版纳微型猪近交系成纤维细胞系的建立及冻存方法研究

The Establishment and Study on Cryopreservation Method of Fibroblast Cell Line of Banna Mini-pig Inbred Line

本试验旨在观察胶原酶对版纳微型猪近交系（Banna mini-pig inbred line,BMI）耳组织的消化效果,探讨不同冻存方法对耳组织成纤维细胞存活率的影响.用胶原酶Ⅳ消化法培养BMI 60日龄仔猪原代耳组织成纤维细胞,建立耳组织成纤维细胞系,比较-80℃条件下分别保存3,5,7,30 d后冻存效果.结果表明：BMI仔猪成纤维细胞具有典型的成纤维细胞特征;生长曲线呈“S”形,倍增时间为37.1h;-80℃冻存3,5,7d的成纤维细胞存活率与液氮保存（93.74％）差异不显著（P＞0.05）,冻存30 d的存活率（79.38％）显著低于其他各组（P＜0.05）,但经48 ～ 72 h培养后仍能贴壁生长.因此,可得出此结论：采用胶原酶Ⅳ消化法可消化BMI耳组织成纤维细胞,而且该细胞在-80℃条件下能进行短期保存.

This experiment was conducted to study the digestion effect of collagenase on Banna minipig Inbred Line （BMI） ear tissue and the effect of different cryopreservation methods on viable rate of fibroblast. We cultured the primary ear fibroblast cell by using collagenase Ⅳ digestion of 60 day old piglet of BMI, and established the ear fibroblast cell line. And then, we compared the effects of cryopreservation under -80 ℃ for different time （3, 5, 7, 30 d）. The results showed that the fibroblast of BMI exhibited typical fibroblast morphology. The growth curve was ＂S＂ shape and the doubling time was 37. 1 h. The survival rate of fibroblast cell in these groups of - 80 ℃ frozen for 3,5, 7 d showed no significant difference with liquid nitrogen preservation group （97.41%） （P 〉0. 05）. The survival rate in the group of -80 ℃ frozen for 30 d was significantly lower than that of other groups （P 〈 0. 05 ）, but these cells could still adherent growth after culture for 48 - 72 h. In conclusion, collagenase Ⅳ could be used to digest ear fibroblast of BMI and these cells could be cryopreserved under -80 ℃ for a short time.