新生猪骨髓间充质干细胞衍生细胞的分离、培养及生物学特性分析

doi:10.13560/j.cnki.biotech.bull.1985.2015.06.027

生物技术通报 ›› 2015, Vol. 31 ›› Issue (6): 170-176.doi: 10.13560/j.cnki.biotech.bull.1985.2015.06.027

新生猪骨髓间充质干细胞衍生细胞的分离、培养及生物学特性分析

阮征^1，2 王莲芳¹ 胡修忠¹ 吴建英² 张四化² 代长云² 华娟¹ 夏瑜¹ 胡小明³ 李杰¹ 黄海军^1，4

1.武汉市畜牧兽医科学研究所，武汉 430208；2. 武汉市重大动物疫病防控中心，武汉 430023；3.华中农业大学猪遗传育种农业部重点实验室&农业动物遗传育种与繁殖教育部重点实验室，武汉 430070；4.武汉市畜牧兽医局，武汉 430023

收稿日期:2014-09-19 出版日期:2015-06-19 发布日期:2015-06-20
作者简介:阮征，男，高级兽医师，研究方向：动物疫病防控；E-mail：paper2006@sina.cn 并列第一作者：王莲芳，女，畜牧师，研究方向：动物科学技术与推广；E-mail：hygene@qq.com
基金资助:
国家自然科学基金项目(31201938)，武汉市晨光计划(201150431078)，武汉市农科院英才计划(YC201101)

Isolation，Culture and Characterization of Derived Cells from Neonatal Porcine Bone Marrow Mesenchymal Stem Cells

Ruan Zheng^1，2, Wang Lianfang¹, Hu Xiuzhong¹, Wu Jianying², Zhang Sihua², Dai Changyun², Hua Juan¹, Xia Yu¹, Hu Xiaoming³, Li Jie¹, Huang Haijun^1，4

1.Wuhan Institute of Animal and Veterinary Science，Wuhan 430208；2. Wuhan Animal Disease Prevention and Control Center，Wuhan 430023；3. Key Laboratory of Swine Breeding and Genetics of Ministry of Agriculture & Key Laboratory of Agricultural Animal Genetics，Breeding and Reproduction of Ministry of Education，Huazhong Agricultural University，Wuhan 430070；4. Wuhan Bureau of Animal Husbandry and Veterinary，Wuhan 430023

Received:2014-09-19 Published:2015-06-19 Online:2015-06-20

摘要/Abstract

摘要： 利用骨髓间充质干细胞(Bone mesenchymal stem cells，BMSCs)治疗疾病已经逐渐成为现实，但是作为被移植的种子细胞，BMSCs体外传代能力非常有限，种子细胞来源极为贫乏。本研究通过差速贴壁筛选的方法分离出一种猪BMSCs的衍生细胞株，命名为猪骨髓间充质干细胞衍生细胞(Bone mesenchymal stem-derived cells，BMSDCs)。分别对BMSDCs与BMSCs细胞进行细胞生物学特性分析，探讨其体外诱导分化特性，并应用流式细胞术测定细胞表面标记物。结果表明，BMSC和BMSDCs细胞倍增时间分别为31.3 h和30.3 h，平均传代时间分别为3-5 d和 2-3 d；两种细胞均阳性表达 CD34、CD90，阴性表达CD44、CD45；经体外诱导后均可分化为成脂细胞和成肌细胞。在传代能力上，前者可传代15至20次，后者可长期传代(200次以上)且维持正常染色体特征。研究认为在适宜的实验条件下，体外培养的猪骨髓间充质干细胞的衍生细胞——BMSDCs能够稳定生存增殖并维持BMSCs多向分化潜能，可作为组织工程的理想种子细胞。

关键词: 骨髓间充质干细胞, 衍生细胞, 猪, 生物学特性, 组织工程

Abstract: Recently it has become possible to use bone marrow mesenchymal stem cells(BMSCs)in the clinical treatment of certain diseases. However, BMSCs, the seed cells used in transplantation, are limited in vitro proliferation capability and can only be obtained from very few sources. In order to acquire a substitute for BMSCs, the method of differential velocity adherent screening was used in this study to isolate a derived strain of porcine BMSCs, named bone marrow mesenchymal stem-derived cells(BMSDCs). The biological characteristics of BMSDCs and BMSCs cells were compared by continuous morphological imaging by inverted microscope, and the growth curves of both cells were monitored by the MTT method. Furthermore, their characteristics of differentiation in vitro were examined, and flow cytometry was used to measure cell surface markers. Our results suggest that the doubling times of BMSCs and BMSDCs are 31. 3 h and 30. 3 h, respectively, and the average passaging time is 3-5 d and 2-3 d, respectively. Cultured BMSCs and BMSDCs are both positive for CD34 and CD90 and negative for CD44 and CD45. Both BMSCs and BMSDCs can differentiate into adipocytes and myoblasts by induced differentiation in vitro. Concerning their passaging abilities, the former can have 15 to 20 passages in vitro, whereas latter can have a longer period of time(more than 200 passages)while normal karyotypes are still maintained. We conclude that, under certain experimental circumstances, cultured porcine BMSDCs in vitro can survive and proliferate while maintaining multi-directional differentiation potential of BMSCs, and potentially it can be an ideal type of seed cell for tissue engineering.

Key words: bone marrow mesenchymal stem cell, derived cell, porcine, biological characteristics, tissue engineering

阮征, 王莲芳, 胡修忠, 吴建英, 张四化, 代长云, 华娟, 夏瑜, 胡小明, 李杰, 黄海军. 新生猪骨髓间充质干细胞衍生细胞的分离、培养及生物学特性分析[J]. 生物技术通报, 2015, 31(6): 170-176.

Ruan Zheng, Wang Lianfang, Hu Xiuzhong, Wu Jianying, Zhang Sihua, Dai Changyun, Hua Juan, Xia Yu, Hu Xiaoming, Li Jie, Huang Haijun. Isolation，Culture and Characterization of Derived Cells from Neonatal Porcine Bone Marrow Mesenchymal Stem Cells[J]. Biotechnology Bulletin, 2015, 31(6): 170-176.

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新生猪骨髓间充质干细胞衍生细胞的分离、培养及生物学特性分析

Isolation，Culture and Characterization of Derived Cells from Neonatal Porcine Bone Marrow Mesenchymal Stem Cells

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