Wang Beibei, Zou Xifeng, Zhang Haiyan, Duan Deyi, Ju Lili, Jiang Xiaohua, Sun Xiaohong, Zhao Chunli, Zhao Huanying, Guo Jin, Xu Changlei, Gao Erjing, Xu Qunyuan
Beijing Institute for Neuroscience, Capital Medical University, Beijing Center of Neural Regeneration and Repair, Key Laboratory of Neurodegenerative diseases of Ministry of Education of China, Beijing 100069, China.
Brain Res. 2008 May 19;1210:63-75. doi: 10.1016/j.brainres.2008.02.062. Epub 2008 Mar 4.
Effective cell replacement therapies for neurological disease require neuron-restricted precursors as grafted cells. The problem of obtaining sufficient grafts for transplantation can be resolved by creating an appropriate immortalized cell line. In the present study, a thermally controlled immortalized GABAergic neuronal progenitor cell line (RMNE6) was established from E13 rat ventral mesencephalon cells immortalized using the temperature-sensitive mutant of SV40 large T antigen (ts-TAg). RMNE6 cells proliferated rapidly and expressed a neuron-like phenotype at the permissive temperature (33 degrees C), but eventually stopped growing at the non-permissive temperature (39 degrees C). Expression of the neuronal markers PSA-NCAM, beta-tubulin III and MAP2 by RMNE6 cells was confirmed by RT-PCR or immunocytochemistry. Furthermore, these cells exhibited functional GABAergic neuron properties, as evidenced by the expression of glutamate decarboxylase (GAD) as well as the synthesis and release of the neurotransmitter GABA in a calcium-dependent manner. Moreover, RMNE6 cells spontaneously expressed and secreted several neurotrophic factors, such as NGF, BDNF, NT-3, NT-4/5, and GDNF. The cells survived well and kept expression of SV40 Tag, GAD65/67 and GABA in the striatum, at least 28 days after being transplanted in the rat brain. Tumorigenesis assays confirmed the safety of the immortalized cell line in vivo. Taken together, the results support the use of RMNE6 cells as an ideal cell model for transplantation research aimed at the treatment and prevention of neurodegenerative disease.
用于神经疾病的有效细胞替代疗法需要将神经元限制性前体细胞作为移植细胞。通过创建合适的永生化细胞系可以解决获得足够移植用移植物的问题。在本研究中，利用SV40大T抗原的温度敏感突变体（ts-TAg）使E13大鼠腹侧中脑细胞永生化，从而建立了一种热控永生化GABA能神经元祖细胞系（RMNE6）。RMNE6细胞在允许温度（33℃）下迅速增殖并表现出神经元样表型，但在非允许温度（39℃）下最终停止生长。通过RT-PCR或免疫细胞化学证实了RMNE6细胞表达神经元标志物PSA-NCAM、β-微管蛋白III和MAP2。此外，这些细胞表现出功能性GABA能神经元特性，谷氨酸脱羧酶（GAD）的表达以及神经递质GABA以钙依赖方式的合成和释放证明了这一点。而且，RMNE6细胞自发表达并分泌几种神经营养因子，如NGF、BDNF、NT-3、NT-4/5和GDNF。在大鼠脑内移植至少28天后，这些细胞在纹状体中存活良好并保持SV40 Tag、GAD65/67和GABA的表达。致瘤试验证实了该永生化细胞系在体内的安全性。综上所述，这些结果支持将RMNE6细胞用作旨在治疗和预防神经退行性疾病的移植研究的理想细胞模型。
