鸡胚精原干细胞体外保存能力的研究

采用二酶3步法获取孵化第19 d的鸡胚精原干细胞（SSCs）,比较在快速冷冻条件下3种冷冻保护剂（DMSO、乙二醇、甘油）在3个浓度5%、10%、15%条件下对鸡胚精原干细胞的冷冻保存效果进行研究。结果显示：（1）当DMSO的浓度为5%、10%及15%时,复苏后细胞存活率分别为73.1%、88.6%和74.8%,三者差异显著（P〈0.05）;而10%DMSO保存精原干细胞的存活率、复苏后培养细胞生长和集落形成均显著高于其它2个浓度;当乙二醇浓度为5%、10%和15%时,复苏后细胞存活率分别为69.4%、83.1%和65.2%,三者差异显著（P〈0.05）;复苏后无论饲养层存在与否,SSCs均能增殖,但未有AKP阳性集落生成;当甘油浓度为5%、10%、15%时,复苏后细胞存活率均小于15%,三者差异不显著（P〉0.05）;复苏后细胞存活时间极短,仅存活12 h左右;（2）当在10%DMSO浓度条件,复苏后SSCs在有饲养细胞层的条件下,培养5 d形成集落,表明10%DMSO是鸡胚精原干细胞适宜的冷冻保护剂。

Cryopreservation Capacity of Chicken Spermatogonial Stem Cells

To isolate spermotogonia using two combinatorial enzyme three step wises digest the testicular tissues from 19 d-old-embryo after hatching,the cryopreserve effect of dimethylsulphoxide(DMSO),glycerol(GLY) and ethylene glycol(EG) cryoprotectants,each at(5%,10%,and 15%) concentration on the 19 d chicken embryonic SSCs was compared with fast freeze method.The results indicated that:(1) When SSCs frozen in 5%,10% and 15% DMSO medium,the viability rate of after thawed SSCs were 73.1%,88.6% and 74.8% respectively,the difference of three concentration were significant(P<0.05).When SSCs frozen in 5%,10% and 15% EG medium,the viability rate of after thawed SSCs were 69.4%,83.1% and 65.2% respectively,and the difference of three concentration were significant(P<0.05).When SSCs frozen in 5%,10% and 15% glycerin medium,the viability rate were less than 15%,and difference of three concentration were not significant(P>0.05);(2) The SSCs were cryopreservated in 10% DMSO for 1-2 weeks,then thawed and seeded on the feed cells to culturing,the chicken embryonic fibroblast cells as feed cells.The results found that the SSCs had formed the colonies which were positive after AKP staining.On the contrary,when the SSCs were cultured without feed cells,the colonies couldn't form. The results showed that the 10% DMSO was suitable freezing media for the 19 d SSCs from chicken embryo.