人卵巢组织玻璃化冻存方案优选的实验研究  

Experimental study on optimal vitrification of human ovary tissue

作　　者：李晓虹[1] 汤惠茹[1] 李雨珊 曾玉翠 魏蔚霞[1] 黄斌[1] 杜辉[1] 吴瑞芳[1,2] LI Xiao-hong;TANG Hui-ru;LI Yu-shan;ZENG Yu-cui;WEI Wei-xia;HUANG Bin;DU Hui;WU Rui-fang(Peking University Shenzhen Hospital,Shenzhen 518036;Shenzhen Key Laboratory on Technology for Early Diagnosis of Major Gynecologic Diseases,Shenzhen 518036)

机构地区：[1]北京大学深圳医院,深圳518036 [2]深圳市女性重大疾病早期诊断技术重点实验室,深圳518036

出　　处：《生殖医学杂志》2021年第1期87-93,共7页Journal of Reproductive Medicine

基　　金：深圳市三名工程(SZSM201412010);深圳市科创委技术攻关项目(JSGG20180703164202084)。

摘　　要：目的评价4种玻璃化冷冻保存液对人卵巢组织的冻存效果,以筛选出较优的玻璃化冷冻保存液配方。方法收集2018年3月至2019年12月期间在北京大学深圳医院妇科因疾病手术治疗的4例患者的卵巢组织,将人卵巢分割为(5~10)mm×10 mm×1 mm大小的组织块进行玻璃化冷冻保存,根据玻璃化冷冻保存液组成成分不同将组织块随机分为4组[A组为20%乙二醇(EG)+20%二甲基亚砜(DMSO)+0.5 mol/L蔗糖+20%人工合成血清替代品(SSS)+M199培养基;B组为38%EG+0.5 mol/L海藻糖+6%SSS+M199培养基;C组为15%EG+15%DMSO+0.5 mol/L蔗糖+20%SSS+M199培养基;D组为商品化Cryotissue Kit VT301/VT302成品]以及对照组(未经冻融处理的新鲜卵巢组织)。采用液氮液滴法在显微镜下观察4种保存液在快速降温和复融时的玻璃化状态;HE染色比较冻存后各组卵巢组织形态学改变;采用免疫组化法检测各组中凋亡标志物细胞色素C(Cytochome C)与半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)的表达情况。结果液滴法检测发现,在液氮中快速降温时A、B和D组冻存液均能保持完全玻璃化状态,C组冻存液呈部分结晶;在复融过程中,A、B和C组冻存液均出现结晶,D组冻存液则绝大部分保持透明的玻璃化状态。HE染色结果发现,A、B、C和D组人卵巢组织中的正常卵泡率与对照组比较均无显著性差异(P>0.05);免疫组化结果显示,D组和对照组卵泡中Cytochome C显著低于C组(P<0.05),C组卵泡Caspase-3阳性率显著高于其他组(P<0.05)。结论通过液氮液滴法观察冻存液在快速降温和复融时的玻璃化状态方便直观,可用于对人卵巢组织玻璃化冷冻液的初步筛选;商品化Cryotissue Kit VT301/VT302冻存液冻融后人卵巢组织细胞凋亡较少,为较优的冻存液配方。Objective:To evaluate the cryopreservation effects of 4 kinds of vitrification cryopreservation solutions on human ovarian tissues to screen out the best vitrification cryopreservation solution formula.Methods:The ovarian tissues of 4 patients who were surgically treated for diseases in the Department of Gynecology,Peking University Shenzhen Hospital from March 2018 to December 2019 were collected. The human ovary were cut into(5-10) mm×10 mm×1 mm tissue blocks for vitrification cryopreservation. According to the composition of the vitrified cryopreservation solution,the tissue blocks were randomly divided into 4 groups:group A was 20% ethylene glycol(EG)+20% dimethyl sulfoxide(DMSO)+0.5 mol/L sucrose+20% artificial synthetic serum substitute(SSS)+M199 medium;group B was 38% EG+0.5 mol/L trehalose+6% SSS+M199 medium;group C was 15% EG+15% DMSO+0.5 mol/L sucrose+20% SSS+M199 medium;group D was the commercial Cryotissue Kit VT301/VT302] and the control group(fresh ovarian tissue without freezing and thawing treatment). The vitrification state of the four preservation solutions during rapid cooling and thawing by liquid nitrogen drop method was observed under the microscope. HE staining was used to compare the morphological changes of ovarian tissues in each group after cryopreservation. The expression of cytochrome C and caspase-3 were detected by immunohistochemistry method.Results:It was found that the cryopreservation solution of group A,B and D could keep complete vitrification when the temperature was rapidly decreased in liquid nitrogen,and the cryopreservation solution of group C was partially crystallized. During the thawing process,the cryopreservation solution of group A,B and C appeared crystallization,while the majority of group D cryopreservation liquid remained transparent and vitrified. HE staining showed that there was no significant difference in the percentage of normal follicles between groups A,B,C and D(P>0.05). Immunohistochemical results showed that cytochrome C in follicles of group D and

关 键 词：卵巢组织 玻璃化冷冻 物理性状 

分 类 号：R361[医药卫生—病理学] R711[医药卫生—基础医学]