南阳牛骨髓间充质干细胞的分离培养及多向分化

doi:10.16431/j.cnki.1671-7236.2015.09.025

《中国畜牧兽医》---唯一指定的官方网站 ›› 2015, Vol. 42 ›› Issue (9): 2377-2383.doi: 10.16431/j.cnki.1671-7236.2015.09.025

南阳牛骨髓间充质干细胞的分离培养及多向分化

朱雪敏, 位兰, 张自强, 邓雯, 孙宇, 赵前宁, 王梦凡

河南科技大学动物科技学院, 洛阳 471000

收稿日期:2015-01-22 出版日期:2015-09-20 发布日期:2015-09-25
通讯作者: 朱雪敏 E-mail:lyzxm7501@163.com
作者简介:朱雪敏(1975-),女,河南洛阳人,副教授,研究方向:组织胚胎学及发育生物学
基金资助:
河南科技大学博士启动基金(402513480062)

Isolation,Culture and Multiple Differentiation Induction of Nanyang Bovine Bone Marrow Mesenchymal Stem Cells

ZHU Xue-min, WEI Lan, ZHANG Zi-qiang, DENG Wen, SUN Yu, ZHAO Qian-ning, WANG Meng-fan

College of Animal Science and Technology, Henan University of Science and Technology, Luoyang 471000, China

Received:2015-01-22 Online:2015-09-20 Published:2015-09-25

摘要/Abstract

摘要： 本研究旨在建立南阳牛骨髓间充质干细胞(bone marrow-derived mesenchymal stem cells,BMSCs)体外分离培养方法,在此基础上研究其生物学特性和多向分化的能力。采用骨髓穿刺法取3月龄小牛的肋骨骨髓,分离培养BMSCs,传代培养并测定其生长曲线,RT-PCR检测Oct4、Nanog、Sox2基因的表达,然后取P3 BMSCs分别向神经和脂肪细胞进行诱导分化,并利用组织学染色技术和RT-PCR技术进行鉴定。结果表明,分离得到的BMSCs大小均匀,多呈梭形的成纤维细胞样生长;RT-PCR可检测到干细胞因子Oct4、Nanog、Sox2的表达;不同代次细胞生长曲线呈S型,一般在第3天时进入指数生长期,第7天后进入平台期;成神经诱导后,甲苯胺蓝染色可见明显的尼氏体结构,RT-PCR检测ENO2和GFAP基因表达呈阳性;成脂肪诱导后,油红O染色后可见大量的脂滴存在,RT-PCR检测Leptin和PPAR基因表达呈阳性。试验证明,成功分离得到了南阳牛BMSCs,且其具有多向诱导分化潜能。

关键词: 南阳牛; 骨髓间充质干细胞; 干细胞培养; 诱导分化

Abstract: The study aimed to build the method of Nanyang bovine bone marrow-derived mesenchymal stem cells (BMSCs) separation culture in vitro, and study its bionomics and the ability of multiple differentiation induction on this basis. Using bone marrow puncture to take rib bone marrow of 3 months calf, isolation and culture BMSCs, subcultured and determined its curve of growth, detected Oct4, Nanog, Sox2 genes expression by RT-PCR technique, then took P3 BMSCs into neural and fat cells to carry out induction differentiation, and took the use of histology staining technique and RT-PCR technique to detect. The results of the BMSCs were uniform sindleshaped in appearance; RT-PCR could dectect the expression of stem cell factor Oct4, Nanog, Sox2. The results showed that an "S" shape growth curve of cell at different generations times, it entered exponential growth stage at the 3rd day generally, and entered plateau after then the 7th day. Into nerve after induction, stained with toluidine blue was apparent throughout the structure of Nissl substance, ENO2 and GFAP genes showed a positive expression. After adipogenic differentiation of ASCs was assessed by oil Red O staining which showed a large number of lipiddroplet, RT-PCR dectected Leptin and PPAR genes showing a positive expression. The tests showed that had successfully isolated from Nanyang bovine BMSCs and it had the potential of multidirectional differentiation.

Key words: Nanyang bovine; BMSCs; stem cell culture; differentiation

中图分类号:

S839.2

朱雪敏, 位兰, 张自强, 邓雯, 孙宇, 赵前宁, 王梦凡. 南阳牛骨髓间充质干细胞的分离培养及多向分化[J]. 《中国畜牧兽医》---唯一指定的官方网站, 2015, 42(9): 2377-2383.

ZHU Xue-min, WEI Lan, ZHANG Zi-qiang, DENG Wen, SUN Yu, ZHAO Qian-ning, WANG Meng-fan. Isolation,Culture and Multiple Differentiation Induction of Nanyang Bovine Bone Marrow Mesenchymal Stem Cells[J]. , 2015, 42(9): 2377-2383.

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南阳牛骨髓间充质干细胞的分离培养及多向分化

Isolation,Culture and Multiple Differentiation Induction of Nanyang Bovine Bone Marrow Mesenchymal Stem Cells

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