SV40LTag诱导鼠骨骺软骨细胞稳定分化的实验研究

Immortalization of rat epiphysis cartilage cells induced by simian virus 40 large T antigen gene transfection

目的建立稳定分化大鼠骨骺软骨细胞株,为细胞替代治疗和基因治疗小儿生长发育迟缓提供稳定的细胞来源。方法利用脂质体介导的基因转染技术将含有猿肾病毒40大T抗原(simian virus 40 large T antigen gene,SV40LTag)基因的质粒pEGFP-IRES2-SV40LTag转染原代培养的新生大鼠骨骺软骨细胞,G418筛选,抗性克隆扩大培养传代。应用II型胶原、X型胶原和SV40LTag抗体进行细胞鉴定,体外检测其分化能力,观察细胞的形态及其生长状况,绘制细胞生长曲线。用RT-PCR、Southern blot和免疫细胞化学法鉴定SV40LTag在转染细胞中的表达。结果转染后获得了阳性细胞克隆,免疫细胞化学证实为具有较强增殖能力和多分化潜能的骨骺软骨细胞。经Southern印迹杂交证实,SV40LTag已稳定转染入骨骺软骨细胞,表达mRNA及其蛋白。结论SV40LTag导入可诱导骨骺软骨细胞稳定分化,为细胞替代治疗和基因治疗小儿生长发育迟缓等疾病提供稳定的细胞来源。

Objective To establish immortalized epiphysis cartilage cell strains in order to provide a stable cell resource for cell substitution and gene therapies of growth retardation. Methods Plasmid pEGFP-IRES2-SV40LTag containing simian virus 40 large T antigen gene was transfected into primarily cultured epiphysis cartilage cells of the newborn rat using the lipefectin transfection method. Colonies were isolated by G418 selection and cultured to immortalized cell strains. Fibroblast growth factor receptor-3 （ FGFR-3 ）, anti-collagen type II and type X antibodies were used to identify cultured cells and to investigate the capability of differentiation of the transfected cells. SV40LTag expression in expanded cell strains was identified by RT-PCR, Southern blot and immunocytochemistry method. Results Anti-G418 cell clone was obtained, which was confirmed as FGFR-3 positive epiphysis cartilage cells with the capability of stable proliferation. mRNA and protein of SV40LTag were expressed in transfected cells after stable transfection. The transfected cells were expanded to immortalized cell strains and named as immortalized epiphysis cartilage cells. The immortalized cells were elliptic or triangular, with two or three short axons. The immortalized epiphysis cartilage cell strains had stable biological characters. Conclusions SV40LTag gene transfection can immortalize epiphysis cartilage cells. The establishment of FGFR-3 positive immortalized epiphysis cartilage cell strains may provide a stable cell resource for cell substitution and gene therapies of growth retardation.