Sato N, Hotta K, Waguri S, Nitatori T, Tohyama K, Tsujimoto Y, Uchiyama Y
Department of Cell Biology and Neuroanatomy, Iwate Medical University, School of Medicine, Japan.
J Neurobiol. 1994 Oct;25(10):1227-34. doi: 10.1002/neu.480251005.
Rat pheochromocytoma PC12 cells die when cultured in serum-free medium. Neurotrophic factors can rescue PC12 cells from cell death, and induce neuronal differentiation. To further investigate the relationship among cell death, survival, and differentiation, the bcl-2 cDNA, which is known to prevent apoptosis in various types of cells, was transfected into PC12 cells. Six monoclonal bcl-2-transfected cell lines were isolated and confirmed to express mRNA and protein product of bcl-2. The wild-type and bcl-2-transfected PC12 cells were kept to adhere to collagen-coated dishes at the initiation of serum-free experiments to avoid cellular damage due to detachment of the cells by trituration. Even under the conditions, the control PC12 cells mostly died within 24 h, when cultured in serum-free medium, whereas those expressing Bcl-2 survived even for 7 days in serum-free medium. Moreover, outgrowth of long processes in the bcl-2-transfected cells was only observed under the condition to keep the cells attached to the dishes in serum-free medium without any additive neurotrophic or growth factors. Neurofilament medium protein, which is a neuron-specific cytoskeletal component, was also expressed in the differentiated cells, suggesting that the long processes in bcl-2-transfected PC12 cells are neurites. However, neuronal differentiation of PC12 cells expressing Bcl-2 was not observed when cultured in serum-containing medium. Accordingly, survival of PC12 cells expressing Bcl-2 under the condition which cells usually die may be accompanied with neuronal differentiation.
大鼠嗜铬细胞瘤PC12细胞在无血清培养基中培养时会死亡。神经营养因子可挽救PC12细胞免于细胞死亡，并诱导其神经元分化。为了进一步研究细胞死亡、存活和分化之间的关系，将已知可防止多种类型细胞凋亡的bcl-2 cDNA转染到PC12细胞中。分离出六个单克隆bcl-2转染细胞系，并证实它们表达bcl-2的mRNA和蛋白质产物。在无血清实验开始时，将野生型和bcl-2转染的PC12细胞保持贴附在胶原包被的培养皿上，以避免因研磨导致细胞脱离而造成细胞损伤。即使在这种条件下，对照PC12细胞在无血清培养基中培养时，大多在24小时内死亡，而表达Bcl-2的细胞在无血清培养基中甚至能存活7天。此外，仅在无血清培养基中使细胞贴附于培养皿且无任何添加的神经营养因子或生长因子的条件下，才观察到bcl-2转染细胞长出长突起。神经丝中蛋白是一种神经元特异性细胞骨架成分，在分化细胞中也有表达，这表明bcl-2转染的PC12细胞中的长突起是神经突。然而，在含血清培养基中培养时，未观察到表达Bcl-2的PC12细胞发生神经元分化。因此，表达Bcl-2的PC12细胞在细胞通常会死亡的条件下存活，可能伴随着神经元分化。
