梅花鹿鹿茸软骨细胞和间充质干细胞永生化细胞株的构建及鉴定

摘要

Isolated sika deer antler chondrocytes and mesenchymal stem cells,constructed immortalization cell lines by SV40LT lentiviral vector and identification.The lentiviral vector containing SV40 LT cDNA was transfected into 293T cells to obtain the virus particles.Virus infected antler chondrocytes and mesenchymal stem cells.Morphological observation,cell proliferation,real-Time PCR,toluidine blue staining and flow cytometry were used to detect the expression ofSV40 LT antigen and cell properties.The immortalized cells established in this experiment can be passage steady and have strong proliferation activity in vitro.The expression of SV40 LT in immortalized cells was higher than primary cells.The immortalized cells have the fundamental properties of primary cell.Immortalized antler chondrocytes and mesenchymal stem cell lines were successful construction.It laid the foundation for the follow-up deer antler growth and development research.%分离梅花鹿鹿茸软骨细胞和间充质干细胞,使用SV40 LT抗原慢病毒载体建立永生化软骨细胞和间充质干细胞系并鉴定.将含有SV40LT基因片段的慢病毒载体,转染人胚肾细胞293T获得包装后的病毒粒子,感染鹿茸软骨细胞及间充质干细胞,连续传代培养,通过形态观察、细胞增殖、real-time PCR、甲苯胺蓝染色法和流式细胞术等方法检测SV40 LT抗原表达以及细胞性质.实验所建立的永生化鹿茸软骨细胞系与间充质干细胞系能够稳定传代并具有较强的体外增值活性.RT-PCR检测到SV40T抗原的表达,通过甲苯胺蓝染色法和流式细胞术鉴定所得细胞系具有原代细胞的基本性质.成功获得永生化的软骨细胞与间充质干细胞系,为后续鹿茸生长及发育研究奠定了基础.