61%相比无差异(P>0.05)，但B组42%阳性率分别低
于A组和C组(P<0.05)。结论 人体大块卵巢组织冻融后仍具有活性，并且两步法冻存效果优于玻璃化冷冻法。
Objective To study effects of cryopreservation of large pieces of human ovarian tissues and assess the follicular
viability after vitrification and two-step freezing. Methods Ovarian tissues from 15 patients were collected. The size of each
tissue block was about 15 mm×15 mm×2mm. The tissue block was cut into three pieces and randomly grouped as fresh(A), vitrification
(B), and two-step freezing (C) groups. All the pieces were cultured in vitro after thawing. The viability and proliferative capacity
of tissues were evaluated by in vitro production of estrogen and progestogen, histological analysis and immunohistochemical
staining of nuclear proliferation-associated antigens, Ki67 and B cell lymphoma/lewkmia-2（Bcl-2）. Results The ratios of normal
follicles were the highest in group A(91.9%), median in group C (82.9%) and the lowest in group B(71.3%) (P<0.05). The ratios of
morphologically normal primordial and primary follicles in group C were 86.8% and 54.4% which were higher than 73.8% and 44.4% in
group B(P<0.05), respectively. Difference of hormonal activity was not found among three groups（P>0.05). Positive expression of
Ki67 of group C（73%）was higher than group B（50%）and group A（55%）(P<0.05), where as that of group A was lower than group B
(P<0.05).There was no difference of positive expression of Bcl-2 staining between group A（57%）and group C（61%）(P>0.05), but
the ratio of positive Bcl-2 expression of group B（42%）was lower than groups A and C (P<0.05). Conclusions The frozen/thawed
large pieces of human ovarian tissues can reserve their vitality and the tissue with two-step freezing is better preserved than
the tissues with vitrification.
卵巢组织 / Ki67 / 冷冻保存 / 玻璃化冻存 / 两步法冷冻 / 人
Ovarian tissue / Ki67 / Cryopreservation / Vitrification / Two-step freezing / Human
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基金项目：人类自体生殖细胞及卵巢组织库建立的初步研究;项目号: 2010GSF10814
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