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 ›› Issue (3) : 415-421. DOI: 10.16098/j.issn.0529-1356.2015.03.022

技术方法

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*

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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*

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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.

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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 https://doi.org/10.16098/j.issn.0529-1356.2015.03.022

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]. Acta Anatomica Sinica. 2015(3): 415-421 https://doi.org/10.16098/j.issn.0529-1356.2015.03.022

参考文献

［1］Siegel R, Ma J, Zou Z, et al. Cancer statistics, 2014［J］. CA Cancer J Clin, 2014, 64(1): 9-29.
［2］Wallace WH, Thomson AB, Saran F, et al. Predicting age of ovarian failure after radiation to a field that includes the ovaries［J］. Int J Radiat Oncol BiolPhys, 2005, 62(3): 738-744.
［3］Macklon KT, Jensen AK, Loft A, et al. Treatment history and outcome of 24 deliveries worldwide after autotransplantation of cryopreserved ovarian tissue, including two new Danish deliveries years after autotransplantation［J］. J Assist Reprod Genet, 2014, 31(11): 1557-1564.
［4］Ernst E, Bergholdt S, Jrgensen JS, et al. The first woman to give birth to two children following transplantation of frozen/thawed ovarian tissue［J］. Hum Reprod, 2010, 25(5): 1280-1281.
［5］Liu J, Van der Elst J, Van den Broecke R, et al. Early massive follicle loss and apoptosis in heterotopically grafted newborn mouse ovaries［J］. Hum Reprod, 2002, 17(3): 605-611.
［6］Imhof M, Bergmeister H, Lipovac M, et al. Orthotopic microvascular reanastomosis of whole cryopreserved ovine ovaries resulting in pregnancy and live birth［J］. Fertil Steril, 2006, 85 (Suppl 1): 1208-1215.
［7］Campbell BK, Hernandez-Medrano J, Onions V, et al. Restoration of ovarian function and natural fertility following the cryopreservation and autotransplantation of whole adult sheep ovaries［J］. Hum Reprod, 2014, 29(8): 1749-1763.
［8］Schiraldi C, Di Lernia I, De Rosa M. Trehalose production: exploiting novel approaches［J］. Trends Biotechnol, 2002, 20(10): 420-425.
［9］Lee YA, Kim YH, Kim BJ, et al. Cryopreservation in trehalose preserves functional capacity of murine spermatogonial stem cells［J］. PloSOne, 2013, 8(1): e54889.
［10］Xu Z, Wang X, Wu Y, et al. Slow-controlled freezing versus speed-cooling for cryopreservation of whole guinea pig ovaries［J］. Theriogenology, 2012, 77(3):483-491.
［11］Revel A, Elami A, Bor A, et al. Whole sheep ovary cryopreservation and transplantation［J］. Fertil Steril, 2004, 82(6): 1714-1715.
［12］Zhang JM, Zhang YC, Ruan LH, et al. Optimizing cryoprotectant perfusion conditions for intact ovary: a bovine model［J］. J Assist Reprod Genet, 2012, 29(11): 1255-1260. 
［13］Katkov II, Kim MS, Bajpai R, et al. Cryopreservation by slow cooling with DMSO diminished production of Oct-4 pluripotency marker in human embryonic stem cells［J］. Cryobiology, 2006, 53(2): 194-205.
［14］Wang HY, Lun ZR, Lu SS. Cryopreservation of umbilical cord blood-derived mesenchymal stem cells without dimethyl sulfoxide［J］. Cryo Letters, 2011, 32(1): 81-88.
［15］Men NT, Kikuchi K, Nakai M, et al. Effect of trehalose on DNA integrity of freeze-dried boar sperm, fertilization, and embryo development after intracytoplasmic sperm injection［J］. Theriogenology, 2013, 80(9): 1033-1044.
［16］Zhao J, Wang S, Bao J, et al. Trehalose maintains bioactivity and promotes sustained release of BMP-2 from lyophilized CDHA scaffolds for enhanced osteogenesis in vitro and in vivo［J］. PloS One, 2013, 8(1): e54645.
［17］Croew LM, Reid DS, Crowe JH. Is trehalose special for preserving dry biomaterials［J］. Biophys J, 1996,71(4):2087-2093.
［18］Younis A, Carnovale D, Butler W, et al. Application of intra-and extracellular sugars anddimethylsulfoxide to human oocyte cryopreservation［J］. J Assist Reprod Genet, 2009, 26(6):341-345.
［19］Zhao Sh, Zhao ShQ, Shi Q. et al. Effects of different freezing methods on the follicular viability of large pieces of human ovarian tissues ［J］. Acta Anatomica Sinica, 2013, 44(3): 427-432.(in Chinese)
赵硕, 赵淑琴, 时庆, 等. 两种冷冻方法对人大块卵巢组织卵泡活性的影响［J］. 解剖学报, 2013, 44(3): 427-432.
［20］Chen LF, Liu JH, Ouyang XL, et al.Effects of trehalose loading on the freeze-dried red blood cells ［J］. Chinese Journal of Blood Transfusion, 2008, 21(1): 28-31.(in Chinese)
陈麟凤, 刘景汉, 欧阳锡林, 等. 海藻糖负载对人红细胞冷冻干燥保存影响的实验研究［J］. 中国输血杂志, 2008, 21(1): 28-31.
［21］Jiang YH, Xu LH, Luo YR, et al.Effects of trehalose on bcl-2 and bax mRNA expression in the cryopreserved sternum ［J］. Chinese Journal of Tissue Engineering Research, 2011, 15(21): 3975-3978.(in Chinese)
江玉华, 徐林浩, 罗宜人，等. 海藻糖对低温保存胸骨中 bcl-2 和bax基因表达的影响［J］. 中国组织工程研究, 2011, 15(21): 3975-3978.
［22］Xia Z, Jiang K, Liu T, et al. The protective effect of Cold-inducible RNA-binding protein (CIRP) on testicular torsion/detorsion: an experimental study in mice ［J］. J Pediatr Surg, 2013, 48(10): 2140-2147.
［23］Li S, Zhang Z, Xue J, et al. Coldinducible RNA binding protein inhibits H2O2 induced apoptosis in rat cortical neurons ［J］. Brain Res, 2012, 1441(2): 47-52.
［24］Sakurai T, Itoh K, Higashitsuji H, et al. Cirp protects against tumor necrosis factor-alpha-induced apoptosis via activation of extracellular signalregulated kinase［J］. Biochim Biophys Acta, 2006, 1763(3): 290-295.