黑白花奶牛乳腺上皮细胞冻存方法的研究

Research on the Cryopreservation Method of Holstein Cow Mammary Gland Epithelial Cells

本试验研究了北方地区黑白花奶牛乳腺上皮细胞的最佳冻存方法。采用内蒙古地区健康黑白花奶牛的新鲜乳腺组织,将组织进行Ⅱ型胶原酶消化后原代培养,培养的细胞经5代纯化后将密度调整为1×106个/mL。采用6种处理方法进行冻存,各设3个重复。液氮冻存1个月后,分别将6种不同方法冻存的细胞复苏后测定细胞活性,包括冻存死亡率、贴壁细胞占活细胞的比率、凋亡率等,同时进行形态学观察。结果显示,处理1组与2、3、5组间冻存死亡率存在极显著差异(P<0.01),处理1、4、6组间冻存死亡率无显著差异(P>0.05),处理2、6组间冻存死亡率存在极显著差异(P<0.01);处理4组的贴比率高于处理2、5、6组,存在极显著差异(P<0.01)。形态学观察结果发现,处理1、4组细胞较其它组生长状态良好。因此,处理4可作为黑白花奶牛乳腺上皮细胞的最佳冻存方法。

The effects of different cryopreservation methods of Holstein cow mammary gland epithelial cells on its viability were studied.Primary bovine mammary gland epithelial cells were passing five generations and adjusting the cell density to 1×106cells/mL,and than being cryopreserved by 6 different methods.The viability of cells include mortality,adhering dish rate,apoptosis rate was measured after being recovered.The results showed that the death rate of cell being cryopreserved by treatments 2,3,5 was significant different from treatment 1（P〈0.01）.The difference of death rate of cell being cryopreserved by treatments 1,4,6 was no significant（P〉0.05）.Death rate of cryopreserved cell from treatments 2 and 6 was significant different（P〈0.01）.Adhering rate of cryopreserved cell from treatment 4 was significant higher than treatments 2,5 and 6（P〈0.01）.Cell growth state of treatments 1 and 4 were better than others.The results indicated that the treatment 4 was the best method in this study.