北京鸭表皮干细胞的分离培养

生物技术通报 ›› 2013, Vol. 0 ›› Issue (11): 93-97.

北京鸭表皮干细胞的分离培养

吕春荣¹, 熊慧^1，2, 宫雪莲¹, 关伟军¹, 马月辉¹

1.中国农业科学院北京畜牧兽医研究所，北京 100193；2. 佳木斯大学，佳木斯 154000

收稿日期:2013-04-24 出版日期:2013-11-14 发布日期:2013-11-14
作者简介:吕春荣，女，硕士，研究方向：动物细胞及分子生物学；熊慧为并列第一作者，E-mail：gzblxinghui@163.com
基金资助:
国家高技术研究发展计划资助项目(“863”计划)(2007AA10Z170)，转基因生物新品种培育科技重大专项(2008ZX08009-003)，国家科技支撑项目(2008BADB2B01)，“十一五”国家科技支撑计划重点项目(2008BAK41B01-5)

Separation and Culture of Beijing Duck Epidermal Stem Cells

Lü Chunrong¹, Xiong Hui^1，2, Gong Xuelian¹, Guan Weijun¹, Ma Yuehui¹

(1. Institute of Animal Science，Chinese Academy of Agricultural Sciences，Beijing 100193；2. Jiamusi University，Jiamusi 154000)

Received:2013-04-24 Published:2013-11-14 Online:2013-11-14

摘要/Abstract

摘要： 旨在研究北京鸭表皮干细胞(epidermal stem cells，ESCs)的分离培养方法及其生物学特性。采用组织块贴壁法和酶消化法分离北京鸭表皮干细胞，对分离得到的细胞进行了免疫荧光鉴定，并进行了细胞凋亡检测与外源基因转染研究。结果表明，细胞为贴壁生长，体积小，核质比高，在显微镜下折光性强、胞体透亮。北京鸭ESCs免疫荧光检测显示，细胞表达β1 integrin与K19。细胞凋亡检测结果显示，北京鸭ESCs凋亡的细胞很少，凋亡率仅为6.5%。脂质体介导pEGFP-N3、pEYFP-N1和pDsRed-N1基因转染北京鸭ESCs，转染后48-96 h阳性细胞明显增多、强度增强，其中转染48 h时的阳性细胞最多，转染率最高可达56%。转染细胞的形态、生长速度无明显改变，转入的荧光蛋白种类不同，转染效率也略有差异。结果显示，体外培养的细胞为表皮干细胞，其凋亡率低，并且能够应用于外源基因转染研究。

关键词: 北京鸭, 表皮干细胞, 分离培养

Abstract: This experiment was conducted to study the separation methods and the biological characteristics of epidermal stem cells(ESCs). Beijing Duck epidermal stem cellswere respectively obtained by enzymic digestion and tissue piece culture, then the cultured cells were identified by immunohistochemistry. We detected the apoptosis of ESCs and studied the transfection efficiency of exogenous genes including pEGFP-N3、pEYFP-N1 and pDsRed-N1. Results showed that the ESCs obtained from the two methods both exhibited adherent growth type, small volume, high nucleus-cytoplasm ratio, high refractivity and transparent cell appearance. Immunofluorescence assay indicated that Beijing Duck ESCs expressed β1 integrin and K19, not expressed K10. The results of apoptosis showed that the apoptosis rate was only 6.5%. The study of transfection experiment indicated that the amount of positive cells and its intensity increased after 48-96 h, among which, 48 h group had the most positive cells with the transfection efficiency up to 56%. The fluorescent protein gene(pEGFP-N3, pDsRed-N1 and pEYFP-N1)expression had no obvious effect on the growth and proliferation of the ESCs. The transfection efficiency was different in the different fluorescent genes. These results indicated that the cultured cells were indeed ESCs. The cells cultured in vitro owned low apoptosis ratio, and can be used in the research of exogenous genes transfection.

Key words: Beijing duck, Epidermal stem cells, Separation and culture

吕春荣, 熊慧, 宫雪莲, 关伟军, 马月辉. 北京鸭表皮干细胞的分离培养[J]. 生物技术通报, 2013, 0(11): 93-97.

Lü Chunrong, Xiong Hui, Gong Xuelian, Guan Weijun, Ma Yuehui. Separation and Culture of Beijing Duck Epidermal Stem Cells[J]. Biotechnology Bulletin, 2013, 0(11): 93-97.

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