中国组织工程研究与临床康复2008，Vol.12Issue(43)：8588-8592,5.
新生大鼠前软骨干细胞株的体外培养分选、鉴定及永生化研究
Separation,identification and immortalization of precartilaginous stem cells from neonatal rats
摘要
Abstract
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.关键词
SV40T/干细胞/软骨/细胞培养/组织构建分类
基础医学引用本文复制引用
胡伟华,郭风劲,陈安民,张树威..新生大鼠前软骨干细胞株的体外培养分选、鉴定及永生化研究[J].中国组织工程研究与临床康复,2008,12(43):8588-8592,5.基金项目
国家自然科学基金面上项目(30571872) （30571872）
