解冻温度和冷冻保护剂对中华鲟精原干细胞超低温冷冻保存效果的影响  

Effects of thawing temperature and cryoprotectant on the ultra-low temperature cryopreservation of spermatogonial stem cells of Chinese sturgeon(Acipenser sinensis)

作　　者：舒雪密 叶欢[1] 仲嘉 张鸣 罗江[1] 刘源 杜浩[1,2] SHU Xuemi;YE Huan;ZHONG Jia;ZHANG Ming;LUO Jiang;LIU Yuan;DU Hao(Key Laboratory of Freshwater Biodiversity Conservation,Ministry of Agriculture and Rural Areas/Yangtze River Fisheries Research Institute,Chinese Academy of Fishery Sciences,Wuhan 430223,China;Chinese Academy of Agricultural Sciences,Beijing 100010,China;College of Fisheries and Life Science,Shanghai Ocean University,Shanghai 201306,China)

机构地区：[1]中国水产科学研究院长江水产研究所,农业农村部淡水生物多样性保护重点实验室,湖北武汉430223 [2]中国农业科学院,北京100010 [3]上海海洋大学水产与生命学院,上海201306

出　　处：《中国水产科学》2024年第7期745-753,共9页Journal of Fishery Sciences of China

基　　金：国家重点研发计划项目(2021YFD120030107);中央级公益性科研院所基本科研业务费专项资金资助项目(2023TD08).

摘　　要：中华鲟(Acipenser sinensis)是我国一级重点保护水生动物,其精原干细胞等种质资源冷冻保护技术尚不完善。本研究在前期建立的匙吻鲟(Polyodon spathula)精原干细胞冷冻保存液配方基础上,研究解冻温度、抗氧化剂和抗冻蛋白对中华鲟精原干细胞冷冻保存效果的影响,以期建立高效的中华鲟精原干细胞超低温冷冻保存方法。研究结果表明,实验设置的解冻温度(20、25、30和40℃)中25℃组冷冻保存效果最佳:解冻后细胞数目为(3.86±0.51)×10^(5),细胞存活率可达(96.36±0.53)%;比较不同浓度组(500、1000和1500 mg/L)的谷胱甘肽、抗坏血酸和α-生育酚等3种抗氧化剂对中华鲟精原干细胞冷冻保存效果的影响,结果显示,冷冻保存液中添加1000 mg/Lα-生育酚的实验组,解冻后得到的细胞数目最多(7.64±0.34)×10^(5),显著高于其他抗氧化剂添加组,细胞存活率可达(92.82±0.72)%;通过比较不同浓度(0.1、1.0和10μg/mL)的两种类型抗冻蛋白(AFPI和AFPIII)对中华鲟精原干细胞的冷冻保存效果,发现添加了1.0μg/mL AFPI的实验组解冻效果最好,解冻后得到的细胞数目为(6.85±0.19)×10^(5),显著高于其他实验组,细胞存活率为(86.89±0.73)%。综上所述,中华鲟精原干细胞冷冻保存的最佳解冻温度为25℃,1000 mg/Lα-生育酚作为抗氧化剂,和1.0μg/mL AFPI作为抗冻蛋白对其冷冻保存效果最佳。The Chinese sturgeon(Acipenser sinensis)is one of the key aquatic animals under first-degree protection in China,but its germplasm resources,such as spermatogonial stem cells,are not well protected by cryoprotection technology.In this study,we investigated the effects of thawing temperature,antioxidants,and antifreeze proteins on the cryopreservation of spermatogonial stem cells of Chinese sturgeon based on the formulation of the spermatogonial stem cell cryopreservation solution used for the American paddlefish(Polyodon spathula).The aim was to establish a highly efficient method for ultra-low-temperature cryopreservation of spermatogonial stem cells of Chinese sturgeon.The best cryopreservation effect was achieved at a thawing temperature of 25℃ among the temperatures tested(20,25,30,and 40℃).The number of cells after thawing was(3.86±0.51)×10^(5),and the cell viability reached(96.36±0.53)%.The effects of different concentrations(500 mg/L,1000 mg/L,and 1500 mg/L)of glutathione,ascorbic acid,andα-tocopherol on the cryopreservation of spermatogonial stem cells of Chinese sturgeon were examined.The results showed that the experimental group with the addition of 1000 mg/Lα-tocopherol to the cryopreservation solution had the highest number of cells(7.64±0.34)×10^(5) after thawing,which was significantly higher than that of the other groups with antioxidants added,and the cell viability reached(92.82±0.72)%.The effects of different concentrations(0.1,1.0,and 10μg/mL)of two types of antifreeze proteins(AFPI and AFPIII)on the cryopreservation of spermatogonial stem cells of Chinese sturgeon were examined.The experimental group with 1.0μg/mL AFPI had the best defrosting effect,and the number of cells obtained after defrosting was(6.85±0.19)×10^(5),which was significantly higher than the number in the other experimental groups,and the cell viability was(86.89±0.73)%.In summary,in the present study,we obtained the optimal thawing temperature for the cryopreservation of spermatogonial stem cells of Chinese

关 键 词：中华鲟 精原干细胞 超低温冷冻保存 解冻温度 抗氧化剂 抗冻蛋白 

分 类 号：S917[农业科学—水产科学]