医学论文-大鼠骨骺干细胞的分离鉴定及其永生化细胞株的构建.docVIP

医学论文-大鼠骨骺干细胞的分离鉴定及其永生化细胞株的构建 【摘要】? 分离、鉴定大鼠骨骺干细胞并建立永生化的大鼠骨骺干细胞株，为细胞移植和转基因治疗提供稳定的细胞来源。［方法］采用Percoll不连续密度梯度离心法分离骨骺干细胞，利用电穿孔转染技术将含有猿肾病毒40大T抗原基因(SV40Tag)的质粒pCMVSV40T／PUR转染骨骺干细胞，经嘌呤霉素筛选，抗性克隆扩大培养。应用FGFR-3抗体和PCNA抗体进行免疫细胞化学染色，观察细胞的形态及其生长状况并绘制细胞生长曲线。用免疫细胞化学法和RT-PCR检测SV40Tag在转染细胞中的表达。［结果］转染后获得一个阳性细胞克隆，免疫细胞化学结果显示FGFR-3抗体染色阳性。SV40Tag抗体染色和RT-PCR结果显示SV40Tag已稳定转染入骨骺干细胞。转染细胞经扩大培养，命名为永生化骨骺干细胞。［结论］成功纯化大鼠骨骺干细胞并构建了SV40Tag永生化的骨骺干细胞株。 【关键词】? 骨骺干细胞 永生化 猿肾病毒40 ????? Abstract：［Objective］To separate rat precartilaginous stem cells (PCSCs) and establish immortalized precartilaginous stem cell(IPCSC) strain which provides stable cell resource for cell-transplantation and gene therapies.［Method］PCSCs were isolated by discontinuous Percoll gradient centrifugation. The specific marker (anti-FGFR-3)? of? PCSCs? was? used? to? identify? the? cultured? cells? by immunocytochemistry. Plasmids pCMVSV40T/PUR containing the simian virus 40 large T antigen gene (SV40Tag) were transfected into the PCSCs by electroporation method. Colonies were isolated by puromycin selection and amplified to cell strain.The expression of SV40Tag in the amplified cell strain was identified by immunocytochemistry and RT-PCR. Anti-FGFR-3 and anti-PCNA were used to identify the cultured cells after puromycin selection.［Result］One anti-puromycin cell clone was obtained, which was confirmed as FGFR-3 positive PCSCs with the capability of proliferation. The mRNA and protein? expression s of SV40Tag were detected in transfected cells after stable transfection. The transfected cells were amplified to immortalized cell strain maintained for more than 30 passages, named immortalized precartilaginous stem cells(IPCSCs). The population doubling time and anti-PCNA positive confirmed the high capability of proliferation.［Conclusion］Rat precartilaginous stem cells were purifued and immortalized precartilaginous stem cell strain was established successfully. It will provide stable cell resource for the basic researches and cell-transplant