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 virus40large 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400bp处有一特异形扩增条带,SV40Tag的RT-PCR结果显示在约560bp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。
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 reveale
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2010年第2期223-226,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
国家自然科学基金项目(30650007)~~
