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Jin-Chung Chen

陳景宗(Jin-Chung Chen)

AppointmentProfessor

LabNeuropharmacology Laboratory

EducationPh.D.

University/NationUniversity of Illinois, USA

Tel: (03)2118800 ext.5282

E-mailJinchen@mail.cgu.edu.tw

Research websitehttp://jcc05282.wixsite.com/cgujcclab

Research interests

The focus of Neuropharmacology lab is to understand the cellular and molecular of several neurological and psychiatric disorders, such as drug addiction, depression, schizophrenia, pain and Parkinson’s disease. The efforts by lab participants during previous years had generated many interesting research findings and now we are continuously working on the following research topics:

 (1)Drug addiction acquisition, extinction and relapse: via behavioral sensitization and conditioned place    preference (CPP) as addiction model conducted on rodents, we intend to explore the neural signals from prefrontal glutamate that project to the midbrain dopamine cell body in the ventral tegmental area (VTA) and discover how stress hormone CRF reinstates the addiction memory and its underlying cellular mechanism.

(2)Brain neuropeptide FF (NPFF)- NPFFR system: via research on home-made NPFFR2 Tg mice, we already found this Tg mice exhibit hypralgesia and depression/anxiety and the outcome of NPFFR2 over-expression on HPA axis, ascending nociceptive pathway, CGRP release and hippocampal neurogenesis. We are now producing NPFFR2 KO mice and will characterize their behavioral and biochemical phenotypes in order to understand the physiological role of brain NPFF-NPFFR system.

(3)Parkinson’s disease and L-dopa-induced dyskinesia (LID): previously, we identified that dopamine D3 receptor stimulation would evoke adult stem/progenitor cell proliferation in the SVZ area and further found systemic D3R agonist treatment could restore the motor dysfunction due to MPTP lesion in the striatum. We are now explore the cellular mechanism of LID via median forebrain bundle 6-OHDA-lesioned mice model, as well as Metabolomic profiling of PD and LID patients.

(4)Psychosis and cognition: via chronic ketamine administration, we intend to explore the cellular mechanism underlying the ketamine addiction and psychosis, focusing on the nucleus accumbens. In addition, via dopamine transporter (DAT) KD mice, we study the role of dopamine D3R on cognitive ability (novel object recognition test) focusing on the neurochemistry of prefrontal cortex.

The expertise in our lab is behavioral manipulation in rodents and in vivo and in vitro biochemical analyses. Experimental techniques routinely performed in our laboratory include western blot, IHC/ICC, brain microdialysis-HPLC, stereotaxic surgery and cannulation, qPCR, cell culture and transfection, behavioral observation (water maze, forced swim test, open-field activity, tail suspension test, NORT, pre-pulse inhibition, CPP, startling, elevated plus maze, von Frey test, etc.). Recently, we established the optogenetic technique and will conduct on GluT-, Gad2-, Pvalb-cre mice for targeting designated neural circuitry. Upon identifying the altered target(s) in in vivo system, a cell culture system would also be adopted for mechanistic study. It is expected that through comprehensive approaches, i.e. from cellular to systemic, we would be able to understand the causes of particular neural disorder and will eventually benefit the translational research.

 

Publications and Books and Chapters:

A. Publications

1.  J.C. Chen, K.K. Rhee, D.M. Beaudry and V.D. Ramirez (1984) In vivo output of dopamine and metabolites from the rat caudate nucleus as estimated with push-pull perfusion on-line with HPLC-EC in unrestrained, conscious rats.  Neuroendocrinology 38: 362-370.

2.  V.D. Ramirez, J.C. Chen, E. Nduka, W. Lin and A.D. Ramirez (1986) Push-pull perfusion (PPP) of the hypothalumus and the caudate nucleus (CN) in conscious, unrestrained animals. Annals of the New York Academy of Sciences 473:434-448.

3.  J.C. Chen and V.D. Ramirez (1988) In vivo dopaminergic activity from nucleus accumbens, substantia nigra and ventral tegmental area in the freely moving rat: basal neurochemical output and prolactin effect. Neuroendocrinology 48:329-335

4.  J.C. Chen and V.D. Ramirez (1988) Comparison of the effect of prolactin on dopamine release from the rat dorsal and ventral striatum and from the mediobasal hypothalamus superfused in vitro. European Journal of Pharmacology 149:1-8.

5.  J.C. Chen, P.B. Crino, P.W. Schnepper and L. Volicer (1989) Increased serotonin efflux by partially oxidized serotonin. Journal of Pharmacology and Experimental Therapeutics 250:141-148.

6.  J.C. Chen and V.D. Ramirez (1989) Effects of prolactin on tyrosine hydroxylase activity of central dopaminergic neurons of male rats: a NSD 1015 study. European Journal of Pharmacology 166:473-479.

7.  P.B. Crino, B.A. Vogt, J.C. Chen and L. Volicer (1989) Neurotoxic effects of partially oxidized serotonin: tryptamine-4,5-dione. Brain Research 504:247-257.

8.  P. Cai, J.K. Snyder, J.C. Chen and L. Volicer (1989) Preparation, reactivity and neurotoxicity of tryptamine-4,5-dione. Tetrahedron Letters, 31:969-972.

9.  J. Fishman, J. Rubins, J.C. Chen and L. Volicer (1991) Selective modification of guanine nucleotide-binding proteins by tryptamine-4,5-dione, a neurotoxic derivative of 5-hydroxytryptamine (serotonin). Journal of Neurochemistry 56:1851-1854.

10. J.C. Chen, J. Tonkiss, J.R. Galler and L. Volicer (1992) Prenatal protein malnutrition enhances serotonin release from hippocampus. Journal of Nutrition 122:2138-2143.

11. J.C. Chen, A. To, P.W. Schnepper and L. Volicer (1992) Neurochemical changes, tryptophan hydroxylase activity and serotonin uptake in rat brain after intraventricular administration of tryptamine-4,5-dione. Neuropharmacology 31:215-219.

12. J.C. Chen, E. Smith and J. Fishman (1992) The opioid receptor binding of dezocine, morphine, fentanyl, butorphanol and nalbuphine. Life Sciences, 52:389-396.

13. M.S. Moolten, J.B. Fishman, J.C. Chen and K.R. Carlson (1993) Etonitazene; an opioid selective for the mu receptor types. Life Sciences, 52:199-203.

14. G.J. Blatt, J.C. Chen, D.L. Rosene, L. Volicer and J.R. Galler (1994) Prenatal protein malnutrition effects on the serotonergic system in the hippocampal formation: an immunocytochemical, ligand binding, and neurochemical study. Brain Research Bulletin 34:507-518.

15. J.C. Chen, G. Turiak, J. Galler and L. Volicer (1995) Effect of prenatal malnutrition on release of monoamines from hippocampal slices. Life Sciences, 57:1467-1475.

 16. J.C. Chen, J. Squepinilli, R. Fine and L. Volicer (1996) Neurotoxicity of free-radical mediated serotonin neurotoxin in cultured embryonic chick brain neurons. European Journal of Pharmacology, 303:109-114

17. J.C. Chen, G.Turiak, J.Galler and L.Volicer (1997) Postnatal changes of brain monoamine levels in prenatally malnorished and control rats. International Journal of Developmental Neurocsience 15: 257-263.

18. J.C. Chen, H.J. Su, L.I. Huang and M.C. Hsieh (1998) The Reductions of binding and functions of D2 dopamine receptors in the rat ventral striatum amphetamine sensitization. Life Sciences 64:343-354.

19. J.Y.Li, C.H.Wong, K.S.Huang, K.W.Liang, M.Y.Lin, P.C.Tan and J.C. Chen (1999) Morphine tolerance in arthritic rats and serotoninergic system. Life Sciences, 64:PL111-116.

20. J.C,Chen, J.-Y. Li, K.-W. Liang and Y.K. Huang (1999) Neuropeptide FF potentiates the behavioral sensitization to amphetamine and alters the levels of neurotransmitters in the median prefrontal cortex. Brain Research 816:220-224.

21. C.L.Huang, H.C.Chen, N.K.Huang, D.M.Yang, L.S.Kao, J.C. Chen, H.L.Lai and YJ Cheren (1999) Modulation of dopamine transporter activity by nicotinic acetylcholine receptors and membrane depolarization in rat phechromocytoma PC12 cells. Journal of Neurochemistry 72:2437-2444.

22. P.C. Tan, J.C. Chen, J.Y.Li, K.W.Liang, C.H.Wong and Eagle Y.K.Huang (1999) Modulation of naloxone-precipitated morphine withdrawal syndromes in rats by neuropeptide FF analogues. Peptides, 20:1211-1217.

23. E.Y.-K. Huang, J.Y. Li, P.P.-C. Tan, C.H. Wong and J.C. Chen (2000) The cardiovascular effects of PFRPamide and PRF(Tic)amide, a possible agonist and antagonist of neuropeptide FF (NPFF). Peptides, 21:205-210.

24. J.C. Chen, K.W. Liang, C.S. Liang and Y.C. Chiang (2001) Significance of glutamate and dopamine neurons in the ventral pallidum during the expression of behavioral sensitization to amphetamine. Life Sciences, 68 (9): 973-983.

25. J.C. Chen, K.W. Liang and Eagle Y.K. Huang (2001) Differential effects of endomorphine-1 and -2 on amphetamine sensitization: neurochemical and behavioral aspects. Synapse, 39 (3): 239-248.

26. J.C. Chen, C.H. Wong, Y.K. Huang, T.C. Tang and J.Y. Li (2001) Modulations of spinal serotonin activity affect the development of morphine tolerance. Anesthesia and Analgesia 92(6):1563-1568.

27. Y.K. Huang, J.Y. Li, C.H. Wong, P.C. Tan and J.C. Chen (2002) Dansyl- PQRamide, a neuropeptide FF receptor analog, induces conditioned place preference. Peptides, 23 (3):489-496.

28. G.C. Yeh, J.C. Chen, H.C. Tsai, H.H. Wu, C.Y. Lin, P.C. Hsu and Y.C. Peng (2002) Amphetamine inhibits the N-methyl-D-aspartate receptor-mediated responses by directly interacting with the receptor/channel complex. Journal of Pharmacology and Experimental Therapeutics 300(3):1008-1016.

29. J.J. Hwang, M.H. Liao, T.C. Yen, S.P. Wey, K.J. Lin, W.H.T. Pan, J.C. Chen and G. Ting (2002) Biodistribution study of [99mTc]TRODAT-1 alone or combined with other dopaminergic drugs in mice with macroautoradiography. Applied Radiation and Isotopes, 57:35-42.

30. H.C. Hsieh, H.Y. Li, M.Y. Lin, Y.F. Chiou, S.Y. Lin, C.H. Wong and J.C. Chen (2002) Spatial and temporal profile of haloperidol-induced immediate early gene expression and phosphoCREB binding in the dorsal and ventral striatum of amphetamine- sensitized rats. Synapse, 45:230-244.

31. J.C. Chen, P.C. Chen and Y.C. Chiang (2003) D3 dopamine receptors are down-regulated in amphetamine-sensitized rats and their putative antagonists modulate the locomotor sensitization to amphetamine. Brain Research, 972(1-2):159-167.

32. Eagle Y.K. Huang, P.L. Tao, J.Y. Li, C.H. Wong and J.C. Chen (2003) Endomorphin-1 and -2 induce naloxone-precipitated withdrawal syndromes in rats. Peptides, 24:477-481.

33. K.J. Lin, T.C. Yen, S.P. Wey, J.J. Hwang, X.X. Ye, K.Y. Tzen, Y.K. Fu and J.C. Chen (2004) Characterization of the binding sites for 123I-ADAM and the relationship to the serotonin transporter in rat and mouse brains using quantitative autoradiography. Journal of Nuclear Medicine, 45:673-681.

34. P.C. Chen and J.C. Chen (2005) Enhanced Cdk5 activity and p35 translocation in the ventral striatum of acute and chronic methamphetamine-treated rats. Neuropsychopharmacology, 30:538-549.

35. J.-S. Wang, Y.S. Li, J.C. Chen, and Y.W. Chen (2005) Effects of exercise training and deconditioning on platelet aggregation induced by alternating shear stress in men. Arteriosclerosis, Thrombosis and Vascular Biology, 25:454-460.

36. T.-H. Yeh, H.M. Hwang, J.C. Chen, Tony Wu, A.H. Li and H.L. Wang (2005) Glutamate transporter function of rat hippocampal astrocytes is impaired following the global ischemia. Neurobiology of Disease, 18:476-483.

37. J.C. Chen, H.C. Hong, J.L. Lu, Y.C. Chiang, H.M. Huang, H.L. Wang, Tony Wu, J.J. Liao and T.S. Yeh (2005) Down-regulation of the glial glutamate transporter GLT-1 in rat hippocampus and striatum and its modulation by a group III metabotropic glutamate receptor antagonist following transient global forebrain ischemia. Neuropharmacology, 49:703-714.

38. Eagle Y.K. Huang, W.Z. Li and J.C.Chen (2006) Rat NPFF1 receptor-mediated signaling: functional comparison of neuropeptide FF (NPFF), FMRFamide and PFR(Tic)amide. Peptides, 27:1005-1014.

39. P.C. Chen, C.L. Lao and J.C. Chen (2007) Dual alteration of limbic dopamine D1 receptor-mediated signaling and Akt/GSK3 pathway in dopamine D3 receptor mutants during the development of methamphetamine sensitization. Journal of Neurochemistry 100:225-241.

40. C.H. Huang, J.C. Cheny, J.C. Chen* and C.P. Tseng* (2007) Evaluation of the role of Disabled-2 in nerve growth factor-mediated neurite outgrowth and cellular signaling, Cellular Signaling, 19:1339-1347. (*corresponding)

41. Y.C. Chiang and J.C. Chen (2007) The role of the cannabinoid type 1 receptor and down-stream cAMP/DARPP-32 signal in the nucleus accumbens of methamphetamine-sensitized rats, Journal of Neurochemistry, 103:2505-2517.

42. P.Y. Chang, C.H. Chuang, J.C. Chen and C.-S. Tung (2008) Behavioral and pharmacological effects of amperozide and serotonin agents on nigrostriatal and mesolimbic dopamine systems, J. Chinese Physiology, 51(2):106-115.

43. H.L. Liu, Y.Y. Wai, W.S. Chen, J.C. Chen, P.H. Hsu, X.Y. Wu, W.C. Huang, T.C. Yen and J.J. Wang (2008) Hemorrhage detection during focused-ultrasound induced blood–brain-barrier opening by using susceptibility-weighted magnetic resonance imaging, Ultrasound in Medicine & Biology, 34(4):598-606.

44. H-L Liu, P-H Hsu, P-C Chu, Y-Y Wai, J.C. Chen, C-R Shen, T-C Yen and J-J Wang (2009) Magnetic resonance imaging enhanced by superparamagnetic iron oxide particles: usefulness for distinguishing between focused ultrasound-induced blood-brain barrier disruption and brain hemorrhage. Journal of Magnetic Resonance Imaging 29:31-38.

45. J.C. Chen, P.C. Chen and Y.C. Chiang (2009) Molecular mechanism of psychostimulant addiction. Chang-Gung Medical Journal, 32:148-154.

46. P.C. Chen, C.L. Lao and J.C. Chen (2009) The D3 dopamine receptor inhibits dopamine release in PC-12/hD3 cells by autoreceptor signaling via PP-2B, CK-1 and Cdk-5, Journal of Neurochemistry, 110:1180-1190.

47. K.J. Lin, H.L. Liu, P.H. Hsu, Y.H. Chung, W.C. Huang, J.C. Chen, S.P. Wey, T.C. Yen and I.T. Hsiao (2009) Quantitative microSPECT/CT for detecting focused ultrasound induced blood-brain barrier opening in the rat, Nuclear Medicine and Biology, 36:853-861.

48. H-L Liu, Y-Y Wai, P-H Hsu, L-A Lyu, J-S Wu, C-R Shen, J-C Chen, T-C Yen and J-J Wang (2010) In vivo assessment of macrophage CNS infiltration during disruption of the blood–brain barrier with bocused ultrasound: a magnetic resonance imaging study, Journal of Cerebral Blood Flow & Metabolism, 30:177-186.

49. C.H. Hsieh, H.Y. Li and J.C. Chen (2010) Nitric oxide and interleukin-1b mediate noradrenergic induced corticotrophin-releasing hormone release in organotypic cultures of rat paraventricular nucleus, Neuroscience, 165:1191-1202.

50. F. Kersanté, J.-Y. Wang, J.-C. Chen, C. Mollereau and Jean-Marie Zajac (2011) Anti-opioid
 effects of neuropeptide FF receptors in the ventral tegmental area, Neuroscience Letters, 
488: 305-309.
51. Y.-L. Shen, J.-C. Chen and R.-M. Liao (2011) Place conditioning and neurochemical responses
 elicited by the aftereffect of acute stressor exposure to an elevated stand, Neuroscience Letters, 
504:156-159.

52. Y.T. Lin and J.C. Chen (2011) The involvement of dorsal root ganglia BDNF-TrkB in inflammatory pain in vivo and in vitro, Journal of Neuroinflammation, 8:126-147.

53. D.-J. Kao, Allen H Li, J.C. Chen, R.S. Luo, Y.L. Chen, J.C. Lu and H.L. Wang (2012) CC chemokine ligand 2 upregulates the current density and expression of TRPV1 channels and Nav1.8 sodium channels in dorsal root ganglion neurons, Journal of Neuroinflammation, 9:189.

54. P.K. Chao, K.T. Lu, Y.L. Lee, J.C. Chen, H.L. Wang, Y.L. Yang, M.Y. Cheng, M.F. Liao, L.S. Ro (2012) Early systemic granulocyte-colony stimulating factor treatment attenuates neuropathic pain after peripheral nerve injury, PLoS ONE, 8:1-13.

55. H.T. Chen, N.Y. Ruan, J.C. Chen* and Z.Y. Lin* (2012), Dopamine D2 receptor-mediated Akt/GSK3b signaling: Initiation by D2S receptor and role in quinpirole-induced behavioral activation, ASN NEURO, 4(6). (*, co-coresponding)

56. C.C. Lin, H.L. Hsieh, R.H. Shih, P.L. Chi, S.E. Cheng, J.C. Chen* and C.M. Yang* (2012) NADPH oxidase 2-derived reactive oxygen species signal contributes to bradykinin- induced matrix metalloproteinase-9 expression and cell migration in brain astrocytes, Cell Communication and Signaling, 10:35. (*, co-coresponding)

57. L.H. Chow, P.L. Tao, J.C. Chen, R.M. Liao, E.P. Chang and E.Y.K. Huang (2013) A possible correlation between oxytocin-induced and angiotensin IV-induced anti-hyperalgesia at the spinal level in rats. Peptides, 39:21-28.

58. T.C. Wu, H.T. Chen, H.Y. Chang, C.Y. Yang, M.C. Hsiao, M.-L. Cheng and J.-C. Chen (2013) Mineralocorticoid receptor antagonist prevents chronic corticosterone induced depression-like behaviors, Psychoneuroendocrinology, 38:871-883.

59. C.L. Lao, C.S. Lu and J.C. Chen (2013) Dopamine D3 receptor activation promotes neural stem / progenitor cell proliferation through AKT and ERK1/2 pathways and expands type-B and type–C cells in adult subventricular zone, Glia, 61:475–489.

60. P-H Hsu, K-C Wei, C-Y Huang, C-J Wen, T-C Yen, C-L Liu, Y-T Lin, J-C Chen, C-R Shen*, and H-L Liu* (2013) Noninvasive and targeted gene delivery into the brain using microbubble-facilitated focused ultrasound, PLoS ONE, 8(2): e57682.

61. C.L. Lao, Y.H. Kuo, Y.T. Hsieh and J.C.Chen (2013), Intranasal and subcutaneous administration of dopamine D3 receptor agonists functionally restores nigrostriatal dopamine in MPTP-treated mice, Neurotoxicity Research, 61:475-489.

62. Y.C. Chiang, Y.N. Lo and J.C. Chen (2013) Dopamine D2 receptors crosstalk with cannabinoid CB1 receptors at ERK1/2 signal and regulate Cnr1 promoter activity, Journal of Neurochemistry, 127:163-176.

63. H.T. Chen and J.C. Chen (2014) Role of the ventral tegmental area in methamphetamine extinction: AMPA receptor-mediated neuroplasticity, Learning and Memory, 22:149-158.

64. S.C. Kao, Y.T. Lin, Y.J. Dey and J.C. Chen (2015) The effects of Neuropeptide FF receptor subtype 2 over-expression on nociception and morphine tolerance, European Journal of Pain, 20:895-906.

65. Y.T. Lin, T.Y. Liu, C.Y. Yang, Y.L. Yu, T.C. Chen, Y.J. Day, C.C. Chang, G.J. Huang and J.C. Chen (2016) NPFFR2 stimulates stress-related depressive behaviors through HPA axis modulation, Psychoneuroendocrinology, 71:73-85.

66. W.Y. Tzeng, H.H. Wu, C.Y. Wang, J.C. Chen, L. Yu and C.F. Cherng (2017) Sex differences in stress and group housing effects on the number of newly proliferated cells and neuroblasts in middle-aged dentate gyrus. Frontiers in Behavioral Neuroscience, 10, 249.

67. Y-T Lin, H-L Liu, Y-J Day, C-C Chang, P-H Hsu and J.C. Chen (2017) Activation of NPFFR2 leads to hyperalgesia through the spinal inflammatory mediator CGRP in mice, Experimental Neurology, 291:62-73.

68. Q-C Moa and J.C. Chen (2017) Reduced neuregulin 1 expression in the medial prefrontal cortex of rat ketamine model for schizophrenia, Neuropsychiatry, 7(3):640-647.

69. S.F. H. Waqas, A.C. Hoang, Y.-T. Lin, G. Ampem, A. Hind, L. Balogh, J.C. Chen, I. Gerling, J.-S. Lim, S. Nam, J. Martinez-Ibanez, S. Paschke, J. Real, R. Quillet, S. Ayachi, S. Frederic, M. Schneider, C. Seroogy , J. Thuroczy, T. Röszer (2017) Neuropeptide-FF increases M2 activation and self-renewal of adipose tissue macrophages. Journal of Clinical Investigation, JCI90152.

70. S.Y. Chang, T.S. Hsieh, W.R. Chen, J.C. Chen and C Chou (2017) Measurement of tissue- radiation dosage using a thermal steady-state elastic shear wave, Journal of Biomedical Optics, 22(8), 085001.

71. Y.T. Lin, Y.L. Yu, W.C. Hong, T.S. Yeh, T.C. Chen and J.C. Chen (2017) NPFFR2 activates the HPA axis and induces anxiogenic effects in rodents, International Journal of Molecular Sciences, 18, 1810.

Y.T. Lin and J.C. Chen (2018) Dorsal root ganglia isolation and primary culture to study neurotransmitter release, JOVE, accepted.

72. P-K Chang, L. Yu and J.C. Chen (2018) Dopamine D3 receptor blockade rescue hyper-dopamine activity-induced deficit in novel object recognition memory, Neuropharmacology, in press.

73. S.H. Huang, W.R. Wu, L.M. Lee, P.R. Huang and J.C.Chen (2018) mTOR signaling in the nucleus accumbens mediates behavioral sensitization to methamphetamine, Progress in Neuro-Psychopharmacology & Biological Psychiatry, accepted.

 

B. Books and Chapters

1.J.C. Chen, A. Ramirez and V.D. Ramirez (1985) Prolactin effect on in vivo striatal

    dopaminergic activity as estimated with push-pull perfusion in male rat. In:

    Prolactin, Basic and Clinical Correlates R.M.  Macleod et al., eds., Liviana press,

    pp.523-532.

2. L. Volicer, J.C. Chen, P.B. Crino, B.A. Vogt, J. Fishman, J. Rubins, P.W. Schnepper

    and N. Wolfe (1989) Neurotoxic properties of a serotonin oxidation product: possible

    role in Alzheimer's disease. In: Progress in Clinical and Biological Research Vol.

    317: Alzheimer's Disease and Related Disorders, Alan R. Liss, Inc., NY, pp. 453-465.

3. 藥理學  1999 楊春茂 林麗慧 陳景宗 余淑美 林光輝 郭詩憲合著 匯華圖書出版有限公

  司 (ISBN 957-594-026-1 C5430)

4. 掌握優勢的關鍵 腦科學知識  2004 曾志朗 洪蘭 賴其萬 胡海國 白明奇 朱迺欣 謝仁俊

   陳景宗合著 東元科技文教基金會 (ISBN9789570829020)

5. 物質濫用 Chapter 5.中樞神經興奮劑  2014 陳景宗 江耀璋 衛生福利部食品藥物管理

  署 (ISBN 978-986-04-3813-0)

6. 彩色圖解藥理學 (Pharmacology: An Illustrated Review) 2017 陳景宗 黃翊恭 江耀璋

    林雅婷 黃信翰 合譯 合記出版社

 

 

 

 

 

 

 

 

 

 

 

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