臺灣博碩士論文加值系統

　　先前的研究顯示，神經性類固醇去氫男性脂酮(DHEA)及硫酸孕烯醇酮(PS)對分佈在腦部神經細胞膜上之胺基酸受體具有調節的作用。由於DHEA及PS對胺基酸受體的作用現已被廣泛地研究，但是其對capsaicin受體的作用卻很少有人做研究。在本實驗中，我們採用快速分離出來的成年大白鼠背根神經節細胞為材料，利用全細胞膜電位固定的電生理記錄方法，探討DHEA及PS對capsaicin受體所媒介電流反應的調控作用。結果顯示DHEA及PS快速且可逆地抑制capsaicin受體所誘發的全細胞內向電流，且抑制作用皆呈現濃度依賴效應，最大抑制作用分別為100 %及65 %，而達到最大抑制作用一半所需的DHEA及PS濃度(EC50)分別為6.7 mM及13 mM。DHEA明顯地增加capsaicin 之EC50但對capsaicin的最大反應沒有作用，表示DHEA對capsaicin受體所媒介反應的抑制作用是屬於競爭型。Capsaicin所誘發的電流及DHEA抑制capsaicin所誘發電流的能力，並不會因為細胞內液添加飽和濃度DHEA而有所降低，表示DHEA對capsaicin所誘發電流的抑制作用，是經由作用到capsaicin受體的細胞外表面位置來達成的。此外，我們發現，DHEA並不是透過活化去磷酸酶1、2A或2B來抑制capsaicin所誘發的電流。並非所有的類固醇對capsaicin所誘發的反應都有抑制作用。黃體激素對capsaicin所誘發的電流幾乎沒有作用，表示DHEA對capsaicin受體的抑制作用具專一性。此外，DHEA的立體異構物3a-DHEA對capsaicin所誘發的反應不但沒有抑制作用，反而有增強的作用，顯示類固醇對capsaicin受體的調控作用具立體結構專一性。此外，capsaicin所誘發的電流及PS抑制capsaicin所誘發電流的能力，並不會因為細胞內液添加飽和濃度PS而有所降低，表示PS對capsaicin所誘發電流的抑制作用，是經由作用到capsaicin受體的細胞外表面位置來達成的。PS對capsaicin的最大反應有抑制作用，但對capsaicin的EC50則無影響作用，顯示PS對capsaicin受體所媒介反應的抑制作用是屬於非競爭型。PS對capsaicin受體所媒介反應的抑制作用既不是膜電位依賴型，也不是作用劑依賴型，表示PS並非capsaicin受體所控制的開離子通道的阻斷劑(open-channel blocker)。另外，我們也利用皮內注射的給藥方式，將capsaicin及其他藥物打入大白鼠的左後爪，測定PS對capsaicin所誘發的疼痛行為反應是否有抑制作用。結果顯示PS對capsaicin所誘發疼痛反應的抑制作用是屬於劑量依賴型。此外，我們發現PS對capsaicin所誘發疼痛反應的抑制作用並不是透過活化opioid或cannabinoid系統來達成的。我們也發現capsaicin的注射並不會引起內生性opioids及cannabinoids的釋放。上述電生理實驗及行為實驗的結果顯示，PS可能藉由直接抑制周邊的capsaicin受體的活性，來達到對capsaicin所誘發疼痛反應的抑制作用。

　Previous studies have shown that neurosteroids dehydroepiandrosterone (5-androsten-3b-ol-17-one; DHEA) and pregnenolone sulfate (5-pregnen-3b-ol-20-one sulfate; PS) modulate amino acid receptor-mediated responses in brain. Compared to the extensive studies of DHEA and PS effect on amino acid receptors, relatively little is known of interaction of DHEA and PS with the capsaicin receptor. In the present study, we investigated the effects of DHEA and PS on the capsaicin receptor-mediated current in acutely dissociated rat dorsal root ganglion neurons using the whole-cell voltage-clamp recording technique. DHEA and PS rapidly and reversibly inhibited the capsaicin-induced current in a concentration-dependent manner, with EC50 values of 6.7 and 13 mM and maximal inhibition of 100 and 65%, respectively. DHEA increased the capsaicin EC50 with little effect on the capsaicin maximal response, suggesting that the blocking action of DHEA was competitive. Neither the capsaicin response nor inhibition of the capsaicin response by extracellularly applied DHEA was significantly affected by inclusion of a saturating concentration of DHEA in the electrode buffer, arguing that DHEA acted at the extracellular surface of the membrane. Moreover, DHEA did not act through protein phosphatases 1, 2A or 2B to inhibit the capsaicin-induced current. Not all steroids inhibited the capsaicin response. Progesterone did not exert any significant effect on the capsaicin-induced current, suggesting that inhibition by DHEA of the capsaicin response was a specific effect. Furthermore, the stereoisomer of DHEA, 5-androsten-3a-ol-17-one (3a-DHEA), failed to inhibit the capsaicin-induced current, producing instead a potentiating effect on the capsaicin response, demonstrating that interaction of steroids with the capsaicin receptor was stereospecific. Neither the capsaicin response nor inhibition of the capsaicin response by extracellularly applied PS was significantly affected by inclusion of a saturating concentration of PS in the electrode buffer, arguing that PS acted at the extracellular surface of the membrane. Furthermore, PS inhibited the capsaicin maximal response with little effect on the capsaicin EC50, demonstrating that the blocking action of PS was noncompetitive. The fact that antagonism of the capsaicin response by PS was neither voltage- nor agonist-dependent, indicating that PS did not act as an open-channel blocker. In addition, we investigated the effects of intradermal administration of PS on the nociceptive response evoked by intradermal injection of capsaicin into the rat hindpaw. Results revealed that PS dose-dependently inhibited the capsaicin-induced nociceptive response. Moreover, PS did not act through opioid- and cannabinoid-dependent mechanisms to reduce the capsaicin-induced nociception. Furthermore, intradermal injection of capsaicin did not cause the release of endogenous opioids and cannabinoids. Our results suggested that PS depressed the capsaicin-induced nociception via direct inhibition of the capsaicin receptor.

總目錄

總目錄 …………………………………………………………… I

表目錄 …………………………………………………………… III

圖目錄 …………………………………………………………… IV

縮寫索引 ………………………………………………………… VIII

中文摘要 ………………………………………………………… IX

英文摘要 ………………………………………………………… XI

壹、緒論 ………………………………………………………… 1

貳、材料與方法

A. 實驗動物 ………………………………………………… 6

B. 藥品 ……………………………………………………… 6

C. 儀器設備 ……………………………………………………… 9

D. 大白鼠背根神經節細胞的製備 ……………………… 10

E. 全細胞膜電位固定記錄法 ……………………………… 11

F. 藥物的給予 …………………………………………………… 13

G. Capsaicin測試 ……………………………………………… 13

H. 實驗設計
(一) DHEA抑制capsaicin受體所媒介電流反應的作用機制 15

(二) PS抑制capsaicin受體所媒介電流反應的作用機制…19

(三) PS抑制capsaicin所誘發疼痛行為的作用機制 …… 22

I. 統計分析 ……………………………………………………26

參、結果 …………………………………………………………27

肆、討論 …………………………………………………………40

伍、參考文獻 ……………………………………………………57

陸、表 ……………………………………………………………74

柒、圖 ……………………………………………………………75

捌、附錄 …………………………………………………………109

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