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研究生(外文):Tsai-Chen Chiang
論文名稱(外文):Studies on the Growth, Differentiation and Function of Neuronal cells by Chinese Herbs
指導教授(外文):Rong-Tsun Wu
外文關鍵詞:neuronal stem cellsanti-aging chinese herbsEGb761field EPSPacetylcholine
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神經系統由負責訊息連結的神經細胞與具有支持與保護作用的神經膠細胞所組成,不同於體內其他器官系統,其擁有獨特的可塑性。外傷、遺傳性或老化造成的神經退化性疾病,目前都沒有良好的治療方式。本實驗的目的在於發展有益腦部再生與活化腦部功能作用的藥物篩選平台,以篩選有效的抗老化中草藥。神經幹細胞擁有自我更新的能力,且能夠分化成神經細胞、神經膠細胞及寡突狀細胞,幫助受損的神經系統及神經退化性疾病的修復。本實驗培養出生一天小鼠腦神經幹細胞,分別處理42種抗老化中草藥,觀察其生長分化的情況,發現黃精、白朮、赤靈芝、冬蟲夏草有刺激神經幹細胞增生的作用;赤靈芝以細胞免疫染色分析發現1 μg/ml赤靈芝處理後幹細胞維持未分化的狀態,100 μg/ml則促進神經膠細胞的分化。學習記憶是神經系統中最重要的特性之一,一氧化氮(NO)在其中扮演重要的角色。本實驗利用腦神經細胞混合培養做為模型,在處理中草藥後偵測NO的產生,觀察藥物對腦部功能的影響。首先以臨床上使用已久對腦部知能有助益的銀杏萃取物EGb761作有效藥物的指標。實驗發現腦部神經細胞混合培養七天後以1 μg/ml EGb761作用72小時,刺激NO的表現達1.46倍。接著利用Liquid Chromatography-Tandem Mass Spectrometry(LC-MS-MS)分析神經傳遞物質的含量了解藥物可能參與何種神經功能的表現,發現1 μg/ml EGb761 能刺激腦神經細胞混合培養細胞內乙烯膽鹼的表現量增加95.3%,而一氧化氮合成酶的抑制劑N-nitro-arginine methyl ester(NAME)可抑制EGb761 造成乙烯膽鹼的增加。於活體動物實驗中餵食100 mg/kg EGb761的小鼠腦部乙烯膽鹼含量相較於對照組多了1.34倍。以反轉錄及多鏈聚合酶連鎖反應分析餵食100 mg/kg EGb761小鼠海馬迴transthyretin mRNA的表現量也明顯增加。電生理活性為神經細胞功能上的重要特性,1 μg/ml EGb761 處理腦活體切片並以高頻率電刺激引發海馬迴區之長期增益,藥物去除後,細胞外興奮性突觸後電位(field EPSP)減少了約20%,顯示EGb761對於長期增益有所影響。由結果顯示,本實驗所開發的平台將可應用於探討抗老化中草藥對腦神經系統生長分化及機能作用的影響。
The nervous system consists of neurons which communicate information and glia which support and protect the neurons in various ways. Synaptic plasticity is the remarkable property of the nervous system and is unique to other systems in the life. Few effective therapies have been developed for brain injury, heredity or aging caused neurodegenerated disorders. Therefore, the present research tried to establish an effective platform, and explore the potential of anti-aging Chinese herbs to improve the nervous system repair and the brain function. Neuronal stem cells(NSCs) have the ability of self-renew, and are capable of differentiating into neurons, astrocytes and oligodendrocytes, which indicates its potential in cell replacement therapy. As an approach for repairing the damaged neurons or preventing neurodegeneration, mouse brain stem cells were cultured and used to screen the capacity of stimulating proliferation and differentiation of neuronal stem cells from 42 anti-aging Chinese herbs. It was found that methanol extract of Rhizoma polygonati, Rhizoma Atractylodis Macrocephalae, Cordyceps sinensis, Ganoderma lucidum(GaLu) enhanced NSCs proliferation in vitro. Nestin of NSCs was expressed after 1 μg/ml GaLu treatment ,and 100 μg/ml GaLu treatment promoted gliaogenesis.Learning and memory is one of the most significant properties of the nervous system. Nitric oxides have been reported that play an important role in this process. We used brain mix culture cells as the in vitro model to monitor NO accumulation after anti-aging Chinese herbs treatment. EGb761 was used as positive control to develop the platform for exploring brain functional improvement. 1 μg/ml EGb761 was treated to mouse brain mix culture cells, and found that it can increase NO production. The amounts of neurotransmitter in the cells were then monitored by liquid chromatography-tandem mass spectrometry(LC-MS-MS). It showed that EGb761 could promote acetylcholine production in the mouse brain mix culture cells. The NO synthase inhibitor, N-nitro-arginine methyl ester, inhibited the increasing activity of acetylcholine after EGb761 treatment. Mice treat with EGb761 for 7 days and analyze the amounts of acetylcholine in the brain by LC-MS-MS. After 100 mg/kg EGb761 treatment, it showed 1.34 fold of acetylcholine expression in the mouse brain cortex compared to control. Reverse transcription-polymerase chain reaction (RT-PCR)analysis showed that the expression of transthyretin was up-regulated in the hippocampus from mice treated with EGb761. We also develop the electrophysiologic experiment to study anti-aging Chinese herbs. Long term potentiation(LTP)can be induced by high frequency electrical stimulation in the brain slice of hippocampus CA1 region. After high frequency stimulation, EGb761 showed enhancing effect on the field excitatory postsynaptic potential(fEPSP). LTP may be influenced by EGb761. In conclusion, we have developed the platform for exploring effective anti-aging Chinese herbs for the repairment of brain injury and the improvement of brain function.
1. Nicholls, J.G., Martin, A.R., and Wallace, B.G., From neuron to brain :A Cellular and Molecular Approach to the Function of the Nervous System. 3 ed. 1992: Congress.
2. Levitan, I.B.a.K., L.K., The Neuron. Cell and Molecular Biology. 2 ed. 1997: Oxford.
3. Mark F. Bear, B.W.C., Michael A. Paradiso., Neuroscience. Exploring The Brain. 2 ed. 2001: Lippincott, Williams & Wilkins,. 140-147.
4. Cavallaro, S., et al., Gene expression profiles during long-term memory consolidation. Eur J Neurosci, 2001. 13(9): p. 1809-15.
5. Leil, T.A., et al., Finding new candidate genes for learning and memory. J Neurosci Res, 2002. 68(2): p. 127-37.
6. Luo, Y., et al., A link between maze learning and hippocampal expression of neuroleukin and its receptor gp78. J Neurochem, 2002. 80(2): p. 354-61.
7. Mayer, B. and B. Hemmens, Biosynthesis and action of nitric oxide in mammalian cells. Trends Biochem Sci, 1997. 22(12): p. 477-81.
8. Pahan, K., et al., Lovastatin and phenylacetate inhibit the induction of nitric oxide synthase and cytokines in rat primary astrocytes, microglia, and macrophages. J Clin Invest, 1997. 100(11): p. 2671-9.
9. Curran, A.D., The role of nitric oxide in the development of asthma. Int Arch Allergy Immunol, 1996. 111(1): p. 1-4.
10. Faraci, F.M. and J.E. Brian, Jr., Nitric oxide and the cerebral circulation. Stroke, 1994. 25(3): p. 692-703.
11. Johns, R.A., EDRF/nitric oxide. The endogenous nitrovasodilator and a new cellular messenger. Anesthesiology, 1991. 75(6): p. 927-31.
12. Garthwaite, J., S.L. Charles, and R. Chess-Williams, Endothelium-derived relaxing factor release on activation of NMDA receptors suggests role as intercellular messenger in the brain. Nature, 1988. 336(6197): p. 385-8.
13. Palmer, R.M., A.G. Ferrige, and S. Moncada, Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature, 1987. 327(6122): p. 524-6.
14. Prast, H. and A. Philippu, Nitric oxide as modulator of neuronal function. Prog Neurobiol, 2001. 64(1): p. 51-68.
15. Kiss, J.P. and E.S. Vizi, Nitric oxide: a novel link between synaptic and nonsynaptic transmission. Trends Neurosci, 2001. 24(4): p. 211-5.
16. Lu, Y.F., E.R. Kandel, and R.D. Hawkins, Nitric oxide signaling contributes to late-phase LTP and CREB phosphorylation in the hippocampus. J Neurosci, 1999. 19(23): p. 10250-61.
17. Mizuno, M., et al., CREB phosphorylation as a molecular marker of memory processing in the hippocampus for spatial learning. Behav Brain Res, 2002. 133(2): p. 135-41.
18. Reynolds, B.A. and S. Weiss, Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science, 1992. 255(5052): p. 1707-10.
19. Temple, S. and A. Alvarez-Buylla, Stem cells in the adult mammalian central nervous system. Curr Opin Neurobiol, 1999. 9(1): p. 135-41.
20. Kuhn, H.G., et al., Epidermal growth factor and fibroblast growth factor-2 have different effects on neural progenitors in the adult rat brain. J Neurosci, 1997. 17(15): p. 5820-9.
21. Okano, H., Stem cell biology of the central nervous system. J Neurosci Res, 2002. 69(6): p. 698-707.
22. Kuhn, H.G., H. Dickinson-Anson, and F.H. Gage, Neurogenesis in the dentate gyrus of the adult rat: age-related decrease of neuronal progenitor proliferation. J Neurosci, 1996. 16(6): p. 2027-33.
23. Magavi, S.S., B.R. Leavitt, and J.D. Macklis, Induction of neurogenesis in the neocortex of adult mice. Nature, 2000. 405(6789): p. 951-5.
24. Mehler, M.F., et al., Bone morphogenetic proteins in the nervous system. Trends Neurosci, 1997. 20(7): p. 309-17.
25. Lim, D.A., et al., Noggin antagonizes BMP signaling to create a niche for adult neurogenesis. Neuron, 2000. 28(3): p. 713-26.
26. Sun, Y., et al., Neurogenin promotes neurogenesis and inhibits glial differentiation by independent mechanisms. Cell, 2001. 104(3): p. 365-76.
27. Brody, L.M., Human pharmacology molecular to clinical. 3 ed: Mosby.
28. Zurn, A.D., H.R. Widmer, and P. Aebischer, Sustained delivery of GDNF: towards a treatment for Parkinson's disease. Brain Res Brain Res Rev, 2001. 36(2-3): p. 222-9.
29. Watanabe, C.M., et al., The in vivo neuromodulatory effects of the herbal medicine ginkgo biloba. Proc Natl Acad Sci U S A, 2001. 98(12): p. 6577-80.
30. Christen, Y. and J.M. Maixent, What is Ginkgo biloba extract EGb 761? An overview--from molecular biology to clinical medicine. Cell Mol Biol (Noisy-le-grand), 2002. 48(6): p. 601-11.
31. Christen, Y., E. Olano-Martin, and L. Packer, EGb 761 in the postgenomic era: new tools from molecular biology for the study of complex products such as Ginkgo biloba extract. Cell Mol Biol (Noisy-le-grand), 2002. 48(6): p. 593-9.
32. Chandrasekaran, K., et al., Neuroprotective effects of bilobalide, a component of the Ginkgo biloba extract (EGb 761), in gerbil global brain ischemia. Brain Res, 2001. 922(2): p. 282-92.
33. Le Bars, P.L., et al., A placebo-controlled, double-blind, randomized trial of an extract of Ginkgo biloba for dementia. North American EGb Study Group. JAMA, 1997. 278(16): p. 1327-32.
34. Paul E. Gold, L.C.a.G.L.W., The Lowdown on Ginkgo Biloba. Scientific American, 2003 April.
35. Bastianetto, S., et al., The Ginkgo biloba extract (EGb 761) protects hippocampal neurons against cell death induced by beta-amyloid. Eur J Neurosci, 2000. 12(6): p. 1882-90.
36. Luo, Y., et al., Inhibition of amyloid-beta aggregation and caspase-3 activation by the Ginkgo biloba extract EGb761. Proc Natl Acad Sci U S A, 2002. 99(19): p. 12197-202.
37. Reynolds, B.A. and S. Weiss, Clonal and population analyses demonstrate that an EGF-responsive mammalian embryonic CNS precursor is a stem cell. Dev Biol, 1996. 175(1): p. 1-13.
38. Sergent-Tanguy, S., et al., Fluorescent activated cell sorting (FACS): a rapid and reliable method to estimate the number of neurons in a mixed population. J Neurosci Methods, 2003. 129(1): p. 73-9.
39. Misko, T.P., et al., A fluorometric assay for the measurement of nitrite in biological samples. Anal Biochem, 1993. 214(1): p. 11-6.
40. Gharavi, N. and A.O. El-Kadi, Measurement of nitric oxide in murine Hepatoma Hepa1c1c7 cells by reversed phase HPLC with fluorescence detection. J Pharm Pharm Sci, 2003. 6(2): p. 302-7.
41. Itoh, Y., et al., Determination and bioimaging method for nitric oxide in biological specimens by diaminofluorescein fluorometry. Anal Biochem, 2000. 287(2): p. 203-9.
42. Reubsaet, J.L., et al., Sample preparation and determination of acetylcholine in corneal epithelium cells using liquid chromatography-tandem mass spectrometry. J Chromatogr Sci, 2003. 41(3): p. 151-6.
43. Zhu, Y., et al., In vivo microdialysis and reverse phase ion pair liquid chromatography/tandem mass spectrometry for the determination and identification of acetylcholine and related compounds in rat brain. Rapid Commun Mass Spectrom, 2000. 14(18): p. 1695-700.
44. Shimono, K., et al., Long-term recording of LTP in cultured hippocampal slices. Neural Plast, 2002. 9(4): p. 249-54.
45. Ohkuma, S., et al., Involvement of peroxynitrite in N-methyl-D-aspartate- and sodium nitroprusside-induced release of acetylcholine from mouse cerebral cortical neurons. Brain Res Mol Brain Res, 1995. 31(1-2): p. 185-93.
46. Bliss, T.V. and G.L. Collingridge, A synaptic model of memory: long-term potentiation in the hippocampus. Nature, 1993. 361(6407): p. 31-9.
47. Volpicelli-Daley, L.A., et al., Altered striatal function and muscarinic cholinergic receptors in acetylcholinesterase knockout mice. Mol Pharmacol, 2003. 64(6): p. 1309-16.
48. Kennea, N.L. and H. Mehmet, Neural stem cells. J Pathol, 2002. 197(4): p. 536-50.
49. Calza, L., et al., Neural stem cells and cholinergic neurons: regulation by immunolesion and treatment with mitogens, retinoic acid, and nerve growth factor. Proc Natl Acad Sci U S A, 2003. 100(12): p. 7325-30.
50. Bhagat, K. and P. Vallance, Nitric oxide 9 years on. J R Soc Med, 1996. 89(12): p. 667-73.
51. Gold, P.E., Acetylcholine modulation of neural systems involved in learning and memory. Neurobiol Learn Mem, 2003. 80(3): p. 194-210.
52. Nitsch, R.M., From acetylcholine to amyloid: neurotransmitters and the pathology of Alzheimer's disease. Neurodegeneration, 1996. 5(4): p. 477-82.
53. Macovschi, O., et al., Effects of an extract of Ginkgo biloba on the 3',5'-cyclic AMP phosphodiesterase activity of the brain of normal and triethyltin-intoxicated rats. J Neurochem, 1987. 49(1): p. 107-14.
54. Hersh, L.B. and M. Shimojo, Regulation of cholinergic gene expression by the neuron restrictive silencer factor/repressor element-1 silencing transcription factor. Life Sci, 2003. 72(18-19): p. 2021-8.
55. Li, G. and A. Wieraszko, Dual effect of sodium nitroprusside on potentials recorded from mouse hippocampal slices. Brain Res, 1994. 667(1): p. 33-8.
56. Bertoni-Freddari, C., et al., Chronic administration of EGb 761 modulates synaptic and mitochondrial plasticity in adult vitamin E-deficient rats. Cell Mol Biol (Noisy-le-grand), 2002. 48(6): p. 709-15.
57. Javerfalk-Hoyes, E.M., et al., Simultaneous analysis of endogenous neurotransmitters and neuropeptides in brain tissue using capillary electrophoresis--microelectrospray-tandem mass spectrometry. Electrophoresis, 1999. 20(7): p. 1527-32.
58. Nathan, P., Can the cognitive enhancing effects of ginkgo biloba be explained by its pharmacology? Med Hypotheses, 2000. 55(6): p. 491-3.
59. Gallagher, M. and P.J. Colombo, Ageing: the cholinergic hypothesis of cognitive decline. Curr Opin Neurobiol, 1995. 5(2): p. 161-8.
60. Oken, B.S., D.M. Storzbach, and J.A. Kaye, The efficacy of Ginkgo biloba on cognitive function in Alzheimer disease. Arch Neurol, 1998. 55(11): p. 1409-15.
61. Diamond, B.J., et al., Ginkgo biloba extract: mechanisms and clinical indications. Arch Phys Med Rehabil, 2000. 81(5): p. 668-78.
62. Solomon, P.R., et al., Ginkgo for memory enhancement: a randomized controlled trial. JAMA, 2002. 288(7): p. 835-40.
63. Nunes, M.C., et al., Identification and isolation of multipotential neural progenitor cells from the subcortical white matter of the adult human brain. Nat Med, 2003. 9(4): p. 439-47.
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