中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (2): 223-226.doi: 10.3969/j.issn.1673-8225.2010.02.008

• 软骨组织构建 cartilage tissue construction • 上一篇    下一篇

永生化人前软骨干细胞株的构建　

尹德龙，陈安民，郭风劲，王俊芳，程  浩   

  1. 华中科技大学同济医学院附属同济医院骨科，湖北省武汉市 430030
  • 出版日期:2010-01-08 发布日期:2010-01-08
  • 通讯作者: 程 浩， 博士，主治医师，华中科技大学同济医学院附属同济医院骨科，湖北省武汉市 430030 chenghao@tjh.tjmu.edu.cn
  • 作者简介:尹德龙☆，男，1982年生，天津市宁河县人，汉族，华中科技大学同济医学院在读博士，主要从事骨肿瘤研究。 yindelong607@sina.com
  • 基金资助:

    课题为国家自然科学基金项目(30650007)。

Construction of immortalized human precartilaginous stem cell lines

Yin De-long, Chen An-min, Guo Feng-jin, Wang Jun-fang, Cheng Hao   

  1. Department of Orthopedics, Tongji Hospital, Tongji Medical College, Huazhong University of Science & Technology, Wuhan   430030, Hubei Province, China
  • Online:2010-01-08 Published:2010-01-08
  • Contact: Cheng Hao, Doctor, Attending physician, Department of Orthopedics, Tongji Hospital, Tongji Medical College, Huazhong University of Science & Technology, Wuhan 430030, Hubei Province, China chenghao@tjh.tjmu.edu.cn
  • About author:Yin De-long☆, Studying for doctorate, Department of Orthopedics, Tongji Hospital, Tongji Medical College, Huazhong University of Science & Technology, Wuhan 430030, Hubei Province, China yindelong607@sina.com
  • Supported by:

     the National Natural Science Foundation of China, No. 30650007*

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摘要：

背景：前软骨干细胞体外培养增殖能力有限，但永生化细胞株可以提供大量性状稳定的永生化细胞，并且猿肾病毒40大T抗原基因(simian virus 40 large tumor antigen，SV40Tag)是较为常用且有效的体外永生化细胞的基因片段之一。
目的：以SV40Tag转染的人前软骨干细胞构建永生化人前软骨干细胞株。
方法：采用酶消化法及免疫磁珠筛选技术分离纯化人胚胎前软骨干细胞。利用脂质体介导基因转染技术将含有SV40Tag的质粒pCMVSV40T/PUR转染原代胚胎前软骨干细胞，以未转染细胞作阴性对照。阳性克隆扩大培养，观察细胞形态学以及传代复苏情况，计算细胞存活率和群体倍增时间，绘制细胞生长曲线。以免疫荧光细胞化学技术检测永生化人前软骨干细胞成纤维生长因子受体3，以RT-PCR检测永生化人前软骨干细胞及人前软骨干细胞中SV40Tag和成纤维生长因子受体3表达。
结果与结论：贴壁培养的永生化人前软骨干细胞形态与原代人前软骨干细胞形态无明显差别。传代、冻存、复苏对人永生化前软骨干细胞的存活率无影响,第6，10代人前软骨干细胞的存活率降低(P < 0.01)。与第6，10代人前软骨干细胞相比，永生化人前软骨干细胞增殖较旺盛，群体倍增时间短、增殖率高(P < 0.01)。第2代永生化人前软骨干细胞成纤维生长因子受体3染色阳性, 成纤维生长因子受体3的RT-PCR结果显示在约400 bp处有一特异形扩增条带，SV40Tag的RT-PCR结果显示在约560 bp处有一特异形扩增条带, 未转染的原代细胞未见条带。提示以免疫磁珠筛选技术及脂质体转染技术成功构建了SV40Tag永生化人前软骨干细胞株。

关键词: 人前软骨干细胞, 永生化, SV40Tag, 软骨组织工程

Abstract:

BACKGROUND: The precartilaginous stem cells are limited regarding in vitro proliferative capacity, but the immortalized cell lines can provide a large number of stable immortalized cells, and simian virus 40 large T antigen gene (SV40Tag) is one of gene fragments which are commonly used and effective in vitro immortalized cells.
OBJECTIVE: To construct human immortalized precartilaginous stem cells (IPSCs) using human precartilaginous stem cells induced by SV40LTAg gene.
METHODS: The human immortalized precartilaginous stem cells were isolated from aborted fetus and purified with enzyme digestion and immunomagnetic beads screening method. By using liposome-mediated gene transfection technology, plasmid pCMVSV40T/PUR containing SV40Tag was transfected in primary embryonic precartilaginous stem cells, while non-transfected cells served as negative controls. Positive clones were cultured to observe the cell morphology and the passage recovery, to calculate cell survival rate and population doubling time, to draw cell growth curve. Immunofluorescence cytochemistry was used to detect the expression of IPSCs fibroblast growth factor receptor 3, the expressions of SV40Tag and fibroblast growth factor receptor 3 in the human precartilaginous stem cells were determined by RT-PCR.
RESULTS AND CONCLUSION: Morphology of human IPSCs seemed coincidence with primary human precartilaginous stem cells. The survival rate of human IPSCs was not influenced by subculture, freezing and recovery, but the survival rate was descended in the human precartilaginous stem cells at the 6th and 10th passages (P < 0.01). Compared with cells at the 6th and 10th passages, the proliferation of human IPSCs was greater, with short population doubling time and high growth rate (P < 0.01). The immunofluorescence showed that fibroblast growth factor receptor 3 was positive in human IPSCs at the second passage, and the RT-PCR results of fibroblast growth factor receptor 3 revealed a specific amplification band at 400 bp, while that of SV40Tag revealed at 560 bp. No band was seen in the primary cells. It is indicated that SV40Tag human IPSCs can be constructed successfully using immunomagnetic bead screening technology and liposome transfection technique.

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