绒山羊皮肤干细胞的初步分离及碱性磷酸酶鉴定

›› 2012, Vol. 39 ›› Issue (10): 154-159.

绒山羊皮肤干细胞的初步分离及碱性磷酸酶鉴定

苏小虎¹, 高峰², 刘迎春¹, 王宏¹, 张焱如¹, 周欢敏¹

1. 内蒙古农业大学生命科学学院,内蒙古呼和浩特 010018;
2. 内蒙古农业大学动物科学学院,内蒙古呼和浩特 010018

收稿日期:2012-03-23 出版日期:2012-10-20 发布日期:2012-10-19
通讯作者: 刘迎春。 E-mail:liuyingchun1994@yahoo.com.cn高峰。E-mail:gaofeng1994@yahoo.com.cn E-mail:liuyingchun1994@yahoo.com.cn; gaofeng1994@yahoo.com.cn
作者简介:苏小虎(1986-),男,山西人,硕士生,研究方向:发育生物学。
基金资助:
国家自然科学基金(30901016,31160445);内蒙古自然科学基金(20080404MS040);中国科学院"西部之光"人才项目资助项目。

Primary Isolation of Stem Cells from Cashmere Goat Skin and Identification of AKP

SU Xiao-hu¹, GAO Feng², LIU Ying-chun¹, WANG Hong¹, ZHANG Yan-ru¹, ZHOU Huan-min¹

1. College of Life Sciences,Inner Mongolia Agricultural University, Hohhot 010018,China;
2. College of Animal Sciences,Inner Mongolia Agricultural University, Hohhot 010018,China

Received:2012-03-23 Online:2012-10-20 Published:2012-10-19

摘要/Abstract

摘要： 以0.02%胰酶4 ℃过夜消化,分离表皮,37 ℃消化30 min,打成单细胞悬液,经100 μg/mL Ⅳ型胶原处理的培养皿黏附10 min,除掉未黏附的细胞,加入培养基(80% DMEM-F12+20% FBS+氢化可的松(25 μg/mL)+青霉素(100 IU/mL)+链霉素(100 μg/mL)+胰岛素(15 μg/mL)+转铁蛋白+EGF(20 μg/mL))培养24 h,而后将此细胞消化接种到经20 μg/mL丝裂霉素C处理4 h的成年绒山羊成纤维细胞滋养层上,培养2 周后有各种形态的克隆状细胞集落出现,用碱性磷酸酶(AKP)染色呈深黑紫色,初步判断细胞呈阳性。本研究旨在分离绒山羊皮肤干细胞,为研究干细胞分化机制,探索绒毛发育机理,培育高产高质绒毛性状奠定分子育种理论基础。

关键词: 绒山羊; 干细胞; 分离; 鉴定

Abstract: The skin sample of cashmere goat was incubated in 0.02% trypsin at 4 ℃ overnight and the epidermis was separated,then they were placed in asterile tube containing 0.25% trypsin at 37 ℃ for 30 minutes and the dissociated cells were adhered with collagen type Ⅳ to culture dishes for 10 minutes. 10 minutes later, the unattached cells were removed and the rapidly adherent epidermal cells were cultured in stem cell growth medium(80% DMEM-F12 + 20% FBS + 25 μg/mL hydrocortisone + 100 IU/mL penicillin + 100 μg/mL streptomycin + 15 μg/mL insulin + transferring + 20 μg/mL EGF) for 24 h. And then dissociated cells were cultured on layer cells which the adult cashmere goat fibroblast were treated with 20 μg/mL mitomycin C for 4 h. After 2 weeks, these cells could form different shapes of clones. Dyed with AKP, it showed deep purple and cells were judged as positive.

Key words: cashmere goat; stem cell; isolation; identification

中图分类号:

S813.1⁺1

苏小虎, 高峰, 刘迎春, 王宏, 张焱如, 周欢敏. 绒山羊皮肤干细胞的初步分离及碱性磷酸酶鉴定[J]. , 2012, 39(10): 154-159.

SU Xiao-hu, GAO Feng, LIU Ying-chun, WANG Hong, ZHANG Yan-ru, ZHOU Huan-min. Primary Isolation of Stem Cells from Cashmere Goat Skin and Identification of AKP[J]. , 2012, 39(10): 154-159.

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