臺灣博碩士論文加值系統

偏頭痛(Migraine)為一種神經血管方面的疾病，發作時通常伴隨著嚴重搏動性的疼痛，其病生理機轉目前仍然未有定論。近來，Genome-wide association (GWAS)研究顯示Transient receptor-potential Melastatin (TRPM8)通道可能參與在偏頭痛的病生理機制中。然而，與TRPM8相關之偏頭痛分子機制仍然不明。現今普遍認為三叉神經系統(Trigeminovascular system)的活化伴隨著Calcitonin gene-related peptide (CGRP)從三叉神經節(Trigeminal ganglion, TG)中被釋放在偏頭痛中扮演著重要的角色。研究亦發現，抑制Calmodulin-dependent protein kinase II (CaMKII)會使從三叉神經細胞中釋放出來的CGRP減少。因此，我們假設TRPM8可能是透過Ca2+ / CaM / CaMKII的路徑促進三叉神經細胞中CGRP的釋放。
本研究利用剛出生的仔鼠建立初代培養三叉神經細胞 (Primary cultured TG neurons)作為實驗材料。我們首先發現TRPM8確實會在TG neurons中表現，且其中約有43.9 %的細胞與CGRP共存，而不表現於其他非神經細胞。其次，酵素免疫分析法(Enzyme-linked immunosorbent assay, ELISA)的結果顯示，給予已知能促進TG中CGRP釋放的KCl及TRPM8的活化劑Icilin皆能使CGRP從TG neurons釋放量增加。然而，即時聚合酶連鎖反應 (qPCR) 顯示，給予10 mM的Icilin卻會降低細胞中CGRP的合成量。另外，給予CaMKII的抑制劑KN-93 則會降低Icilin引發的CGRP釋放量。最後，細胞在沒有鈣離子的情況下，並不會被Icilin所誘發而促進CGRP的釋放，顯示此路徑為鈣離子依賴的反應。由以上研究結果可知，TRPM8可能在三叉神經節中扮演調節CGRP釋放的角色。

Migraine is a disabling neurovascular disease, commonly characterized by severe throbbing pain and pulsing sensation, while its underlying pathophysiology remains to be debated. Recent genome-wide association studies have suggested a role for transient receptor potential melastatin 8 (TRPM8) channel in migraine pathophysiology. However, the molecular mechanisms contributed to TRPM8-associated migraine pathophysiology remain unclear. Release of CGRP from trigeminal gnaglia (TG) following activation of trigeminovascular system has been considered to play a critical role in migraine pathophysiology. In addition, blockade of calmodulin-dependent protein kinase II (CaMKII) is known to reduce capsaicin-induced CGRP release from TG neurons. Thus, we hypothesized that TRPM8 may promote CGRP release from TG neurons via activation of Ca2+/CaM/CaM-dependent kinase II pathway.
The present study was carried out in primary cultured TG neurons of the neonatal rat. We firstly found that TRPM8 is expressed in TG neurons and approximately 43.9 % of TRPM8-positive cells coexisted with CGRP. There are no co-localization of TRPM8 with Non-neuron cells. Result from Enzyme-linked immunosorbet assay (ELISA) suggested that both well-known stimulator KCl and TRPM8 agonist Icilin are able to increase CGRP release from TG neurons. However, results from qPCR indicated that 10 M Icilin tends to decrease intracellular CGRP synthesis. In addition, our results also showed that inhibition of CaMKII by KN-93 block CGRP release under TRPM8 activation. Also, the release of CGRP from TG neurons attributing by Icilin is significantly decreased under calcium-free condition, which indicated it is a calcium-dependent pathway. Our findings from this study reveal that TRPM8 may play a mediating role in CGRP release in TG.

致謝 2
中文摘要 i
英文摘要 ii
目錄 iii
圖目錄 v
縮寫對照表 vii
第一章 研究背景 1
1.1 偏頭痛的病生理機轉 1
1.2 Calcitonin gene-related peptide (CGRP)在偏頭痛的病生理角色 2
1.3 Transient receptor-potential melastatin 8 (TRPM8)與偏頭痛的關聯性 4
1.4 TRPM8調控CGRP釋放的可能性 5
第二章 研究動機與目的 7
第三章 材料與方法 8
3.1實驗動物 8
3.2實驗材料 8
3.3實驗方法 9
3.3.1 三叉神經細胞株之建立 9
3.3.2 SRB (Sulforhodamine B colorimetric) assay 10
3.3.3 蛋白質定量及分析 10
3.3.4 組織學實驗 12
3.3.5 CGRP 酵素免疫分析測定 12
3.3.6 即時定量聚合酶連鎖反應(Real-time polymerase chain reaction, Real-time PCR) 13
3.4 統計分析 14
3.5 研究設計 14
3.5.1 實驗流程 14
3.5.2 實驗分組 14
第四章 結果 16
4.1 三叉神經細胞株的建立 16
4.1.1 製作初代培養三叉神經節細胞動物年齡之選擇 16
4.1.2 神經細胞在初代培養三叉神經節細胞中的分布 16
4.1.3 CGRP在三叉神經節神經細胞中的分布與數量 16
4.1.4 初代三叉神經節細胞釋放CGRP之能力 17
4.2 TRPM8的表現 17
4.2.1 TRPM8在初代培養三叉神經節細胞之表現 17
4.2.2 TRPM8與CGRP在三叉神經節細胞中之表現 17
4.3 活化三叉神經節的TRPM8促進胞內CGRP的釋放 18
4.3.1 TRPM8作用劑濃度對三叉神經節細胞存活率的影響 18
4.3.2 TRPM8活化對三叉神經細胞胞內鈣離子濃度變化的影響 18
4.3.3 TRPM8 活化對三叉神經節細胞CGRP釋放量的影響 18
4.4 胞外鈣離子在TRPM8促進三叉神經節細胞CGRP釋放的角色 19
4.5 CaMKII在TRPM8促進三叉神經節細胞CGRP釋放中扮演的角色 19
4.5.1 TRPM8活化誘發三叉神經節細胞中CaMKII之活化 20
4.5.2 抑制CaMKII可減緩TRPM8促進三叉神經節細胞CGRP的釋放 20
4.6 TRPM8作用劑減少三叉神經節細胞中CGRP的表現量 20
第五章 討論 22
第六章 結論 26
參考文獻 27
附圖 35

圖 1初代大鼠三叉神經節細胞型態 36
圖 2 初代大鼠三叉神經節細胞之神經元比例 37
圖 3 初代大鼠三叉神經節細胞之細胞種類 38
圖 4 初代大鼠三叉神經節細胞之神經細胞中CGRP之分布情形 39
圖 5 初代大鼠三叉神經節細胞CGRP釋放能力之確認 40
圖 6 初代大鼠三叉神經節細胞中TRPM8的表現情形 41
圖 7 初代大鼠三叉神經節TRPM8與CGRP的表現情形 42
圖 8 TRPM8作用劑對細胞存活率的影響 43
圖 9 TRPM8作用劑對細胞內鈣離子濃度的影響 44
圖 10 TRPM8作用劑對三叉神經節細胞釋放CGRP之影響 45
圖 11胞外鈣離子在TRPM8作用劑引發CGRP釋放量之角色 46
圖 12 TRPM8活化對三叉神經節細胞中磷酸化CaMKII蛋白表現量之影響 47
圖 13 CaMKII在TRPM8活化對三叉神經細胞釋放CGRP之角色 48
圖 14 TRPM8作用劑對大鼠三叉神經節合成CGRP之影響 49

表格 1本研究使用之primer序列 50
表格 2本研究使用之抗體 51

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