达氏鲟精巢细胞消化分离和超低温冷冻保存

摘要

In this study, to obtain high recovery ratio of living cells after thawing for Dabry′s sturgeon ( Acipenser dabrya-nus) testicular cells, we evaluate the effect of enzymatic dissociation on testicular cells, and the influence of cryoprotecta-nts, freezing ratio, sugars and lecithin on testicular cells cryopreservation. Testes were dissociated and digested with 0. 25% trypsin or a combination of 2 mg/mL collagenase H and 500 U/mL Dispase II. The number of testicular cells was counted respectively when tests were dissociated by these two enzymes for 2, 3, 4 and 5 h. The result showed that there was no significant difference between those enzymes. Per gram testis can obtain 1. 27 × 107 living cells dissociated by 0. 25% trypsin and 1. 07 × 107 by the combined enzymes on average. We also investigate the effect of cryoprotectants, freezing rate, sugars and lecithin on testicular cells cryopreservation. Cells was diluted in 50 mM D-trehalose, 5 mg/mL Bovine serum albumin, 200 mL/L fetal bovine serum (pH 8. 0) supplemented with 10% dimethyl sulfoxide (DMSO), ethylene glycol ( EG) or methanol ( MET) respectively, freezing on a cooling protocol from 0 ℃ to -80 ℃at a rate of -1 ℃/min, or transferring into liquid nitrogen ( LN2 ) directly. The results demonstrated that cryopreservation with those cryoprotectants significantly influenced recovery rate of living cells, in the following sequence: EG(51. 70% ± 5. 24%) >MET(45. 09% ± 3. 15%) >DMSO(40. 18% ± 3. 90%). And freezing on slow rate, -1 ℃/min, showed obviously high rate of living cells after thawing. Based on the previous study, D-trehalose in the extenders was replaced by D-su-crose, and supplemented 0%, 5%, 8%, 11% lecithin. Almost the same viability rates were obtained with no signifi-cant differences among tested sugars, but the differences performed obviously on lecithin. The results showed that (93. 55 ± 2. 56)% living cells can be obtained after thawing when 8% lecithin was supplemented. The number of testicular cells increased 3. 19 times for 10 days culture.%通过研究两种酶对精巢细胞的消化效果，探究抗冻剂、降温程序、糖类和卵磷脂对达氏鲟( Acipenser dab-ryanus)精巢细胞冻存效果的影响，获得消化冻存后高存活率的细胞。实验使用0．25%胰蛋白酶和2 mg/mL胶原酶 H+500 U/mL中性酶II的组合酶对达氏鲟精巢细胞进行消化，获得不同消化时间内的活细胞数量。另外，还研究了冷冻稀释液中分别添加10%二甲基亚砜( DMSO)、乙二醇( EG)、甲醇( MET)作为抗冻剂，采用-1℃/min慢速降温以及直接投入液氮中的快速降温方法冻存，冷冻稀释液采用D-海藻糖或同浓度D-蔗糖，以及添加5%、8%、11%卵磷脂对冻存效果的影响。结果显示：两种消化酶在同一时间消化所得的活细胞数和活细胞率无显著差异，并且都在3 h获得最多活细胞。慢速降温的冻存效果极显著地好于快速降温( P=0．01)。不同抗冻剂的保存效果差异显著，复苏后细胞相对存活率 EG (51．70%±5．24%)>MET (45．09%±3．15%)>DMSO (40．18%±3．90%)。不同糖对达氏鲟精巢细胞冻存效果无显著影响；不同浓度的卵磷脂冻存效果有极显著差异。含8%卵磷脂的冻存液对细胞的冻存效果最好，细胞存活率可达(93．55±2．56)%，培养10 d后细胞数目为0 d时的3．19倍。