PC-12細胞由於具有被神經生長因子﹝NGF﹞誘導分化為類神經細胞的特性，長期以來作為神經科學相關研究的體外模型被廣泛運用。然而在最終所運用於實驗的類神經PC-12細胞中由於分化率的限制，未分化PC-12 細胞對實驗結果所可能造成之難以避免雜訊，一直以來是一個困擾的問題。本實驗利用細胞培養環境的改變，以提高已分化類神經 PC-12細胞的分化程度、降低未分化 PC-12 細胞的增殖能力、提升類神經 PC-12 細胞在整體細胞中的比例，並用此模型作為實驗平台，通過觀察核內基因轉錄因子的變化，進行神經活化後訊息傳遞的研究。在神經活化的模型上，我們選擇麩胺酸(Glutamic acid)活化 NMDA受體引發胞內分子訊息傳遞的模型，對已知 345 個轉錄因子進行轉錄因子-核酸矩陣列實驗之系統性研究。在麩胺酸刺激後之類神經細胞轉錄因子的觀察中，我們發現除了過去較為已知數個轉錄因子具有親和力(affinity)或含量的提升，尚有更多的轉錄因子也具有兩倍以上的提升量。另外在神經生長因子誘導下，意外發現多數轉錄因子在誘導分化之後呈現親和力或含量的下降。在我們的研究結果中，我們不但建立了一個類神經 PC-12 細胞分化程度相對較高的神經細胞模型，並且也確認了其NMDA受體對麩胺酸刺激的專一性能力。本研究結果為對神經細胞內分子訊息傳遞研究建構了一個可實際應用的實驗平台，為將來更進一步的細胞內分子生物模型研究打下良好的基礎。

Cloned rat pheochromocytoma (PC-12) cell has been used as in vitro model of neuroscience research for a long time since it could differentiate both morphologically and
biologically into neuronal cells. However, the ratio of undifferentiated to differentiated PC-12 cells can have noises that affect the outcomes of the experiments. In this study, we attempt to enhance the degree of PC-12 cell differentiation, repress the proliferative ability of
undifferentiated PC-12 cells and promote the ratio of differentiated PC-12 to whole cells by changing the cell culture condition. In addition, we apply this model to screen the variation of the nuclear transcription factors (TFs). We choose the Glutamate induced NMDA receptor
model in our study of neuronal activity signaling pathway and systematically studied 345 TFs by protein/DNA array. Our result indicates that except for the several known TFs, which have been shown to increase the affinity or content, there were more TFs, which were uncovered
and also have good affinity. In this experiment, we established a neuron-like cell model, which has a relative higher degree of differentiation and confirmed the specificity of NMDA receptor to Glutamate.

1. Ji, R.R., et al., "Central sensitization and LTP: do pain and memory share similar mechanisms?" Trends Neurosci, 2003. 26(12): p. 696-705.
2. Tischler, A.S. and L.A. Greene, "Nerve growth factor-induced process formation by cultured rat pheochromocytoma cells". Nature, 1975. 258(5533): p. 341-2.
3. Nankova, B.B., et al., "Nicotinic induction of preproenkephalin and tyrosine hydroxylase gene expression in butyrate-differentiated rat PC12 cells: a model for adaptation to gut-derived environmental signals". Pediatr Res, 2003. 53(1): p. 113-8.
4. Mazzio, E., et al., "Effect of antioxidants on L-glutamate and N-methyl-4-phenylpyridinium ion induced-neurotoxicity in PC12 cells". Neurotoxicology, 2001. 22(2): p. 283-8.
5. Crisanti, P., et al., "Aspirin prevention of NMDA-induced neuronal death by direct protein kinase Czeta inhibition". J Neurochem, 2005. 93(6): p. 1587-93.
6. Lee, E., et al., "Effects of NMDA receptor inhibition by phencyclidine on the neuronal differentiation of PC12 cells". Neurotoxicology, 2006. 27(4): p. 558-66.
7. Song, Y., et al., "Minocycline protects PC12 cells against NMDA-induced injury via inhibiting 5-lipoxygenase activation". Brain Res, 2006. 1085(1): p. 57-67.
8. Santos, S.D., P.J. Verveer, and P.I. Bastiaens, "Growth factor-induced MAPK network topology shapes Erk response determining PC-12 cell fate". Nat Cell Biol, 2007. 9(3): p. 324-30.
9. Segal, R.A. and M.E. Greenberg, "Intracellular signaling pathways activated by neurotrophic factors". Annu Rev Neurosci, 1996. 19: p. 463-89.
10. Kaplan, D.R. and F.D. Miller, Signal transduction by the neurotrophin receptors. Curr Opin Cell Biol, 1997. 9(2): p. 213-21.
11. Groot, M., L.M. Boxer, and G. Thiel, "Nerve growth factor- and epidermal growth factor-regulated gene transcription in PC12 pheochromocytoma and INS-1 insulinoma cells". Eur J Cell Biol, 2000. 79(12): p. 924-35.
12. Khokhlatchev, A.V., et al., "Phosphorylation of the MAP kinase ERK2 promotes its homodimerization and nuclear translocation". Cell, 1998. 93(4): p. 605-15.
13. Aziz, N., et al., "c-Myb and v-Myb are differentially phosphorylated by p42mapk in vitro". Oncogene, 1993. 8(8): p. 2259-65.
14. Bijur, G.N. and R.S. Jope, "Opposing actions of phosphatidylinositol 3-kinase and glycogen synthase kinase-3beta in the regulation of HSF-1 activity". J Neurochem, 2000. 75(6): p. 2401-8.
15. Davis, R.J., "The mitogen-activated protein kinase signal transduction pathway". J Biol Chem, 1993. 268(20): p. 14553-6.
16. Gille, H., A.D. Sharrocks, and P.E. Shaw, "Phosphorylation of transcription factor p62TCF by MAP kinase stimulates ternary complex formation at c-fos promoter". Nature, 1992. 358(6385): p. 414-7.
17. Hunter, T., "Protein kinases and phosphatases: the yin and yang of protein phosphorylation and signaling". Cell, 1995. 80(2): p. 225-36.
18. Kaplan, D.R. and F.D. Miller, "Neurotrophin signal transduction in the nervous system". Curr Opin Neurobiol, 2000. 10(3): p. 381-91.
19. Whitmarsh, A.J. and R.J. Davis, "Transcription factor AP-1 regulation by mitogen-activated protein kinase signal transduction pathways". J Mol Med, 1996. 74(10): p. 589-607.
20. Platika, D., et al., "Neuronal traits of clonal cell lines derived by fusion of dorsal root ganglia neurons with neuroblastoma cells". Proc Natl Acad Sci U S A, 1985. 82(10): p. 3499-503.
21. Sanchez, J.F., et al., "GABAergic lineage differentiation of AF5 neural progenitor cells in vitro". Cell Tissue Res, 2006. 324(1): p. 1-8.
22. Brewer, G.J., et al., "Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination". J Neurosci Res, 1993. 35(5): p. 567-76.
23. Platt, S.R., "The role of glutamate in central nervous system health and disease--a review". Vet J, 2007. 173(2): p. 278-86.
24. Huettner, J.E., "Kainate receptors and synaptic transmission". Prog Neurobiol, 2003. 70(5): p. 387-407.
25. Bonsi, P., et al., "Modulatory action of metabotropic glutamate receptor (mGluR) 5 on mGluR1 function in striatal cholinergic interneurons". Neuropharmacology, 2005. 49 Suppl 1: p. 104-13.
26. Chu, Z. and J.J. Hablitz, "Quisqualate induces an inward current via mGluR activation in neocortical pyramidal neurons". Brain Res, 2000. 879(1-2): p. 88-92.
27. Endoh, T., "Characterization of modulatory effects of postsynaptic metabotropic glutamate receptors on calcium currents in rat nucleus tractus solitarius". Brain Res, 2004. 1024(1-2): p. 212-24.
28. Hinoi, E., et al., "Characterization with [3H]quisqualate of group I metabotropic glutamate receptor subtype in rat central and peripheral excitable tissues". Neurochem Int, 2001. 38(3): p. 277-85.
29. Kutsuwada, T., et al., "Molecular diversity of the NMDA receptor channel". Nature, 1992. 358(6381): p. 36-41.
30. Mori, H. and M. Mishina, "Structure and function of the NMDA receptor channel". Neuropharmacology, 1995. 34(10): p. 1219-37.
31. Mothet, J.P., et al., "D-serine is an endogenous ligand for the glycine site of the N-methyl-D-aspartate receptor". Proc Natl Acad Sci U S A, 2000. 97(9): p. 4926-31.
32. Choi, D.W., "Glutamate neurotoxicity and diseases of the nervous system". Neuron, 1988. 1(8): p. 623-34.
33. Lipton, S.A., "Paradigm shift in neuroprotection by NMDA receptor blockade: memantine and beyond". Nat Rev Drug Discov, 2006. 5(2): p. 160-70.
34. Lipton, S.A. and P. Nicotera, "Calcium, free radicals and excitotoxins in neuronal apoptosis". Cell Calcium, 1998. 23(2-3): p. 165-71.
35. Lipton, S.A. and P.A. Rosenberg, "Excitatory amino acids as a final common pathway for neurologic disorders. N Engl J Med, 1994. 330(9): p. 613-22.
36. Meldrum, B. and J. Garthwaite, "Excitatory amino acid neurotoxicity and neurodegenerative disease". Trends Pharmacol Sci, 1990. 11(9): p. 379-87.
37. Cole, A.J., et al., "Rapid increase of an immediate early gene messenger RNA in hippocampal neurons by synaptic NMDA receptor activation". Nature, 1989. 340(6233): p. 474-6.
38. Hevroni, D., et al., "Hippocampal plasticity involves extensive gene induction and multiple cellular mechanisms". J Mol Neurosci, 1998. 10(2): p. 75-98.
39. Lanahan, A. and P. Worley, "Immediate-early genes and synaptic function". Neurobiol Learn Mem, 1998. 70(1-2): p. 37-43.
40. Krichevsky, A.M. and K.S. Kosik, "Neuronal RNA granules: a link between RNA localization and stimulation-dependent translation. Neuron, 2001. 32(4): p. 683-96.
41. Xie, J. and D.L. Black, "A CaMK IV responsive RNA element mediates depolarization-induced alternative splicing of ion channels". Nature, 2001. 410(6831): p. 936-9.
42. Cruzalegui, F.H. and H. Bading, "Calcium-regulated protein kinase cascades and their transcription factor targets". Cell Mol Life Sci, 2000. 57(3): p. 402-10.
43. West, A.E., et al., "Calcium regulation of neuronal gene expression". Proc Natl Acad Sci U S A, 2001. 98(20): p. 11024-31.
44. West, A.E., E.C. Griffith, and M.E. Greenberg, "Regulation of transcription factors by neuronal activity". Nat Rev Neurosci, 2002. 3(12): p. 921-31.
45. Sala, C., S. Rudolph-Correia, and M. Sheng, "Developmentally regulated NMDA receptor-dependent dephosphorylation of cAMP response element-binding protein (CREB) in hippocampal neurons". J Neurosci, 2000. 20(10): p. 3529-36.
46. Brown, C.E., et al., "The many HATs of transcription coactivators". Trends Biochem Sci, 2000. 25(1): p. 15-9.
47. Sucher, N.J., et al., "Expression of endogenous NMDAR1 transcripts without receptor protein suggests post-transcriptional control in PC12 cells". J Biol Chem, 1993. 268(30): p. 22299-304.
48. Zhou, M. and M. Baudry, "Developmental changes in NMDA neurotoxicity reflect developmental changes in subunit composition of NMDA receptors". J Neurosci, 2006. 26(11): p. 2956-63.
49. Wang, J.Q., E.E. Fibuch, and L. Mao, "Regulation of mitogen-activated protein kinases by glutamate receptors". J Neurochem, 2007. 100(1): p. 1-11.
50. Belsham, D.D. and P.L. Mellon, "Transcription factors Oct-1 and C/EBPbeta (CCAAT/enhancer-binding protein-beta) are involved in the glutamate/nitric oxide/cyclic-guanosine 5''-monophosphate-mediated repression of mediated repression of gonadotropin-releasing hormone gene expression". Mol Endocrinol, 2000. 14(2): p. 212-28.
51. West, A.E., E.C. Griffith, and M.E. Greenberg, Regulation of transcription factors by neuronal activity. Nat Rev Neurosci, 2002. 3(12): p. 921-31.
52. Edwards, M.A., et al., "Lack of functional expression of NMDA receptors in PC12 cells". Neurotoxicology, 2007. 28(4): p. 876-85.
53. Mandel, G., et al., "Selective induction of brain type II Na+ channels by nerve growth factor". Proc Natl Acad Sci U S A, 1988. 85(3): p. 924-8.