摘要
藏猪是青藏高原特有的生态猪种,是我国珍贵的地方种质资源。为了保护藏猪种质资源,本试验通过组织块贴壁法和胰酶消化法对藏猪耳部成纤维细胞进行分离培养、传代与冻存,绘制其生长曲线,使用台盼蓝染色法进行细胞活力检测,并进行真菌细菌、病毒和支原体污染检测。结果显示,两种方法均长出梭形、纺锤形或星形的成纤维细胞,发现使用组织块贴壁法培养成纤维细胞比胰酶消化法更易生长、形态学特征和活力更好,冻存复苏的细胞可以正常贴壁生长和传代;其生长曲线为典型的“S”型,潜伏期存在3 d,对数生长期从第4 d开始持续到第8 d,从第9 d开始进入生长抑制期;台盼蓝染色检测活力发现冻存前后成纤维细胞的生长活率在90%以上,且发现不存在真菌细菌、病毒和支原体污染。本试验成功对藏猪耳组织成纤维细胞进行分离纯化培养与冻存,可为体外藏猪克隆提供材料,并为完善藏猪成纤维细胞种质资源库奠定基础。
The Xizang pig is a unique ecological pig breed in Qinghai-Xizang Plateau and a precious local germplasm resource in China.In order to protect the germplasm resources of Xizang pigs,the ear fibroblasts of Xizang pigs were isolated,cultured,subcultured and cryopreserved by tissue block adhesion method and trypsin digestion method,their growth curve was drawn.Trypan blue staining was used to detect cell viability and fungal bacteria,virus and mycoplasma contamination.The results showed that spindle-shaped,spindle-shaped or star-shaped fibroblasts were grown by both methods.It was found that fibroblasts cultured by tissue attachment method were easier to grow,morphological characteristics and vitality were better than trypsin digestion method.Cryopreserved resuscitated cells can grow and passage normally.The growth curve was typical“S”type,the incubation period existed for 3 days,the logarithmic growth period lasted from the 4th day to the 8th day,and entered the growth inhibition period from the 9th day.Trypan blue staining showed that the growth rate of fibroblasts was more than 90%before and after cryopreservation,and there was no fungal bacteria,virus and mycoplasma contamination.The successful isolation,purification,culture and cryopreservation of Xizang pig ear fibroblasts in this experiment can provide materials for Xizang pig cloning in vitro and lay a foundation for perfecting the germplasm bank of Xizang pig fibroblasts.
作者
刘博华
王运路
尼玛群宗
刘楠
次仁德吉
加布
徐业芬
罗桑多杰
LIU Bohua;WANG Yunlu;Nima qunzong;LIU Nan;Ciren deji;Jiabu;XU Yefen;Luosang duojie(College of Animal Science,Xizang Agricultural and Animal Husbandry College,Linzhi Xizang,860000,China;Xizang Autonomous region Animal Husbandry General Station,Lasa Xizang,850000,China;Agricultural and Rural Bureau of Gongbujiangda County,Linzhi Xizang,860200,China;Agriculture and Animal Husbandry Service Center,Dongru Township,Ritu County,Ali Prefecture Xizang,859000,China)
出处
《高原农业》
2024年第5期533-539,共7页
Journal of Plateau Agriculture
基金
第三次全国畜禽遗传资源普查项目
西藏自治区科技厅区域协同创新专项(QYXTZX-NQ2022-04)
西藏自治区科技厅重点项目(XZ202001ZY0044N)。
关键词
藏猪
耳部组织成纤维细胞
分离培养
活力测定
生长曲线
Xizang pigs
Ear tissue fibroblasts
Isolation and culture
Activity determination
Growth curve
