承德无角山羊耳成纤维细胞系的建立与冷冻保存研究

Establishment and Cryopreservation of Chengde Hornless Goat Ear Fibroblast Cell Line

【目的】试验以甜菜碱作为冷冻保护剂对承德无角山羊耳成纤维细胞系进行冷冻保存,旨在优化冷冻保护效果,以期在细胞水平上长期保存这一地方品种家畜的遗传资源。【方法】采用组织块培养法建立承德无角山羊耳成纤维细胞系,利用甜菜碱作为冷冻保护剂对其进行玻璃化冷冻,以不同浓度(5%、10%、15%、20%及25%)甜菜碱为试验组,以不同浓度(10%和20%)二甲基亚砜(DMSO)为对照组,对承德无角山羊耳成纤维细胞进行冷冻,复苏后检测细胞的存活率、增殖活力以及细胞中B淋巴细胞瘤-2 (Bcl-2)/Bcl-2相关X蛋白(Bax)基因表达量,同时评估甜菜碱的毒性。【结果】本研究建立的承德无角山羊耳成纤维细胞系生长态势良好,细胞纯度较高,细菌、真菌和支原体3类微生物检测结果均为阴性,生长曲线呈S型,达到了建立成纤维细胞系的要求;当甜菜碱浓度为15%时细胞存活率最高,达90.86%,极显著高于对照组(P<0.01),细胞增殖活力及Bcl-2/Bax基因表达量均显著高于对照组(P<0.05),且甜菜碱的毒性远低于传统冷冻保护剂DMSO(P<0.05)。【结论】试验成功以甜菜碱作为单独的冷冻保护剂对承德无角山羊耳成纤维细胞系进行低温冷冻保存,从而保护了承德无角山羊的遗传资源。

【Objective】In this study,betaine was used as a cryoprotectant to cryopreserve Chengde Hornless goat ear fibroblast cell lines for optimizing the cryopreservation effect,so as to preserve the genetic resources of this local breed at the cellular level.【Method】The ear fibroblast cell line of Chengde Hornless goat was established by tissue block culture method.Vitrification of fibroblasts with betaine as a cryoprotectant,different concentrations of betaine(5%,10%,15%,20%and 25%)were used as the experimental group,and different concentrations of dimethyl sulfoxide(DMSO,10%and 20%)were used as the control group.Cryopreservation of Chengde Hornless goat ear fibroblasts was performed,the cell survival rate,cell proliferative activity and the expression of B-cell lymphoma-2(Bcl-2)/Bcl-2 related X protein(Bax)genes were measured after cell resuscitation,and the toxicity of betaine was also assessed.【Result】Chengde Hornless goat ear fibroblast cell line established in this study had good growth trend and high cell purity.The result of bacteria,fungi and mycoplasma were negative,and the growth curve was S type,which met the requirements of establishing fibroblast cell line.When the concentration of betain was 15%,the survival rate of cells was the highest,reaching 90.86%,which was extremely significantly higher than that of control group(P<0.01).The cell proliferation activity and the expression of Bcl-2/Bax genes were significantly higher than those in control group(P<0.05),and the toxicity of betain was much lower than that of DMSO(P<0.05).【Conclusion】In this study,betaine was successfully used as a cryoprotectant to cryopreserve the ear fibroblast cell line of Chengde Hornless goat,so as to protect the genetic resources of Chengde Hornless goat.