三种染色方法检测冻融后兔卵巢组织的活力和质量

目的建立一种简单的评价冷冻前后卵巢组织活力与质量的方法。方法 10只性成熟雌性大耳白兔,取卵巢组织制成皮质片,随机均分成2组(n=5):新鲜组织和冷冻组织。台盼蓝、Calcein AM/EH及中性红染色检测分离卵泡的存活率并对卵巢组织中卵泡的存活情况进行染色。激素释放试验检测冷冻前后兔卵巢组织的功能。结果应用台盼蓝、Calcein AM/EH及中性红对卵巢组织分离卵泡活力的评价效果相似;中性红能较好的评价卵巢组织中卵泡的存活情况。经冷冻解冻后,卵泡的活力下降,差异有统计学意义(P<0.05);体外培养过程中,培养液E2水平不断增加,培养12 d后,E2浓度在新鲜及冷冻复苏组织分别达1054、961 pg/m L。孕酮水平分别为2.31、1.9 ng/m L。结论中性红可有效地评价卵巢组织卵泡的活性;激素释放试验可以提供冷冻解冻卵巢组织存在活性的佐证。

Objective To establish a simple evaluated methods to assess the vitality and quality of ovarian tissue after freezing-thawing procedures. Methods Ovarian tissues from 10 female rabbits were randomly divided into two groups：fresh group and frozen group. The follicles viability were assessed by trypan blue staining,calcein AM/ethidium homodimer-1 staining and neutral red staining. The function of ovarian tissues were detected by hormone-releasing assays. Results Trypan blue, Calcein AM/EH and neutral red staining have the similar results about ovarian follicle viability assessment. The viability of follicles decreased after freezing and thawing （P〈0.05）;E 2 and Progesterone levels increased after cultured 12 days. The supernatants showed 17-βestradiol concentrations of 1054 and 961 pg/mL, and progesterone concentrations of 2.31 and 1.9 ng/mL respectively in fresh and frozen groups. Conclusion Neutral red staining can effectively evaluate the follicle viability;Hormone-releasing test can provide the evidence that the freezing-thawing ovarian tissue has higher developmental potential.