摘要
旨在研究高效鸡囊胚细胞(Blastodermal cells,BCs)冷冻保存技术,为利用BCs保存禽类雌性遗传资源以及胚胎干细胞研究奠定基础。采用解冻后二乙酰荧光素(Fluorescein diacetate,FDA)染色以及培养24h后细胞贴壁率双重检测BCs活力方法,比较二甲基亚砜(DMSO)不同浓度、不同冷冻与解冻速率等对BCs的冷冻保存效果。结果表明:(1)DMSO不同浓度,20%组解冻后活力最高(0.58),与15%组差异不显著(P>0.05),但与5%、10%和25%组差异极显著(P<0.01);细胞复苏后贴壁率,20%组最高(30.5%),与15%组差异不显著(P>0.05),与25%组差异显著(P<0.05),与5%和10%组差异极显著(P<0.01)。(2)冷冻速率,使用三步法冷冻,以-1℃·min-1的速率降温至-7℃,保持10min,继续降温分别至-15、-35、-55、-75℃后,投入液氮中保存,-75℃组解冻后活力最高(0.53),但与-35℃、-55℃组差异不显著(P>0.05);贴壁率,各组间差异极显著(P<0.01),其中-35℃组最高(36.6%)。(3)37℃水浴解冻,10s、1min、2min、3min解冻后,各组间活力差异不显著(P>0.05),其中10s组最高(0.60),解冻后贴壁率,37℃水浴1min组结果最好(43.9%),与水浴2min组差异不显著(P>0.05),但与水浴10s组差异显著(P<0.05),与水浴3min组差异极显著(P<0.01)。50℃水浴5s、20s、40s、1min解冻,5s组活力最高(0.70),与20s组差异显著(P<0.05),与40s、1min组差异极显著(P<0.01),解冻后贴壁率,各组间差异均极显著(P<0.01),其中50℃水浴5s组最高(36.9%),50℃水浴1min组最低(5.6%)。综上表明,鸡BCs使用DMSO浓度为20%的冷冻保护剂进行细管冷冻,以-1℃·min-1的速率降温至-7℃保持10min,继续以此速率降温至-35℃后,投入液氮保存,37℃水浴1~2min解冻,BCs通过FDA染色与体外培养贴壁率双重检测均可获得较好效果。
For developing efficient cryopreservation technology of chicken blastodermal cells(BCs)and storing female poultry genetic resources,the BCs was frozen using dimethyl sulfoxide(DMSO)and straw,and the different DMSO concentration,freezing and thawing rate were compared by calculating the cell viability(CV)using fluorescein diacetate(FDA)and cell adherent rate cultivated for 24h(CAR).Results showed that:(1)For the concentration of DMSO,the highest CV was in 20% group(0.58)and had no significant difference with group 15%(P>0.05),but had extremely significant difference with group 5%,10% and 25%(P<0.01).Thehighest CAR was 30.5%in group 20%,and had no significant difference with group 15%(P>0.05),but had significant difference with group 25%(P<0.05),and had extremely significant with group 5%,10%(P<0.01).(2)The 3steps to freeze BCs was used,and the first step was cooling at a rate of-1 ℃·min-1 to-7 ℃ and kept 10 min,the second step was to continue cooling the temperature at-15,-35,-55or-75 ℃ respectively,the third step was to plunge the straws into liquid nitrogen.The highest CV was 0.53 of group-75℃,and had no significant difference(P>0.05)to group-35or-55 ℃.The CAR had extremely significant difference within each groups(P<0.01),and the highest was 36.6%in group-35 ℃.(3)The thawing rate at 37 ℃ water bath for 10 s,1min,2min and 3min,the CV of each groups had no significant difference(P>0.05),the highest one was 0.60 in 10sgroup.The CAR was 43.9%in group 1min,and it had no significant difference(P>0.05)with group 2 min,had significant difference(P<0.05)with the group 10 sand had extremely significant difference(P<0.01)with group 3min.The thawing rate of 50℃ water bath for 5s,20 s,40sor 1min,the highest CAR was 0.7in group 5s,and it had significant difference(P<0.05)with group 20 s,had extremely significant difference(P<0.01)with group 40 sand 1min.The CAR thawed at 50℃ had extremely significant difference within the 4groups(P<0.01).The highest CAR was 36.9%in group 5s,and the lowest was 5.6%in group 1min.In conclusion,the BCs frozen using 20% DMSO and straw,cooling at-1 ℃·min-1 to-7 ℃ for 10 min,continuing to cool at-35 ℃ at the same ratio and plunging into liquid nitrogen,then thawed by 37 ℃ water bath for 1-2 min,could get not only good CV,but also CAR.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2015年第10期1775-1783,共9页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
国家国际科技合作专项(2013DFG31810)
湖南省科技计划(2014WK2008)
长沙市科技计划(K1307134-24)
关键词
鸡
囊胚细胞
DMSO
细管
冷冻保存
chicken
blastodermal cells
DMSO
straw
cryopreservation
