活体肺癌组织玻璃化保存的研究  

Study on Vitrification of Living Lung Cancer Tissue

作　　者：李嘉慧 杜羽琨 余科科[2] 邢杰 周新丽[1] LI Jia-Hui;DU Yu-Kun;YU Ke-Ke;XING Jie;ZHOU Xin-Li(Institute of Biothermal Science,University of Shanghai for Science and Technology,Shanghai 200093,China;Shanghai Chest Hospital,Shanghai Jiao Tong University,Shanghai 200030,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 [2]上海交通大学附属胸科医院,上海200030

出　　处：《生物化学与生物物理进展》2021年第9期1096-1103,共8页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金(51376132)资助项目。

摘　　要：保存活体的肺癌组织将为肺癌发病基因筛查和靶向药物筛选等体外实验研究提供更完整的样本信息.本文对活体肺癌组织的玻璃化保存方法进行研究,首先采用针浸法玻璃化保存单块肺癌组织,对所需低温保护剂的浓度和平衡时间进行了优化;其次采用冻存管对多块肺癌组织样本进行玻璃化保存,对低温保护剂溶液体积以及平衡时间进行了优化;最后对慢速冷冻、不加低温保护剂快速冷冻、玻璃化冷冻3种冷冻方法的冻存效果进行比较并通过低温显微分析其冰晶损伤机理.结果表明,20%EG+20%DMSO+0.5 mol/L海藻糖作为低温保护剂,在平衡溶液和玻璃化溶液分别加载3 min和1 min时,针浸法和0.25 ml冻存管内玻璃化冻存,复苏后组织活力最高,分别约为79.96%与80.44%.免疫组化显示玻璃化保存肺癌组织经过复苏后,相比慢速冷冻和无保护剂快速冷冻,组织结构损伤较小,组织内细胞TUNEL阳性表达较少.低温显微结果表明,玻璃化保存组织内部及周围只出现少量细小冰晶,而慢速冷冻、快速冷冻组织皆出现明显冰晶.Preservation of living lung cancer tissues will provide more complete sample information for in vitro experimental studies, such as lung cancer genetic screening and targeted drug screening. This article studied the vitrification method of living lung cancer tissue. Firstly, the needle immersion method was used to vitrify a single piece of lung cancer tissue, and the concentration and equilibrium time of the required cryoprotectant were optimized. Secondly, multiple lung cancer tissues were vitrified in cryotubes, and the volume of cryoprotectant solution and equilibrium time were optimized. Finally, the effects of the traditional slow freezing, rapid freezing without cryoprotectant, and vitrification methods were compared, and low-temperature microscope was used to analyze the damage mechanism of ice crystal. The results showed when 20% EG+20% DMSO+0.5 mol/L trehalose was used as the cryoprotectant, the equilibration solution and the vitrification solution are loaded for3 min and 1 min, respectively, the needle immersion method and vitrification in a 0.25 ml cryotube have the highest tissue viability(79.96% and 80.44%) after recovery. Immunohistochemistry showed that the tissue structure of lung cancer after vitrification was less damaged, only a few positive expression of TUNEL in the cells, compared with slow freezing and fast freezing without cryoprotectants. Low-temperature microscopy showed that only a few small ice crystals appeared in and around the vitrified tissue, while obvious ice crystals appeared in slow freezing and rapid freezing.

关 键 词：肺癌组织 玻璃化 保护剂加载 冷冻载体 

分 类 号：Q6-33[生物学—生物物理学] Q813