摘要
目的 构建温度敏感型永生化的骨髓间充质干细胞（ tsSV40LTag - BMSCs）.观察不同温度条件下,tsSV40LTag - BMSCs的增殖情况.方法 分离、培养大鼠原代骨髓间充质干细胞（BMSCs）,FITC标记CD29、CD31、CD44、CD45、CD90抗体,CD34单克隆抗体荧光标记大鼠BMSCs,流式细胞仪检测其表面标志.pRNS -1质粒转染BMSCs以及阳性克隆的筛选和扩增.RT - PCR检测BMSCs中tsSV40LTag的表达.测定33℃和37C两种温度下,tsSV40LTag - BMSCs生长曲线.结果 成功构建tsSV40LTag - BMSCs.33℃时,tsSV40LTag - BMSCs生长增殖速度较快,在体外培养条件下可连续传代增殖；37℃时,细胞生长增殖速度明显减慢,甚至停止生长.结论 tsSV40LTag- BMSCs的成功构建,为其移植治疗颅脑创伤奠定了实验基础.
Objective Bone marrow mesenchymal stem cells of temperature sensitive simian virus 40 large T antigen gene （tsSV40LTag - BMSCs ） was constructed successfully.The proliferation of tsSV40LTag - BMSCs at different temperatures was observed.Method Isolation and cultivation of primary BMSCs from SD rats was performed.Antibodies of CD29,CD31,CD44,CD45 and CD90 marked by FITC and CD34 were added to the cell suspensions respectively.Identification of surface markers of BMSCs was performed by flow cytometry.pRNS - 1 plasmid transfection,screening and positive amplification were done.RT - PCR was used to explore the expression of tsSV40LTag in BMSCs.Growth curve of tsSV40LTag -BMSCswas determined at temperature of 33℃ and 37℃.Results TsSV40LTag was transfected to BMSCs of SD rats with pRNS - 1 plasmid.TsSV40LTag - BMSCs grew fast at 33℃,serial passage was available in vitro culture.But cells grew significantly slowly at 37℃,even stopped growing.Conclusions The model of tsSV40LTag- BMSCs is set up successfully,which provides experimental proof for tsSV40LTag - BMSCs transplantation treatment against TBI.
出处
《中华神经外科杂志》
CSCD
北大核心
2012年第1期88-92,共5页
Chinese Journal of Neurosurgery
基金
天津市科技发展计划项目（06YFSZSF01200）
国家自然科学基金（30872668）
关键词
骨髓
间充质干细胞
转染
Bone marrow
Mesenchymal stem cells
Transfection
