小鼠睾丸组织慢速冷冻保存工艺的优化研究  

Study on Optimization of Slow Freezing Protocol of Mouse Testis

作　　者：谭佳 郭莹莹 周新丽[1] TAN Jia;GUO Ying-Ying;ZHOU Xin-Li(Institute of Biothermal Science,University of Shanghai for Science and Technology,Shanghai 200093,China)

机构地区：[1]上海理工大学生物系统热科学研究所,上海200093

出　　处：《生物化学与生物物理进展》2022年第10期2054-2062,共9页Progress In Biochemistry and Biophysics

基　　金：上海市促进市级医院临床技能与临床创新能力三年行动计划重大临床研究项目(SHDC2020CR3077B)资助。

摘　　要：目的冷冻保存睾丸组织用于后期移植,是除精子冻存以外保持男性生育力的另一有效途径。本文对块状睾丸组织常用的慢速冷冻降温程序进行了改进。方法通过缩短保护剂加载时间、提高第一阶段的冷却速率、第二阶段直接投入液氮等方法对小鼠睾丸组织进行冷冻保存。在不同温度对小鼠睾丸组织冻存体系进行诱导冰晶成核并冻存,降低睾丸组织慢速冷冻保存所需保护剂浓度。结果改进的两步法冻后组织内生殖细胞的凋亡阴性率均较高,其中精原细胞98.4%、精母细胞99.2%、精子细胞88.4%、支持细胞98.1%,显著高于常用慢速冷冻组,与对照组均无显著性差异。相比于未置核组,-10℃置核可显著提高5%DMSO保护剂慢速冷冻保存的冻后效果,生殖细胞的凋亡阴性率为精原细胞82.9%、精子细胞92.1%、精母细胞93.2%及支持细胞88.9%,与较高浓度保护剂10%DMSO组冻存结果无显著性差异,说明置核能够降低所需保护剂的浓度,降低毒性损伤。结论本研究通过改进两步法和置核提高了小鼠睾丸组织冻后的质量,为临床上人睾丸组织的冻存提供参考。Objective Cryopreservation of testicular tissue for later transplantation is another effective way to maintain male fertility.Methods In this paper,the procedure of slow freezing of massive testicular tissue was optimized by shortening the loading time of cryoprotectant(CPA),increasing the freezing rate at the first stage,and directly plunging into liquid nitrogen at the second stage.The mouse testis was cryopreserved by modified two-step freezing.In addition,ice seeding procedure was applied at different temperatures in order to reduce CPA concentration required for cryopreservation of testicular tissue.Results The results showed that negative rate of apoptosis of germ cells in frozen tissues with modified two-step method was significantly higher than commonly used slow freezing method,and had no significant difference with control group.Among them,the negative rate of spermatogonial cells was 98.4%,that of spermatoblast cells was 99.2%,that of sperm cells were 88.4%,and that of sertoli cells was 98.1%.Compared with non-seeding group,seeding at-10℃can improve the survival rate of testicular tissue that frozen with 5%DMSO.The negative rate of apoptosis were 92.1%(spermatozoa),93.2%(spermatocytes)and 88.9%(Sertoli),which are not significantly different from that of 10%DMSO group.This indicates ice seeding can reduce the CPA concentration and toxic damage.Conclusion Modified two-step freezing and ice seeding improve the quality of mouse testicular tissue after freezing,and provide a reference for freezing of human testis in clinical.

关 键 词：睾丸组织 慢速冷冻 两步法冷冻 置核 

分 类 号：Q2[生物学—细胞生物学] Q3Q81