兔胎儿成纤维细胞的分离培养和鉴定

doi:10.11843/j.issn.0366-6964.2016.06.025

畜牧兽医学报 ›› 2016, Vol. 47 ›› Issue (6): 1272-1279.doi: 10.11843/j.issn.0366-6964.2016.06.025

兔胎儿成纤维细胞的分离培养和鉴定

魏如雪，赵学明，郝海生，杜卫华，朱化彬^*

(中国农业科学院北京畜牧兽医研究所，北京 100193)

收稿日期:2015-12-14 出版日期:2016-06-23 发布日期:2016-06-23
通讯作者: 朱化彬，博士，研究员，主要从事家畜胚胎工程与繁殖研究，E-mail：zhuhuabin@caas.cn
作者简介:魏如雪（1991-），女，山东德州人，硕士生，主要从事动物遗传育种与繁殖研究，E-mail：704446770@qq.com
基金资助:
家畜胚胎工程与繁殖创新团队（ASTIP-IAS06-2016）；国家自然科学基金面上项目（31472100）

Isolation，Culture and Identification of Rabbit Embryonic Fibroblasts

WEI Ru-xue，ZHAO Xue-ming，HAO Hai-sheng，DU Wei-hua，ZHU Hua-bin^*

（Institute of Animal Science，Chinese Academy of Agricultural Sciences，Beijing 100193，China）

Received:2015-12-14 Online:2016-06-23 Published:2016-06-23

摘要/Abstract

摘要：

本研究旨在建立稳定传代的兔胎儿成纤维细胞系及其鉴定方法。取配种后14~15 d的兔胎儿，0.25%胰酶消化胎儿皮肤获得兔胎儿成纤维细胞，体外传代培养；通过观察细胞形态、测定生长曲线、免疫荧光染色、核型分析等鉴定兔胎儿成纤维细胞，PCR扩增SRY基因鉴定2株细胞系性别。研究结果表明，兔胎儿成纤维细胞呈梭形，可在体外快速生长，符合“潜伏期-对数期-平台期”的生长规律；免疫荧光染色结果显示，波形蛋白与纤维连接蛋白染色为阳性，细胞角蛋白染色为阴性，表明兔胎儿成纤维细胞纯度高，活性好；染色体检测结果显示，在传至第5代时，80%的成纤维细胞核型正常，为2n=44，属正常范畴；PCR法检测的2株细胞系均来源于兔雄性胎儿。综上表明，本研究建立了兔胎儿成纤维细胞系体外培养、传代、鉴定及其性别鉴定方法，可为转基因兔、克隆兔、兔诱导性多能性干细胞等研究提供充足、可靠的成纤维细胞来源。

Abstract:

This research was conducted to establish the stable rabbit embryonic fibroblast cell lines and identification methods. Rabbit embryonic fibroblasts were isolated from 14-15 d rabbit fetus by trypsin enzyme digesting skin tissues, and passaged in vitro steadily. Morphology observation, growth curve measurement, immunofluorescence and karyotype analysis were applied to cell lines identification. The sex of 2 cell lines were determined by amplifying core sequence of rabbit SRY gene. The results showed that rabbit embryonic were spindle shaped and fibroblasts grew fast in vitro, conforming to the pattern of “latent phase-logarithmic growth phase-stagnate phase”. Immunofluorescence assay showed that vimentin and fibronectin staining were positive, while cytokeratin staining was negative, indicated the high purity and activity of rabbit embryonic fibroblast cells. The result of chromosome analysis showed that 80% ploidy of one cell lines maintained normal (2n=44) when cultured up to passage 5. For cell lines sexing, SRY-PCR analysis suggested the 2 determined cell lines were male. These results indicated that our research could be used to establish rabbit embryonic fibroblast cell lines and determine sex, providing adequate and reliable fibroblast cells for transgenesis, cloning and iPS rabbit research.

中图分类号:

魏如雪，赵学明，郝海生，杜卫华，朱化彬. 兔胎儿成纤维细胞的分离培养和鉴定[J]. 畜牧兽医学报, 2016, 47(6): 1272-1279.

WEI Ru-xue，ZHAO Xue-ming，HAO Hai-sheng，DU Wei-hua，ZHU Hua-bin. Isolation，Culture and Identification of Rabbit Embryonic Fibroblasts[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(6): 1272-1279.

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兔胎儿成纤维细胞的分离培养和鉴定

Isolation，Culture and Identification of Rabbit Embryonic Fibroblasts

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