不同冷冻保护剂在羊卵巢整体冻存中的效果比较

doi:10.16098/j.issn.0529-1356.2015.03.022

解剖学报 ›› 2015, Vol. ›› Issue (3): 415-421.doi: 10.16098/j.issn.0529-1356.2015.03.022

不同冷冻保护剂在羊卵巢整体冻存中的效果比较

杜天奇¹迟令龙⁴赵淑芹⁵时庆² 秦睿羲³ 孙艳艳¹ 李栋² 邓晓惠^1*

1.山东大学齐鲁医院不孕不育诊疗中心; 2.低温医学研究室; 3.病理科，济南 250012；4.山东省齐鲁干细胞有限公司，济南 250004; 5.山东省枣庄市妇幼保健院，山东枣庄 277100

收稿日期:2014-10-15 修回日期:2015-02-24 出版日期:2015-06-06 发布日期:2015-06-06
通讯作者: 邓晓惠 E-mail:dxh0122@163.com
基金资助:
山东省科技攻关项目;国家自然科学基金面上项目

Comparison of different cryoprotectants in whole sheep ovary cryopreservation

DU Tian-qi¹ CHI Ling-long⁴ ZHAO Shu-qin⁵SHI Qing² QIN Rui-xi³ SUN Yan-yan¹ LI Dong² DENG Xiao-hui ^1*

1.Infertility Center; 2.Cryomedicine Laboratory; 3.Department of Pathology, Qilu Hospital of Shandong University, Ji’nan 250012, China; 4.Shandong Qilu Stem Cells Co.Ltd, Ji’nan 250000, China; 5.Zaozhuang Maternal and Child Care Service Hospital, Shandong Zaozhuang 277100, China

Received:2014-10-15 Revised:2015-02-24 Online:2015-06-06 Published:2015-06-06
Contact: DENG Xiao-hui E-mail:dxh0122@163.com

摘要/Abstract

摘要：

目的应用自制梯度浓度混合-程控降温器官灌注仪，灌注羊完整卵巢，并行程序化冷冻，探讨冻融羊完整卵巢的效果并筛选最佳冷冻保护剂组合。方法将收集的28个羊完整卵巢，随机分配到新鲜对照组（A组）、海藻糖组（B组）、甘油组（C组）,二甲基亚砜（DMSO）组（D组）。冻融后组织切片行HE染色及原位缺口末端转移酶标记技术（TUNEL）检测，并通过RT-PCR技术检测组织Bcl-2相关X蛋白(Bax)及冷诱导RNA结合蛋白（CIRP）的mRNA表达。结果 B组正常卵泡形态比率与A组差异无显著性(P>0.05)，C组及D组正常形态卵泡比率显著低于A组，差异有统计学意义(P<0.05)。 B、C、D 3组冻存组基质细胞密度均低于A组，其中D组最低，差异具有统计学意义(P<0.05)。冻存组中，B组TUNEL反应阳性的细胞数目显著少于C组及D组，差异具有统计学意义(P<0.05)，且均显著多于新鲜组。A组及B组bax基因 mRNA的表达量明显低于C组及D组（P<0.05）；B组CIRP基因 mRNA的表达量明显高于C组及D组，差异均具有统计学意义(P<0.05)。结论羊卵巢整体冻存后可较好保存组织学形态，海藻糖组的冷冻保护剂在组织结构保存及抑制细胞凋亡方面优于甘油组及DMSO组。

Abstract:

Objective To study the effects of freezing sheep intact ovary by perfusing different cryoprotectants with concentration gradient blended programmed organ cooling perfusor, and to obtain the best cryoprotectants combination for cryopreservation of the whole sheep ovary. Methods Twenty-eight ovaries collected from 6-8 months non-pregnant female sheeps were randomly distributed into fresh group (group A), trehalose group (group B), glycerin group (group C) and DMSO group (group D). The morphology, cell apoptosis (by HE staining and TUNEL assay) and mRNA transcript of Bcl-2 associated X protein and cold inducible RNA-binding protein (by real-time PCR) of thawed sheep ovaries were tested to established the criterion for appraising cryopreservation results. Results The percentage of normal follicles in group B was comparable with group A (P＞0.05). The value in group C and group D were significant lower than that in group A (P<0.05). Quantitative assessment of stromal cell density indicated that the values in group D significantly lower than values in group A while group B and group C had values similar to those of group A. The TUNEL assay showed that the number of positive cells in group A were the lowest of all groups followed by group B, group C and group D (P<0.05). The level of BAX transcripts significantly increased in group C and group D (P<0.05). The level of CIRP transcripts increased highest in group B followed by group C and group D (P<0.05). Conclusion The whole ovary can be appropriately preserved after cryopreservation, and cryoprotectant combination of group B appears to be better for cryopreservation of whole sheep ovary in aspect of histology and anti-apoptosis.

杜天奇迟令龙赵淑芹时庆秦睿羲孙艳艳李栋邓晓惠*. 不同冷冻保护剂在羊卵巢整体冻存中的效果比较[J]. 解剖学报, 2015, (3): 415-421.

DU Tian-qi CHI Ling-long ZHAO Shu-qin SHI Qing QIN Rui-xi SUN Yan-yan LI Dong DENG Xiao-hui*. Comparison of different cryoprotectants in whole sheep ovary cryopreservation[J]. AAS, 2015, (3): 415-421.

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不同冷冻保护剂在羊卵巢整体冻存中的效果比较

Comparison of different cryoprotectants in whole sheep ovary cryopreservation

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