Whittemore S R, White L A
Miami Project, University of Miami School of Medicine, FL 33136.
Brain Res. 1993 Jun 25;615(1):27-40. doi: 10.1016/0006-8993(93)91111-5.
Following infection of dissociated embryonic day 13 rat medullary raphe cells with a retrovirus encoding the temperature-sensitive mutant of SV40 large T antigen, a clonal cell line, RN33B, was isolated by serial dilution. At 33 degrees C, RN33B cells divide with a doubling time of 48 h and show T antigen, vimentin, nestin, diffuse neuron-specific enolase, and low and medium molecular weight neurofilament immunoreactivities. RN33B cells are immortal, but not transformed, as they will not grow in soft agar. At non-permissive temperature (38.5 degrees C), T antigen expression is markedly decreased and RN33B cells cease mitotic activity and differentiate with phase bright cell bodies and 'neuritic-like' processes. Differentiated RN33B cells express enhanced neuronal-specific protein expression but do not synthesize astrocytic or oligodendrocytic-specific proteins. Moreover, differentiated RN33B cells returned to 33 degrees C re-express T antigen, but do not de-differentiate or begin dividing. Co-culture with embryonic hippocampus and cerebral cortex, but not medullary raphe or spinal cord, resulted in significantly greater survival, more complex neuronal morphology, and enhanced expression of neuronal-specific antigens. Immunohistochemical and Northern blot analysis revealed high levels of low affinity NGF receptor protein and mRNA in differentiated RN33B cells. PCR analysis demonstrated the presence of trkB, but not trkA or trkC, mRNA in both undifferentiated and differentiated RN33B cells. These data suggest that the observed target regulation of RN33B cell neuronal differentiation in co-culture may be mediated by neurotrophin(s).
用编码SV40大T抗原温度敏感突变体的逆转录病毒感染解离的胚胎第13天大鼠中缝髓质细胞后，通过连续稀释分离出克隆细胞系RN33B。在33℃时，RN33B细胞以48小时的倍增时间分裂，并显示T抗原、波形蛋白、巢蛋白、弥漫性神经元特异性烯醇化酶以及低分子量和中分子量神经丝免疫反应性。RN33B细胞是永生的，但未发生转化，因为它们在软琼脂中不会生长。在非允许温度（38.5℃）下，T抗原表达明显降低，RN33B细胞停止有丝分裂活动，并分化为具有明亮相细胞体和“神经样”突起的细胞。分化的RN33B细胞表达增强的神经元特异性蛋白，但不合成星形胶质细胞或少突胶质细胞特异性蛋白。此外，分化的RN33B细胞回到33℃时会重新表达T抗原，但不会去分化或开始分裂。与胚胎海马和大脑皮层共培养，但不与中缝髓质或脊髓共培养，会导致显著更高的存活率、更复杂的神经元形态以及神经元特异性抗原表达增强。免疫组织化学和Northern印迹分析显示，分化的RN33B细胞中低亲和力NGF受体蛋白和mRNA水平很高。PCR分析表明，未分化和分化的RN33B细胞中均存在trkB mRNA，但不存在trkA或trkC mRNA。这些数据表明，共培养中观察到的RN33B细胞神经元分化的靶标调节可能由神经营养因子介导。
