背景:前软骨干细胞虽然具有较强的自我增殖能力和多向分化潜能,但生物学性状不稳定,易分化.导入外源性基因能使其永生化,且不改变细胞的表型和性状.目的:建立永生化大鼠前软骨干细胞株,为以后精确调控前软骨干细胞的增殖与分化打下基础.设计、时间及地点:单一样本观察,于2005-10/2006-09在华中科技大学同济医学院附属同济医院骨科实验室完成.材料:出生＜24 h的新生SD大白鼠,雌雄不限,用于取前软骨干细胞.方法:用LipofectamineTM2000介导基因转染,将含有SV40T抗原基因的真核表达载体pCMVSV40T/PUR导入经免疫磁珠分选出的原代前软骨干细胞进行稳定表达,用嘌呤霉素筛选出阳性克隆并扩大培养.主要观察指标:①前软骨干细胞的生物学性状.②质粒鉴定.③用免疫细胞化学方法和反转录聚合酶链反应鉴定SV40T抗原基因在转染细胞中的表达.④反转录聚合酶链反应结果.⑤细胞生长曲线.结果:①免疫磁珠分离获得细胞阳性克隆,用免疫组织化学证实FGFR-3表达阳性.②双酶切质粒,电泳证实pCMV为3 kb,SV40T基因为2.3 kb.③嘌呤霉素分离获得转化后细胞阳性克隆,用免疫组织化学证实FGFR-3表达阳性.④提取RNA后用反转录-聚合酶链反应法成功扩增出588 bp的片段.转染细胞经扩大培养,命名为永生化前软骨干细胞.⑤贴壁培养的转染细胞群体倍增时间为(22.98±2.77) h,传代、冻存和复苏对细胞形态及生长无明显影响.结论:在体外培养条件下,可以从新生大鼠干骺端中分离、培养出前软骨干细胞.pCMVSV40T/PUR转染能使其永生化.%BACKGROUND: Precartilaginous stem cells (PSCs) have strong proliferation ability and differentiation potential,but they are instable and prone to differentiate.Importing exogenous gene could immortalize them and leave phenotype character unchanged.OBJECTIVE: To establish immortalized precartilaginous stem cells (PSCs) from neonatal SD rats in vitro for the further related research about the differentiation mechanism and clinical application of precartilaginous stem cells.DESIGN,TIME AND SETTING: Single sample observation.The study was carried out in the Department of Orthopedics.Tongji Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology from October 2005 to September 2006.MATERIALS: Neonatal SD rats,irrespective of gender,24-hour old,were used for prepare PSCs.METHODS: By using LipofectamineTM 2000,a gene transfection reagent,plasmid pCMVSV40T/PUR containing the simian virus 40 large T antigene gene (SV40Tag) was transfected into the primary cultured PSCs isolated by immuniomagnetic beads coasted with the second antibody.Colonies were isolated by puromycin selection and expanded by many passages.MAIN OUTCOME MEASURES: Biological character of PSCs; plasmid identification; biological character of transfected cells and identification; RT-PCR; growth curve.RESULTS: Immunomagnetic beads separation system obtains PSCs,which was confirmed as fibroblast growth factor receptor-3 (FGFR-3) positive PSCs.Double restriction enzyme was cut,electrophoresis confirmed pCMV was 3 kb,SV40T was 2.3 kb.A particular anti-puromycin cell clone was acquired,which was confirmed as FGFR-3 positive PSCs.The total RNA was isolated from the positive cell clones,and a 588 bp fragment,which was specific for the SV40T antigene gene,was amplified.The transfected cells were expanded to immortalized cell strain,named as immortalized precartilaginous stem cells (IPSCs).Thepopulation doubling time of IPSCs was (22.98±2.77) hours,no significant effect of subculture,freezing and recovering had been found.CONCLUSION: Precartilaginous stem cells could be isolated from neonatal SD rats,cultured in vitro,and immortalized through the transfection of pCMVSV40T/PUR.
展开▼
