抑制酸敏感受體TDAG8或ASIC3基因表現降低坐骨神經慢性壓迫性損傷所誘發的機械性痛覺過敏現象

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研究生：	鄧志宇 Chih-Yu Teng
論文名稱：	抑制酸敏感受體TDAG8或ASIC3基因表現降低坐骨神經慢性壓迫性損傷所誘發的機械性痛覺過敏現象 Inhibition of TDAG8 or ASIC3 gene expression reduces mechanical hyperalgesia induced by chronic constriction injury of the sciatic nerve
指導教授：	孫維欣 Wei-Hsin Sun
口試委員:
學位類別：	碩士 Master
系所名稱：	生醫理工學院 - 生命科學系 Department of Life Science
論文出版年：	2016
畢業學年度：	104
語文別：	中文
論文頁數：	85
中文關鍵詞：	酸敏感受體
相關次數：	點閱：9 下載：0
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根據全球市場調查從2007年到2015年全球將會有1.5億人將受到長期性疼痛所困
擾，而其中就有3到4.5%是屬於神經性疼痛的部分。在臨床神經性疼痛的治療上目前
扔然受到巨大的挑戰。為了協助治療以及藥物的開發了解神經性疼痛的相關機制是必不可少的。而在前人的實驗中發現，神經性疼痛的小鼠會造成一個長達八天的異常性疼痛，並且也會增加ASIC3基因的表現。且在其他研究中表示，中性粒細胞在小鼠受傷後8小時內有增加的趨勢並且在24小時達到高峰。雖然發炎性疼痛與神經性疼痛的臨床特點有很大的不同，但是有研究說到在周邊的神經局部發炎現象在神經性疼痛的發展中扮演著重要的角色。在實驗中我利用在坐骨神經慢性壓迫性損傷模型(CCI)發現可以造成小鼠一個長期的機械性痛覺過敏現象現象並且能維持4個月之久。在此同時我們也發現了神經的退化現象。我們利用降低基因表現以及基因剔除的方法在小鼠上加以研究。我們的研究發現降低TDAG8的表現量可延遲由CCI所導致的機械性痛覺過敏現象，但特別的是在ASIC3基因被剔除的小鼠中卻能抑制這種由CCI所造成的機械性痛覺過敏現象。另外在坐骨神經上，在降低TDAG8基因的表現後可增加由CCI所導致的前兩週免疫細胞數量，但是在ASIC3剔除的小鼠中卻能抑制這種由CCI所導致的發炎現象。而且另一方面我們也確定了蛋白激酶A及蛋白激酶Cε也參與了由CCI所誘發的機械性痛覺過敏感現象。詳細的機制將進一步進行探討。

According to global market analysis in 2007-2015, 1.5 billion people suffer from chronic pain with 3-4.5 % of population in neuropathic pain. The treatment of neuropathic pain continues to be a major management challenge in clinical practice. Understanding of the molecular mechanism of neuropathic pain is essential to identify potential drug targets for clinical treatments. Previously mice with neuropathic pain showed allodynia for 8 days, and ASIC3 immunoreactivity is up-regulated in DRG neurons. In other studies, neutrophils around the injury site increases substantially within 8 hours, and peaks at 24 hours. These studies suggest that the local inflammation of peripheral nerves and proton-sensing genes may play an important role in neuropathic pain. In this study, I used the model of peripheral mononeuropathy by chronic constriction injury (CCI) of the sciatic nerve, we have found that CCI mice developed long-term (at least 4 month) mechanical hypersensitivity and inflammation. Nerve degeneration was also found. We then generated TDAG8 knockdown(KD) and ASIC3 knockout(KO) mice to study the roles of the genes in neuropathic pain. We found that knockdown of TDAG8 delayed the onset of mechanical hyperalgesia induced by CCI. ASIC3 KO mice inhibited the late phase of CCI-induced mechanical hyperalgesia. Granulocytes cells were increased in TDAG8 KD mice in the first two weeks of CCI. Macrophages were decreased in ASIC3 KO mice in the late phase of CCI.

第一章  緒論    1
1 疼痛 (Pain)    2
2 神經性疼痛 (Neuropathic pain)    3
3 沃勒變性反應 (Wallerian degeneration and regeneration)    3
4 神經發炎反應 (Neuroinflammation)    4
5 神經性疼痛的治療 (Treatment of neuropathic pain)    5
6 酸敏感受體 (Proton sensing receptors)    5
6.1 酸敏感離子通道3 (Acid-sensing ion channel 3, ASIC3)    6
6.2 辣椒素受體1 (Transient receptor potential vanilloid channel 1, TRPV1)    6
6.3 TDAG8受體 (T-cell death-associated gene 8)    7
7 研究目的與動機    8

第二章  材料與方法    9
1 實驗材料    10
1.1 實驗動物 (Experimental animal)    10
1.2 核糖核酸干擾 (RNA interference)    10
1.3 實驗藥品 (Experimental drugs)    10
1.4 實驗器具 (Experimental tools)    11
2 實驗方法    11
2.1 小鼠基因判定 (Genotyping)    11
2.2 質體DNA製備 (Midi-prep)    12
2.3 瓊酯醣膠電泳製備及分析 (Preparation and electrophoresis of agar gel)    13
2.4 慢性壓迫損傷神經性疼痛模型 (Chronic Constriction Injury model, CCI model)    14
2.5 機械性痛覺行為實驗 (Mechanical behavior test)    14
2.6 熱痛覺行為實驗 (Hargreavs’ test)    15
2.7 蘇木素-伊紅染色 ( Hematoxylin and Eosin stain, H&E stain)    16
2.8腳掌及神經之pH值測定(pH value test)    16
2.9 統計分析    16

第三章  結果    17
1 CCI小鼠可誘發長期的機械性及惡痛絕過敏感現象    18
2 CCI小鼠在坐骨神經上的改變    18
3 在坐骨神經上的慢性壓迫損傷造成的長期發炎現象    19
4 TRPV1剔除的小鼠並不能抑制由CCI所造成的機械性痛覺過敏感現象    20
5 在14週的坐骨神經上TRPV1敲除的小鼠可減少粒細胞但是增加巨噬細胞的數量      20
6 ASIC3剔除的小鼠抑制由CCI所造成的機械性痛覺過敏感現象    21
7 剔除ASIC3基因可減緩在坐骨神經上的發炎現象    21
8 降低TDAG8基因的表現可延遲由CCI所導致的機械性痛覺過敏感現象    22
9 降低TDAG8基因表現量的CCI小鼠在坐骨神經上的變化    23
10 降低TDAG8基因的表現量可增加由CCI導致的前兩週免疫細胞的數量    23
11 蛋白激酶A及蛋白激酶Cε參與在CCI動物模型所誘發的機械性痛覺過敏現象    24

第四章  討論    26
1 慢性壓迫損傷模型誘發痛覺過敏和發炎反應    27
1.1 痛覺過敏感現象    27
1.2 神經上的發炎現象    27
2 ASIC3參與調控由CCI動物模型所引起的機械性痛覺過敏感現象    28
3 TDAG8參與調控由CCI動物模型所引起的機械性痛覺過敏現象    29
4 總結    30

第五章  參考文獻    31
附錄Ⅰ(引子序列及實驗藥品)    55
附錄Ⅱ    56
附錄Ⅲ(聚合酶連鎖反應條件)    58
附錄Ⅳ(免疫細胞總數)……………………..…………………………………………………………………60

                                

1. A. Patapoutian, S. Tate, C.J. Woolf. Transient receptor potential channels: targeting pain at the source. Nat. Rev. Drug Discov., 8 (2009), pp. 55–68.

2. A.M. Patwardhan, P.E. Scotland, A.N. Akopian, K.M. Hargreaves. Activation of TRPV1 in the spinal cord by oxidized linoleic acid metabolites contributes to inflammatory hyperalgesia. Proc. Natl. Acad. Sci. USA, 106 (2009), pp. 18820–18824.

3. Bennett GJ, Xie YK. (1988). A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man. Pain. 33:87–107.

4. Baron, R., Binder, A. and Wasner, G. (2010). Neuropathic pain: diagnosis, pathophysiological mechanisms, and treatment. Lancet Neurol 9(8): 807-19.

5. Clatworthy AL, Illich PA, Castro GA, Walters ET. (1995). Role of peri-axonal inflammation in the development of thermal hyperalgesia and guarding behavior in a rat model of neuropathic pain. Neurosci Lett 184: 5–8.

6. Choi JW., Lee SY., Choi Y. (1996). Identification of a putative G-protein-coupled receptor induced during activation-induced apoptosis of T cells. Cell Immunol. Fed 25;168(1):78-84.

7. Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D. (1997). The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature;389:816–824.

8. Campbell, J. N. and Meyer, R. A. (2006). Mechanisms of neuropathic pain. Neuron 52(1): 77-92.

9. Canessa CM. Structural biology: Unexpected opening. Natrue. (2007);449(7160):293-294.

10. Costigan M, Scholz J, Woolf CJ. (2009). Neuropathic pain: a maladaptive response of the nervous system to damage. Annu Rev Neurosci 32:1–32.

11. Chan, Y. J., C. W. Huang, C. S. Lin, W. H. Chang and W. H. Sun. Expression and Function of Proton-Sensing G-Protein-Coupled Receptors in Inflammatory Pain. Mol Pain 5, (2009): 39.

12. Clark A. K., Old E. A., Malcangio M. (2013). Neuropathic pain and cytokines: current perspectives.J. Pain Res. 6, 803–814 10.

13. David, S., & Aguayo, A. J. (1981). Axonal elongation into PNS "bridges" after CNS injury in adult rats. Science, 214, 931-933.

14. Dworkin RH, O’Connor AB, Backonja M, Farrar JT, Finnerup NB, Jensen TS, Kalso EA, Loeser JD, Miaskowski C, Nurmikko TJ, Portenoy RK, Rice AS, Stacey BR, Treede RD, Turk DC, Wallace MS. (2007). Pharmacologic management of neuropathic pain: evidence-based recommendations. Pain.132:237–251.

15. Dubovy P. Wallerian degeneration and peripheral nerve conditions for both axonal regeneration and neuropathic pain induction. Ann Anat. 2011;194(4):267–75.

16. Gaudet AD, Popovich PG, Ramer MS. (2011). Wallerian degeneration: Gaining perspective on inflammatory events after peripheral nerve injury, Journal of Neuroinflammation. 8: 110.

17. Hargreaves K, Dubner R, Brown F, Flores C, Joris J. (1988). A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain.32:77–88.

18. Huang CW, Tzeng JN, Chen YJ, Tsai WF, Chen CC, Sun WH. (2007). Nociceptors of dorsal root ganglion express proton-sensing G-protein-coupled receptors. Mol Cell Neurosci. Oct; 36(2):195-120.

19. Ishii S, Kihara Y, Shimizu T. (2005). Identification of T cell death-associated gene 8 (TDAG8) as a novel acid sensing G-protein-coupled receptor. J Biol Chem. Mar 11;280(10):9083-7.

20. Ihara, Y., Y. Kihara, F. Hamano, K. Yanagida, Y. Morishita, A.Kunita, T. Yamori, M. Fukayama, H. Aburatani, T. Shimizu and S. Ishii. The G Protein-Coupled Receptor T-Cell Death-Associated Gene 8 (TDAG8) Facilitates Tumor Development by Serving as an Extracellular Ph Seneor. Proc Natl Acad Sci U S A 107, no. 40 (2010): 17309-14.

21. Jensen, T. S., Gottrup, H., Sindrup, S. H. and Bach, F. W. (2001). The clinical picture of neuropathic pain. Eur J Pharmacol 429(1-3): 1-11.

22. Kim SH, Chung J-M. (1992). An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat. Pain 50:355–363.

23. M. A. Thacker, Clark, A. K., Marchand, F., and McMahon, S. B. (2007). Pathophysiology of peripheral neuropathic pain: immune cells and molecules. Anesth Analg 105, 838-847

24. Mazzuca M, Heurteaux C, Alloui A, Diochot S, Baron A, Voilley N, Blondeau N, Escoubas P, Gélot A, Cupo A, Zimmer A, et al. A tarantula peptide against pain via ASIC1a channels and opioid mechanisms. Nat Neurosci. 2007;10(8):943–945.

25. Moalem, G; Tracey, DJ; (2006) Immune and inflammatory mechanisms in neuropathic pain. Brain Research Reviews , 51 (2) pp. 240-264.

26. Ohtori S, Inoue G, Koshi T, Ito T, Doya H, Saito T, Moriya H, Takahashi K. Up-regulation of acid-sensing ion channel 3 in dorsal root ganglion neurons following application of nucleus pulposus on nerve root in rats. Spine. 2006;31(18):2048–2052.

27. Reeh, P. W., and Stenn, K. H. (1996). Tissue acidosis in nociception and pain. Brain Research, 113, 143-151.

28. Radu CG, Yang LV, Riedinger M, Au M, Witte ON (2004). T cell chemotaxis to lysophosphatidylcholine through the G2A receptor. Proc Natl Acad Sci U S A. Jan 6;101(1):245-50.

29. Seltzer, Z., Dubner, R. and Shir, Y. (1990). A novel behavioral model of neuropathic pain disorders produced in rats by partial sciatic nerve injury. Pain , 43, 205-218.

30. Scholz J, and Woolf CJ. (2002). Can we conquer pain? Net Neurosci. Nov;5 Suppl:1062-7

31. Stockand JD, Staruschenko A, Pochynyuk O, Booth RE, Silverthorn DU. (2008). Insight toward epithelial Na+ channel mechanism revealed by the acid-sensing ion channel 1 structure. IUBMB Life. 60(9):620–628.

32. Tosa N1, Murakami M, Jia WY, Yokoyama M, Masunaga T, Iwabuchi C, Inobe M, Iwabuchi K, Miyazaki T, Onoe K, Iwata M, Uede T. (2003). Critical function of T cell death-associated gene 8 in glucocorticoid-induced thymocyte apoptosis. Int Immunol. 2003 Jun;15(6):741-9

33. von Frey M (1896). Untersuchunger über die Sinnesfunctionen der menschlichen Haut. Bandes der Abhandlungen der mathematisch-physischen Classe der Königl. Sächsischen Gesellschaft der Wissenschaften, 23: 175-266.

34. van Zwieten R, Wever R, Hamers MN, Roos D. (1981). Extracellular proton release by stimulated neutrophils. J Clin Invest 68:310-313.

35. Voilley N, de Weille J, Mamet J, Lazdunski M. (2001). Nonsteroid anti-inflammatory drugs inhibit both the activity and the inflammation-induced expression of acid-sensing ion channels in nociceptors. J Neurosci. 21(20):8026–8033.

36. van der Wal Selina, Cornelissen Lisa, Behet Marije, Vaneker Michiel, Steegers Monique, Vissers Kris. (2015) Behavior of neuropathic pain in mice following chronic constriction injury comparing silk and catgut ligatures. SpringerPlus 4: 225. doi:10.1186/s40064-015-1009-4.

37. Woolf CJ and Mannion RJ. (1999). Pain: neuropathic pain: aetiology, symptoms, mechanisms and managment. Lancet 353: 1959–64

38. Walker KM, Urban L, Medhurst SJ, Patel S, Panesar M, Fox AJ, McIntyre P (2003) The VR1 Antagonist Capsazepine Reverses Mechanical Hyperalgesia in Models of Inflammatory and Neuropathic Pain. J Pharmacol Exp Ther 304: 56-62

39. Wang JQ, Kon J, Mogi C, Tobo M, Damirin A, Sato K, Komachi M, Malchinkhuu E, Murata N, Kimura T, Kuwabara A, Wakamatsu N, Koizumi H, Uede T, Tsujimoto G, Kurose H, Sato T, Harada A, Misawa N, Tomura H, Okajima F. (2004). TDAG8 is a proton-sensing and psychosine-senstive G-protein-coupled receptor. J Biol Chem. Oct 29;279(44):45626-33.

40. Wemmie JA, Price MP, Welsh MJ. (2006). Acid-sensing ion channels: Advances, questions and therapeutic opportunities. Trends Neurosci. 29(10): 578–586.

41. Xu B, Descalzi G, Ye HR, Zhuo M, Wang YW. (2012). Translational investigation and treatment of neuropathic pain. Mol Pain. 8:15.

42. Zhao X, Wang C, Cui WG, MA Q, Zhou WH. (2015) Fisetin exerts antihyperalgesic effect in a mouse model of neuropathic pain: engagemen of spinal serotonergic system. Sci Rep. 2015 Mar 12;5:9043. doi: 10.1038/srep09043.

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