Article(id=1239230399931543586, tenantId=1146029695717560320, journalId=1238823019242635269, issueId=1239230393547804821, articleNumber=null, orderNo=null, doi=10.12465/j.issn.0253-4339.2025.03.151, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1702051200000, receivedDateStr=2023-12-09, revisedDate=1710691200000, revisedDateStr=2024-03-18, acceptedDate=1711987200000, acceptedDateStr=2024-04-02, onlineDate=1773385151130, onlineDateStr=2026-03-13, pubDate=1750003200000, pubDateStr=2025-06-16, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1773385151130, onlineIssueDateStr=2026-03-13, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1773385151130, creator=13701087609, updateTime=1773385151130, updator=13701087609, issue=Issue{id=1239230393547804821, tenantId=1146029695717560320, journalId=1238823019242635269, year='2025', volume='46', issue='3', pageStart='1', pageEnd='166', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1773385149609, creator=13701087609, updateTime=1773385254705, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1239230834402717933, tenantId=1146029695717560320, journalId=1238823019242635269, issueId=1239230393547804821, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1239230834402717934, tenantId=1146029695717560320, journalId=1238823019242635269, issueId=1239230393547804821, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=151, endPage=157, ext={EN=ArticleExt(id=1239230400195784750, articleId=1239230399931543586, tenantId=1146029695717560320, journalId=1238823019242635269, language=EN, title=Effect of Ice Seeding on Slow Freezing of Ovarian Tissue, columnId=null, journalTitle=Journal of Refrigeration, columnName=null, runingTitle=null, highlight=null, articleAbstract=

Ovarian tissue cryopreservation is an important method for female fertility preservation. Slow freezing of ovarian tissue results in poor follicular survival and low retransplantation efficiency. This study optimized the ovarian tissue cooling procedure by ice seeding, and the effects of ice seeding temperature and cooling rate after seeding on ovarian tissue cryopreservation were analyzed. The programmed cooling apparatus was combined with an ultrasonic device to achieve the ultrasonic seeding of ice crystals, and the ultrasonic intensity was screened. The ovarian survival and histology were assessed after rewarming. The results revealed that the optimized cooling procedure with ice seeding reduced the damage to ovarian tissues. When ice seeding was triggered at -11 ℃ with a cooling rate of 1 ℃/min after nucleation, follicle survival was 88.02%. Ultrasonic nucleation equipment enabled contactless ice seeding of the samples, reducing the risk of contamination and improving the success rate of ice seeding. Furthermore, the follicle survival rate of frozen ovarian tissue increased to 88.38%. The optimization of the procedure and the improvement of the equipment improved the effect of ovarian tissue cryopreservation, reduced the risk of introducing contamination during the cryopreservation process, and provided a new method for the slow cryopreservation of ovarian tissues in clinics.

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Zhou Xinli, female, professor, Institute of Biothermal Science, University of Shanghai for Science and Technology, 86-21-55270218, E-mail: . Research fields: cryopreservation technology for cells, tissues and organs.
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卵巢组织冻存是女性生育力保存的重要方法,慢速冷冻卵巢组织卵泡存活率较低,自体移植效率低。通过诱发成核对卵巢组织降温程序进行优化,分析了成核温度和成核后降温速率对卵巢组织冷冻保存效果的影响。将程序降温仪与超声波诱发成核设备结合,实现超声波诱发成核并优化了超声波强度,对复温后的卵巢存活率和组织学形态进行评估。结果表明:引入诱发成核的降温程序对卵巢组织损伤减小,当在-11 ℃诱发成核,成核后降温速率为1 ℃/min时,卵泡存活率达88.02%。超声波成核设备可对样品进行无接触式诱发成核,降低了污染风险,提高了成核的成功率,冻存卵巢组织卵泡存活率达88.38%。通过程序的优化以及设备的改进提高了卵巢组织冷冻保存效果,降低了冷冻保存过程中引入污染的风险,为临床上卵巢组织慢速冷冻保存提供了新的方法。

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周新丽,女,教授,上海理工大学生物系统热科学研究所,021-55270218,E-mail:。研究方向:细胞、组织、器官的低温保存技术。
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诱发成核对卵巢组织慢速冷冻的影响
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叶娜 , 徐强 , 刘晨曦 , 曹裕坤 , 周新丽
制冷学报 | 2025,46(3): 151-157
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制冷学报 | 2025, 46(3): 151-157
诱发成核对卵巢组织慢速冷冻的影响
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叶娜, 徐强, 刘晨曦, 曹裕坤, 周新丽
作者信息
  • 上海理工大学生物系统热科学研究所 上海 200093

通讯作者:

周新丽,女,教授,上海理工大学生物系统热科学研究所,021-55270218,E-mail:。研究方向:细胞、组织、器官的低温保存技术。
Effect of Ice Seeding on Slow Freezing of Ovarian Tissue
Na Ye, Qiang Xu, Chenxi Liu, Yukun Cao, Xinli Zhou
Affiliations
  • Institute of Biothermal Science and Technology, University of Shanghai for Science and Technology, Shanghai, 200093, China
出版时间: 2025-06-16 doi: 10.12465/j.issn.0253-4339.2025.03.151
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卵巢组织冻存是女性生育力保存的重要方法,慢速冷冻卵巢组织卵泡存活率较低,自体移植效率低。通过诱发成核对卵巢组织降温程序进行优化,分析了成核温度和成核后降温速率对卵巢组织冷冻保存效果的影响。将程序降温仪与超声波诱发成核设备结合,实现超声波诱发成核并优化了超声波强度,对复温后的卵巢存活率和组织学形态进行评估。结果表明:引入诱发成核的降温程序对卵巢组织损伤减小,当在-11 ℃诱发成核,成核后降温速率为1 ℃/min时,卵泡存活率达88.02%。超声波成核设备可对样品进行无接触式诱发成核,降低了污染风险,提高了成核的成功率,冻存卵巢组织卵泡存活率达88.38%。通过程序的优化以及设备的改进提高了卵巢组织冷冻保存效果,降低了冷冻保存过程中引入污染的风险,为临床上卵巢组织慢速冷冻保存提供了新的方法。

卵巢组织  /  冷冻保存  /  慢速冷冻  /  超声植冰

Ovarian tissue cryopreservation is an important method for female fertility preservation. Slow freezing of ovarian tissue results in poor follicular survival and low retransplantation efficiency. This study optimized the ovarian tissue cooling procedure by ice seeding, and the effects of ice seeding temperature and cooling rate after seeding on ovarian tissue cryopreservation were analyzed. The programmed cooling apparatus was combined with an ultrasonic device to achieve the ultrasonic seeding of ice crystals, and the ultrasonic intensity was screened. The ovarian survival and histology were assessed after rewarming. The results revealed that the optimized cooling procedure with ice seeding reduced the damage to ovarian tissues. When ice seeding was triggered at -11 ℃ with a cooling rate of 1 ℃/min after nucleation, follicle survival was 88.02%. Ultrasonic nucleation equipment enabled contactless ice seeding of the samples, reducing the risk of contamination and improving the success rate of ice seeding. Furthermore, the follicle survival rate of frozen ovarian tissue increased to 88.38%. The optimization of the procedure and the improvement of the equipment improved the effect of ovarian tissue cryopreservation, reduced the risk of introducing contamination during the cryopreservation process, and provided a new method for the slow cryopreservation of ovarian tissues in clinics.

ovarian tissue  /  cryopreservation  /  slow freezing  /  ultrasonic ice seeding
叶娜, 徐强, 刘晨曦, 曹裕坤, 周新丽. 诱发成核对卵巢组织慢速冷冻的影响. 制冷学报, 2025 , 46 (3) : 151 -157 . DOI: 10.12465/j.issn.0253-4339.2025.03.151
Na Ye, Qiang Xu, Chenxi Liu, Yukun Cao, Xinli Zhou. Effect of Ice Seeding on Slow Freezing of Ovarian Tissue[J]. Journal of Refrigeration, 2025 , 46 (3) : 151 -157 . DOI: 10.12465/j.issn.0253-4339.2025.03.151
随着医学的进步,癌症患者的治愈率逐渐提高,越来越多的年轻患者存在生育力保存的需求[1-3]。对女性患者而言,可供选择的方法包括胚胎冻存、卵母细胞冻存以及卵巢组织冻存。胚胎冻存仅适用于有配偶的已婚女性,无法为青春期或急需治疗的恶性肿瘤患者保存生育力,同时对于激素敏感型肿瘤患者而言,可能存在潜在的促进肿瘤生长的转移风险[4]。卵母细胞冻存需要预先进行促排卵、取卵等一系列操作,不仅有感染等风险,还会延后治疗周期。此外,促排需要激素注射,也无法为激素敏感型患者提供生育力保存[5]。卵巢组织冷冻保存无需促排,不会延误原发病治疗,适用于青春期前的女性患者。在原发病治愈后,自体移植复苏的卵巢组织有望恢复患者的内分泌和生育功能,对于年轻的女性患者极为有利。
目前用于卵巢组织冷冻保存的方法包括玻璃化冷冻保存和慢速冷冻。两者在临床上均有应用,各有利弊。玻璃化冷冻所需时间短,简单易操作,不依赖昂贵的仪器,但该方法使用的高浓度保护剂对卵巢组织有一定的毒性,同时会产生氧化应激反应和细胞凋亡损伤[6]。不仅如此,有研究表明玻璃化后的卵巢组织中部分卵泡基因表达降低,包括AMH(anti-Mullerian hormone,抗苗勒管激素)和Bmp15(bone morphogenetic protein 15,骨形态发生蛋白4)等。慢速冷冻使用低浓度的冷冻保护剂,毒性降低,冻后组织无明显的变形,且凋亡基因表达显著降低[7]。1994年,N. I. Boland等[8]使用二甲基亚砜作为冷冻保护剂完成了羊卵巢皮质的冷冻自体移植。直至2000年,K. Oktay等[9]报道了1例人卵巢组织自体移植成功,该团队将卵巢皮质制成了2 mm×2 mm至5 mm×10 mm的薄片,使用1.5 mol/L丙二醇慢速冷冻保存了6个月,解冻后移植成功。4年后,J. Donnez等[10]首次报道了1例人卵巢组织的慢速冻存,腹腔镜原位移植后活产,这是人类史上第1次卵巢组织冷冻保存活产。2021年8月31日,阮祥燕教授团队也报道了中国第1例卵巢组织慢速冷冻保存活产病例[11]。虽然慢速冷冻保存已有活产病例,但慢速冷冻在降温过程中,通常会产生较大的过冷度,导致不可控的结晶形成,造成细胞或组织的机械损伤[12],复温后卵巢组织中卵泡存活率低,卵巢组织功能受损。因此,通过改变卵巢组织的降温程序防止冰结晶造成组织损伤的研究是十分必要的。
为解决过冷损伤,目前最常用的方法就是诱发成核。诱发成核的方式有很多种,包括预冷探针接触成核、超声波诱发成核、冰成核剂诱发成核、电脉冲诱发成核等。对于卵巢组织冷冻保存而言,目前所用的诱发成核方式主要是预冷探针接触成核,即使用预冷的镊子或棉签轻触保护剂液面或冻存管,产生冰核[13]。该方式在某种程度上会引入外部的污染源,诱发的冰核均一性差。此外,在操作上需要拧开冻存管的盖子造成样品的温度波动,且无法批量操作。超声波辅助诱发成核作为机械置核的一种,已应用于生物样本的冷冻保存以及食品冷冻保存等领域。M. D. Luque de Castro等[14]研究表明超声对结晶过程的积极影响表现在诱导期,过饱和条件和亚稳区宽度的显著缩短,可以通过适当选择超声条件“定制”冰晶的尺寸分布甚至形状。R. Chow等[15]研究表明在超声存在的情况下,成核始终在较高的温度下发生,且超声波诱发成核的机理可能与空化气泡相关。Ma Xuan等[16]使用多频超声冷冻保存大黄鱼,结果表明多频超声能够显著提高大黄鱼冷冻样品的冷冻速率,并能较好地保存大黄鱼冷冻样品的品质。李维杰等[17]提出一种可用于L-02肝细胞植冰保存过程的超声波植冰系统,通过超声波诱发成核显著提高了冷冻保存肝细胞的存活率,达到(97±2.6)%,并降低了保护液中Me2SO用量。目前,将超声波诱导成核应用于卵巢组织的冷冻保存中的研究还未见报道。
本文在卵巢组织慢速冷冻过程中进行诱发成核,分析了成核温度和成核后降温速率对卵泡存活率的影响;按照冻存管尺寸定制了超声波传递架,并将超声设备置于程序降温仪内,搭建了卵巢组织超声波诱发成核降温系统;最后优化了诱发成核的超声波强度,以达到最优的卵巢组织冻存效果。
选取6~8周ICR系SPF级雌鼠,脱颈致死,迅速置于无菌手术台,腹部备皮。使用镊子与眼科剪小心剖开腹部,顺着输卵管找到双侧卵巢,剔除多余脂肪,取出输卵管与卵巢结合体。将结合体放入预热好的PBS(phosphate buffered saline,磷酸缓冲盐溶液),小心分离出整个卵巢,使用手术刀将卵巢一切为二,2个半卵巢再次均分为4块卵巢组织。得到的卵巢组织筛选出大小相等的组织块进行后续操作。
文中涉及保护剂的百分数若无特殊说明均指体积分数。将卵巢组织块浸入低温保护剂(10%二甲基亚砜+20%胎牛血清+0.1 mol/L蔗糖+PBS)中渗透10 min,移入含有0.5 mL低温保护剂的1.8 mL冻存管中,每个冻存管保存2块卵巢组织。将冻存管放入预冷至4 ℃的程序降温仪(CryoMed Freezer 7453,Thermo Fisher Scientific,USA)中,固定在可升降的杆上,如图1所示。启动降温仪进行程序降温,样品以2 ℃/min的降温速率降至诱发成核温度,当达到诱发成核温度后平衡5 min,提起升降杆,打开冻存管管帽,使用液氮预冷的镊子轻触保护剂液面,完成诱发成核。再次平衡5 min,保证冰核稳定生长。随后以特定的降温速率降至-40 ℃,再以10 ℃/min降至-100 ℃,取出冻存管迅速投入液氮。不置核的对照组冻存管固定在升降杆后,以2 ℃/min的降温速率降至-7 ℃平衡10 min,随后以0.3 ℃/min的降温速率降至-40 ℃,再以10 ℃/min的降温速率降至-100 ℃,取出冻存管迅速投入液氮。
为研究不同的诱发成核温度和成核后的降温速率对卵巢组织慢速冷冻保存效果的影响,设置诱发成核温度分别为-7、-9、-11 ℃,设置成核后的降温速率分别为0.3、1、10 ℃/min,实验组设计如图2所示。
复温过程,将冻存管取出置于37 ℃水浴复温3 min,取出卵巢组织块,分别移入洗脱液1(0.5 mol/L蔗糖+20%胎牛血清+PBS),洗脱液2(0.25 mol/L蔗糖+20%胎牛血清+PBS)和洗脱液3(20%胎牛血清+PBS)洗脱3 min。洗脱后的卵巢组织在显微镜下使用注射针头机械剥离其中的卵泡,PBS洗涤一次后,移入培养皿中,滴入50 μL Calcein-AM/PI染料(2 μmol/L Calcein-AM+1.5 μmol/L PI),于培养箱中37 ℃避光孵育45 min。染色结束后使用荧光显微镜观察卵泡颜色,以判断卵泡的存活。显绿色荧光为阳性即卵泡存活,显红色荧光为阴性即卵泡死亡。卵泡存活率计算如式(1)所示:
式中:V为卵泡存活率;G为被AM标记为绿色的活细胞数,个;R为被PI标记为红色的死细胞数,个。
洗脱后的卵巢组织,使用4%的多聚甲醛固定24 h。然后使用梯度浓度的酒精进行洗脱用时9.5 h,脱水后需对组织进行透明化处理。透明后的组织浸蜡包埋,切片烘烤3 h后脱蜡。脱蜡后的切片分别用Harris氏苏木素染液和1%水溶性伊红染液染色。染色结束切片放入无水乙醇中脱水,放入二甲苯中透明,风干后用中性树胶封片。封好的胶片置于显微镜下,观察卵巢组织致密程度,判断各级卵泡及卵母细胞细胞核形态是否正常。
为了在程序降温过程中采用超声波诱发成核,将定制的超声波传递架置于程序降温仪(CryoMed Freezer 7455,Thermo Fisher Scientific,USA)内,并连接外部的超声波发生器(LH-600,佰斯特超高科技有限公司,中国),如图3所示。超声波传递架材质为不锈钢,整体尺寸为12 cm×12 cm×10 cm,传递架下方焊接超声波传递震子,上方为定制圆孔,孔径为12 mm。超声波发生器可调频率为25、28、40 kHz,最大功率为600 W。
对于超声波诱发成核卵巢组织慢速冷冻,将卵巢组织块浸入低温保护剂(10%二甲基亚砜+20%胎牛血清+0.1 mol/L蔗糖+PBS)中渗透10 min,移入含有0.5 mL低温保护剂的1.8 mL冻存管中,每个冻存管保存2块卵巢组织。将冻存管嵌入超声传递架圆孔,整体置于程序降温仪中,关闭舱门。启动降温仪进行程序降温,样品以2 ℃/min的降温速率降至诱发成核温度,当达到-11 ℃时,恒温5 min后启动超声发生器,诱发成核完成再次恒温5 min稳定生成冰核。随后以1 ℃/min的降温速率降至-40 ℃,再以10 ℃/min的降温速率降至-100 ℃,取出冻存管迅速投入液氮。为研究不同强度超声波诱发成核对慢速冷冻保存卵巢组织的影响,设置了不同功率的超声波诱发成核,由高至低分别为400、270、160 W,复温过程与1.2节相同。
数据采用GraphPad软件的ANOVA(analysis of variance)进行分析。每组实验重复3次,数据以平均值±SD表示,以P<0.05作为显著性差异评判标准,具有统计学意义。
为研究诱发成核温度和成核后的降温速率对卵巢组织慢速冷冻保存效果的影响,设置了10组程序对卵巢组织进行冷冻保存,复温后卵巢组织中卵泡存活率如图4所示。
图4可知,当成核温度为-7 ℃时,成核后以0.3、1、10 ℃/min的降温速率降温,冻存后卵泡存活率分别为79.98%、77.51%、57.41%,而不成核组的卵泡存活率为69%,0.3 ℃/min组存活率最高。当成核温度为-9 ℃时,成核后以0.3、1、10 ℃/min的降温速率降温,冻存后卵泡存活率分别为66.41%、79.56%、66.27%,1 ℃/min组存活率最高。当成核温度为-11 ℃时,成核后以0.3、1、10 ℃/min的降温速率降温,冻存后卵泡存活率分别为77.71%、88.02%、57.92%,1 ℃/min组存活率最高。
预冷探针诱发成核能够使保护剂在局部形成较大的过冷度[18],出现冰核,从而降低保护剂整体的过冷度减小损伤,因此诱发成核更有利于卵巢组织慢速冷冻保存。这与研究者在其他细胞冻存过程中得出的结论相同。Huang Haishui等[19]结合海藻糖预脱水和诱发成核实现了细胞的无CPA(cryoprotective agent,冷冻保护剂)冻存,0.33 mol/L海藻糖用于预脱水和-4 ℃诱发成核的组合可以使人红细胞在无任何渗透性CPA的情况下低温保存后获得高细胞活力,细胞回收率最高为78.1%±4.8%,实验证实了诱发成核的重要作用。Huang Zhiyong等[20]针对人T淋巴细胞的冷冻保存诱发成核温度进行了研究,发现诱发成核温度会影响人T淋巴细胞的冷冻保存,即使成核后的降温速率(90 ℃/min)远高于人体T淋巴细胞的最佳冷却速率,保存效果也很理想。在-14、-16、-18 ℃诱发置核时,细胞的相对存活率比不成核分别提高了97.3%、98.8%、97.0%。实验结果不仅证实了诱发成核的必要性,也证实了诱发成核的温度对冻存效果是有影响的。
从冻存后卵巢组织中卵泡存活率来看,诱发成核温度与成核后的降温速率之间有一定的匹配关系。对于较高的诱发成核温度(-7 ℃),缓慢的降温速率0.3 ℃/min更有利于卵巢组织的保存,对于较低的成核温度(-9 ℃和-11 ℃),增加降温速率至1 ℃/min则显示了更好的冻存效果。该情况可能与冻存过程中冰核的形成与生长有关。成核温度越高产生的过冷度越小,形成的晶核体积大且数量少[21]。同时,降温速率快有利于冰晶生长。即高成核温度结合更快的降温速率会使大晶核快速生产,从而对卵巢组织产生较大的机械损伤。因此,高成核温度成核后,更适合以较低的冷却速率继续降温。较低的成核温度(-9 ℃和-11 ℃)产生的过冷度大,冰核体积小而数量多,同时较大的过冷度会增加溶液的黏度,导致水分子扩散难度越来越大,不利于晶核的生长。在这种条件下增加降温速率至1 ℃/min,不会使小晶核快速长大,不仅可以保证生物样本受到较小的晶核损伤,同时可以减少降温时间,降低降温过程中的高浓度保护剂对生物样本的毒性损伤。当降温速率增至10 ℃/min,无论在什么温度下诱发成核,均会导致胞内自由水没有充足时间渗透到胞外,从而在内部形成胞内冰,造成组织损伤。
预冷探针诱发成核冻存卵巢组织HE染色如图5所示,A组和H组的均较好地保存了卵巢组织的形态结构,虽然与新鲜组相比,各组均有损伤,但各级卵泡形态正常,卵母细胞核保持为圆形或椭圆形,少见核固缩。
A组的降温程序为-7 ℃诱发成核,成核后以0.3 ℃/min的降温速率降至-40 ℃,再以10 ℃/min的降温速率降至-100 ℃,然后迅速投入液氮。这是当前卵巢组织慢速冷冻保存最为常用的降温程序,卵泡存活率达到79.98%。该程序最初应用于卵母细胞的冷冻保存,借鉴至卵巢组织的冷冻保存且效果较好,于是一直沿用。H组的降温程序为-11 ℃诱发成核,成核后以1 ℃/min的降温速率降至-40 ℃,再以10 ℃/min的降温速率降至-100 ℃,然后迅速投入液氮。H组是9个对比组中卵泡存活率最高的组别,达到了88.02%,组织形态学评估结果也较为理想,可以作为卵巢组织慢速冻存的优化程序。
为研究不同强度的超声波诱发成核的效果,使用不同功率的超声波进行诱发成核后冻存,得到复温后卵巢组织卵泡存活率如图6所示,图中不同字母表示单因素方差分析法中具有显著性差异(P<0.05)。
不进行超声波诱发成核,超声波实验组降温速率为1 ℃/min时,卵泡存活率为41.03%。不进行预冷探针诱发成核,预冷探针实验组的降温速率为0.3 ℃/min时,卵泡存活率为69%,如图6所示。可以看出,当降温速率更快时,不进行诱发成核对冻存效果影响显著。当超声波功率为160 W和280 W时,卵泡存活率分别为85.46%和88.38%。当超声波功率为280 W时,卵泡存活率与-11 ℃预冷探针诱发成核降温速率为1 ℃/min的实验组(88.02%)无显著性差异。当超声波强度为400 W时,卵泡存活率大幅下降,仅为25.76%。这是因为超声波功率过大,造成了卵巢组织的机械损伤。M. Saclier等[22]对超声空化引发的冰成核过程进行了模拟,结果表明在空化泡崩溃的最后阶段发生的压力增加了水的平衡冻结温度,结果是局部过冷水平提高,这是冰成核的驱动力。因此,即使在低过冷水平下,也可以在适度的声压振幅下开始成核。N. Lyczko等[23]针对超声波诱发成核辅助冻结硫酸钾的研究表明超声能够在低于无超声的过饱和下触发成核,同时可以避免诱发成核,避免外来污染引入溶液。Hu Fen等[24]研究表明当超声波达到足够高的强度时,才会出现大量充满活力的气泡,气泡的破裂能降低界面能,进而诱发成核。研究发现160 W和280 W的超声波辅助置核对卵泡存活率的影响无显著差异,但280 W的超声波可以更稳定地诱发成核。
超声波诱发成核冻存卵巢组织的组织形态学评估如图7所示。超声波诱发成核组更好地保护了卵泡的结构和卵巢组织内部结构。卵泡周围很少产生空腔,卵巢组织的结构更加致密。不成核组可以明显观察到大量空腔,部分卵泡形状发生了改变,可能会影响卵泡的进一步发育。
本文优化了卵巢组织慢速冷冻保存工艺,包括成核温度和成核后的降温速率;搭建了程序降温与超声波诱发成核结合的系统,并确定了安全且能稳定成核的超声波强度,为临床上卵巢组织冷冻保存提供了新的方案。得到结论如下:
1)相比于通用的-7 ℃诱发成核0.3 ℃/min降温的冷冻程序,优化后的-11 ℃诱发成核1 ℃/min降温的冷冻程序,复温后的卵巢组织卵泡存活率为88.02%,组织形态保持较好。更重要的是该程序缩短了降温时间,能够更快地完成卵巢组织的冻存。
2)将程序降温仪与超声诱发成核相结合,仅需启动降温仪外部的超声发生器即可在内部通过传递架进行超声波诱发成核,无需将样品取出完成诱发成核。该方法避免了预冷探针诱发成核时样品从降温仪中取出温度的急速上升,更稳定地完成整个冷冻过程,并保证了无外源污染的引入。
3)优化了超声波诱发成核的相关参数,在超声波频率为40 kHz,功率为160 W和280 W时诱发成核,卵巢组织冻存效果最好。其中功率为280 W时诱发成核成功率更高,且复温后卵泡存活率达88.38%。
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2025年第46卷第3期
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doi: 10.12465/j.issn.0253-4339.2025.03.151
  • 接收时间:2023-12-09
  • 首发时间:2026-03-13
  • 出版时间:2025-06-16
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  • 收稿日期:2023-12-09
  • 修回日期:2024-03-18
  • 录用日期:2024-04-02
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    上海理工大学生物系统热科学研究所 上海 200093

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周新丽,女,教授,上海理工大学生物系统热科学研究所,021-55270218,E-mail:。研究方向:细胞、组织、器官的低温保存技术。
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2种不同金属材料的力学参数

Family
属数
Number of
genus
种数
Number of
species
占总种数比例
Percentage of
total species (%)

Genus
种数
Number of
species
占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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