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玻璃化冷冻保存技术对鱼类卵细胞的冷冻保存仍少见报道[1]。鱼类的卵与一般细胞相比由于其体积大,相对比表面积小,含水率高,卵黄多等特点,限制了水分的渗出和抗冻剂渗入的速率。同时由于卵细胞具有高度的冷冻敏感性,容易造成冷冻损伤[2]。因此,探索适合于鱼类卵细胞玻璃化冷冻保存技术,可为建立鱼卵细胞库提供技术与方法。本研究以鲫鱼Carassius cuvieri未成熟的卵细胞为材料,对几种毒性相对较低的渗透性抗冻剂的混合比例、玻璃化冻存对卵细胞存活率和琥珀酸脱氢酶(SDH)活性的影响进行研究,以期为鱼类卵细胞冷冻保存及损伤机制的研究提供实验依据。
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选取性发育成熟的雌性鲫鱼,用体积分数为75%乙醇消毒,剪开腹部,取出两侧卵巢,置于D-hank’s液中。去除卵巢周围的脂肪、结缔组织及血块等,撕开卵巢外膜并轻轻抖动,用移液管轻轻吹打卵细胞5~10次至卵细胞单个分离。用D-hank’s 溶液洗涤分离好的卵细胞2~3次[6]。
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实验根据本课题组已成功冻存胚脑[4]、软骨[5]等细胞的玻璃化溶液为基础进行改良。根据Plachinta等[6]、Zhang 等[7]的研究结果,选用了对鱼卵细胞毒性较低的1,2-丙二醇(PG)为主因子,辅以二甲基亚砜、甲醇、乙酰胺、蔗糖、海藻糖和聚乙二醇(平均分子量为4000)等保护剂成分,再根据章龙珍等[8]和 Zhang 等[9]研究的耐受极限浓度,通过预实验确定各组分的浓度范围,然后进行均匀设计,以是否含有海藻糖分成2 组,共11 种玻璃化溶液(表 1~2),并根据Guan等[1]、章龙珍等[10]和田永胜等[11]的方法进行玻璃化形成能力检测,各组均能形成玻璃化。
表 1 不含海藻糖的玻璃化溶液
Table 1. Vitrification solutions without trehalose
玻璃化溶液 渗透性保护剂 非渗透性保护剂 1,2-丙二醇/(g.L-1) 二甲基亚砜/(g.L-1) 甲醇/(g.L-1) 乙酰胺/(g.L-1) 蔗糖/(mol.L-1) 聚乙二醇/(g.L-1) VS1 100.0 120.0 140.0 150.0 0.4 50.0 VS2 120.0 160.0 120.0 140.0 0.7 20.0 VS3 140.0 200.0 100.0 130.0 0.3 60.0 VS4 160.0 100.0 150.0 120.0 0.6 30.0 VS5 180.0 140.0 130.0 110.0 0.2 70.0 VS6 200.0 180.0 110.0 100.0 0.5 40.0 表 2 含海藻糖的玻璃化溶液
Table 2. Vitrification solutions with trehalose
玻璃化溶液 渗透性保护剂 非渗透性保护剂 1,2-丙二醇/(g.L-1) 甲醇/(g.L-1) 海藻糖/(mol.L-1) 聚乙二醇/(g.L-1) VSa 120.0 140.0 0.05 60.0 VSb 140.0 130.0 0.15 20.0 VSc 160.0 120.0 0 80.0 VSd 180.0 110.0 0.10 40.0 VSe 200.0 150.0 0.20 100.0 -
将鲫鱼卵细胞在0 ℃下进行预平衡,采用三步平衡法:取100粒卵在1∶2稀释的玻璃化液中平衡10 min,再在1∶1稀释的玻璃化液中,平衡10 min后,取出转入玻璃化液中平衡10 min,分装入2.0 mL冷冻保存管中。
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将上述冷冻管进行标记,直接投入到液氮(-196 ℃)中保存,冻存时间为:1,2,3,4,6和8 d。
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从液氮中取出的冷冻管直接投入37 ℃的水浴中,充分振荡使其快速复温融化。
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复温融化后样品迅速转移到无菌离心管中。然后加入2倍体积的1.5 mol·L-1的蔗糖溶液,摇匀、静置10 min,去上清液,再加入2倍体积的0.5 mol·L-1的蔗糖溶液重悬浮,然后静置10 min,去上清液,加2倍体积的D-hank’s液重悬浮3次,去上清,加D-hank’s液摇匀备用。
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以台盼蓝拒染法检测细胞存活率。计算公式为: 细胞的存活率=(洗脱后的活细胞数/洗脱后的细胞数)×100%。
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根据Mosmann的方法[12]稍加改进,取冻存后洗脱的卵细胞于16孔无菌培养板中,加入1.0 mL 培养液,再加入5.0 g·L-1的 MTT溶液100 μL,放入37 ℃的50.0 mL·L-1二氧化碳培养箱中孵育240 min,使MTT充分被还原,然后加入1.0 mL二甲基亚砜,摇匀30 min,使结晶物充分溶解后,在酶标仪上测定570 nm处的吸光值。
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以上方法均随机选取多个样本,每个样本多次重复。采用Excel和SPSS软件对数据进行处理,利用单因素方差分析(one-way ANOVA)进行分析,比较结果用字母a,b,c,d,e,f在图中标记,图中在表示标准误差的误差线上方的字母相同表示差异不显著(P>0.05),字母不同表示差异显著(P<0.05)。
Effects of vitrification in crucian carp(Carassius cuvieri) oocytes
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摘要: 以鲫鱼Carassius cuvieri未成熟的卵细胞为材料,研究了玻璃化冻存对鲫鱼卵细胞的存活率及琥珀酸脱氢酶(SDH)活性的影响。结果表明:鲫鱼卵细胞存活率在1~6 d下降明显,6~8 d下降趋势减弱,保持相对稳定;琥珀酸脱氢酶活性呈显著下降(P<0.05)。玻璃化冻存液VSd组的冻存效果最好,其组分为180.0 g·L-1 1,2-丙二醇,110.0 g·L-1甲醇,0.1 mol·L-1 海藻糖和40.0 g·L-1 聚乙二醇。该玻璃化冻存液配方及方法在一定时间内保存鲫鱼未成熟卵细胞具有一定的应用价值。Abstract: The objectives were to analyze the effects of vitrification on the survival rate and succinate dehydrogenase(SDH) activity with immature oocytes in crucian carp(Carassius cuvieri). Results showed that the survival rate decreased significantly within 6 d after vitrification (P>0.05),and SDH activity continued to decrease(P<0.05). Glycol-based vitrification solutions(180.0 g·L-1 propylene glycol,110.0 g·L-1 methanol, 0.1 mol·L-1 trehalose and 40.0 g·L-1 polyethylene glycol) were selected for vitrification due to survival rates and SDH activity. This indicated that with this vitrification solution and method,cryopreservation for immature oocytes of crucian carp was practical.
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Key words:
- zoology /
- oocytes /
- vitrification solution /
- survival rate /
- succinate dehydrogenase (SDH) /
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表 1 不含海藻糖的玻璃化溶液
Table 1. Vitrification solutions without trehalose
玻璃化溶液 渗透性保护剂 非渗透性保护剂 1,2-丙二醇/(g.L-1) 二甲基亚砜/(g.L-1) 甲醇/(g.L-1) 乙酰胺/(g.L-1) 蔗糖/(mol.L-1) 聚乙二醇/(g.L-1) VS1 100.0 120.0 140.0 150.0 0.4 50.0 VS2 120.0 160.0 120.0 140.0 0.7 20.0 VS3 140.0 200.0 100.0 130.0 0.3 60.0 VS4 160.0 100.0 150.0 120.0 0.6 30.0 VS5 180.0 140.0 130.0 110.0 0.2 70.0 VS6 200.0 180.0 110.0 100.0 0.5 40.0 表 2 含海藻糖的玻璃化溶液
Table 2. Vitrification solutions with trehalose
玻璃化溶液 渗透性保护剂 非渗透性保护剂 1,2-丙二醇/(g.L-1) 甲醇/(g.L-1) 海藻糖/(mol.L-1) 聚乙二醇/(g.L-1) VSa 120.0 140.0 0.05 60.0 VSb 140.0 130.0 0.15 20.0 VSc 160.0 120.0 0 80.0 VSd 180.0 110.0 0.10 40.0 VSe 200.0 150.0 0.20 100.0 -
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链接本文:
https://zlxb.zafu.edu.cn/article/doi/10.11833/j.issn.2095-0756.2014.01.013