摘要
目的建立温度敏感型永生化神经前体细胞系,鉴定其细胞生长特点及表达抗原的特性。方法利用逆转录病毒感染原代培养的胚胎13d SD大鼠中脑神经前体细胞,建立温度敏感型永生化神经前体细胞系RMN,利用免疫细胞化学、Western blot和流式细胞分析方法测定RMN细胞的特征。结果在33℃条件下,RMN细胞呈多边形,有较短突起;细胞增殖迅速,群体倍增时间为48 h,可连续传代和冷冻保存;细胞表达SV40大T抗原,nestin蛋白和Nurr1蛋白;RMN细胞中未发现染色体倍体的异常,裸鼠接种20周后未观察到肿瘤的形成。在39℃条件下,RMN细胞增殖速度减缓,细胞形成较长突起。在含1%胎牛血清的培养基中培养1周后,RMN细胞可表达-βIII-tubulin,GFAP和Gal C等抗原,但SV40抗原表达减弱。结论RMN细胞系是SV40/tsA58基因永生化的大鼠神经前体细胞系,是具有温度敏感性及多分化潜能的前体细胞系。
Objective To establish immortalized neural precursors with a temperature-sensitive mutant of SV40 large T antigens and characterize the properties of the ceils. Methods Permanent rat neural precursor cell line （RMN） was developed following infection of primary cultures from embryonic day- 13（ E- 13） Sprague- Dawley rat ventral mesencephalon with a retrovirus encoding the temperature-sensitive mutant of SV40 large T antigens. RMN ceils were characterized in vitro for cellular and properties using immunocytochemistry, Western blotting etc. Results At 33 ℃, the permissive temperature, RMN cells divided with a doubling time of 48h and showed to be polygonal with short processes. Co-expression of SV40 large T antigen and nestin, one of the neural stem cell markers in the cells was determined by immunocytochemistry. As well, Nurrl was confirmed in the cells by Western blotting. These cells could be stored for long term in liquid nitrogen. The RMN cells showed a normal karyotype tested by FACS analysis of propidium iodide-stained DNA and no tumor mass was detected at 20 weeks after the cells were subcutaneously transplanted into nude mice. At 39℃ , the nonpermissive temperature, the processes of the cells increased in length, their morphology became more complex, and their rate of growth was decreased. After treatment with medium contained with 1% fetal bovine serum,these cells expressed neural differentiation markers, β-Ⅲ-tubulin, GFAP and Gal C, the expression of SV40 large T antigen was decreased. Conclusion The RMN cell line is an immortalized neural precursor cell line with temperature-sensitivity.
出处
《解剖学报》
CAS
CSCD
北大核心
2005年第5期491-497,共7页
Acta Anatomica Sinica
基金
国家重点基础研究计划(G1999054008)
北京市自然科学基金重点项目(7011001)
北京市科委重点项目(9555101200)
