鸡胚胎原始生殖细胞对不同冷冻体系适合性的研究

COMPARISON ON THE EFFECTIVENESS OF DIFFERENT CRYOPROTECTANTS WITH CHICKEN EMBRYO PRIMORDIAL GERM CELLS

目的研究鸡胚胎原始生殖细胞(EPGCs)对不同冷冻体系的适合性。方法对发育至第19期和第28期胚胎性腺PGCs,采用二甲基亚砜(DMSO)、乙二醇、聚乙二醇为冷冻保护液,在不同组合、不同浓度、不同的冷冻程序下进行超低温冷冻保存。结果1.采用同一种冷冻保护液,10%浓度与15%浓度之间,除DMSO+乙二醇(冷冻保护液Ⅳ)外,其他类型的冷冻液对EPGCs保存后存活率的影响差异不显著(P>0.05);20%浓度的各种冷冻保护液对EPGCs保存后的存活率最低,且与10%和15%浓度相比差异显著(P<0.05)或极显著(P<0.01);2.采用同一种冷冻保护液,浓度为10%时,冷冻程序Ⅰ(2~4℃平衡45~60 min,-4^-6℃诱发结晶45 min,悬挂液氮面1 h以上,投入液氮保存)和冷冻程序Ⅱ(2~4℃平衡90~120 min,悬挂液氮面1 h以上,投入液氮保存)对EPGCs冷冻后存活率差异显著(P<0.05)或极显著(P<0.01);3.采用冷冻程序Ⅰ,浓度为10%时,不同的冷冻保护液对EPGCs保存后的存活率影响显著(P<0.05)或极显著(P<0.01),DMSO+乙二醇(冷冻保护液Ⅳ)显示出最好的保护效果;4.第19期和第28期性腺中的EPGCs在相同的冷冻条件下冷冻后,存活率差异不显著(P>0.05)。结论采用冷冻程序Ⅰ,在10%的DMSO+乙二醇(冷冻保护液Ⅳ)的保护下对鸡EPGCs冷冻较为适合。

Objective This paper focused on comparing the effectiveness of different cryoprotectants for chicken embryo primordial germ cells （EPGCs）, Methods To isolate EPGCs from gonads at stage 19 and stage 28 by ficoll density-gradient centrifugation, Then the PGCs were freezed in various freezing media, concentration and program. Results The results showed 1.using the same program, concentration, the viability of the frozen-thawed PGCs showed significant difference （ P 〈 0.05） or very significant difference （ P 〈 0.01 ） among cryoprotectant Ⅰ , cryoprotectant Ⅱ , cryoprotectant Ⅲ , cryoprotectant Ⅳ , cryoprotectant Ⅴ , cryoprotectant Ⅵ . 2. In the same cryoprotectant, the viability of the frozen-thawed EPGCs showed no significant difference between the concentration of 10% and 15% （ P 〉 0.05） except cryoprotectant Ⅳ . When the concentration was 20% , the viability of the frozen-thawed PGCs was the lowest, and showed significant difference（ P 〈 0.05） or very significant difference （ P 〈 0.01） compared to other concentration.3. Under the concentration of 10%, in the same cryoprotectant, the viability of the frozen-thawed EPGCs showed significant difference（ P 〈 0.05） or very significant difference（ P 〈 0.01 ） between the different frozen program. 4. In the same frozen program and freezing media, the viability of the frozen-thawed EPGCs showed no significant difference between stage 19 and stage 28.Conclusion The 10% cryoprotectant Ⅳ with the program Ⅰ was suitable for freezing chicken EPGCs