在組織受到傷害或者發生發炎反應時，通常會造成疼痛感覺，同時組織四周圍會有組織酸化的現象，組織酸化被認為是造成疼痛的主要原因。在已知可接受酸刺激的受體中，酸敏感受體3被認為是最主要負責接受酸而引發疼痛的受體，然而令人驚訝的是，酸敏感受體3基因剔除小鼠對於一些中高強度刺激所引起的疼痛更敏感，這似乎代表了有另外一些分子彌補了酸敏感受體3的缺失，使得基因剔除小鼠對高強度有害刺激變得更敏感。在實驗室先前的G蛋白偶合受體微陣列分析中，我們發現血清素受體2B是所有血清素受體中唯一表現量上升的基因。然而血清素受體2B在痛覺上的功能及它和酸敏感受體3基因之間的相互作用仍然未知，本篇論文主要的目的是想了解血清素受體2B和酸敏感受體3之間的相互作用。首先，我先確認微陣列分析的結果是否正確，即血清素受體2B是否在酸敏感受體3基因剔除小鼠的背根神經節中，有表現量上升的情形，此外我也要觀察受體2B在背根神經節中的分佈情形。實驗結果發現血清素受體2B表現在所有的組織中，並且在酸敏感3基因剔除小鼠的背根神經節中表現量增加2倍。利用原位雜合反應實驗，我發現血清素受體2B主要分佈在背根神經節中直徑較小的神經元中，而這些直徑較小神經纖維主要負責痛覺訊息傳遞，血清素受體2B不但表現在酸敏感受體3基因剔除小鼠的背根神經節中直徑較小的神經元，且分佈細胞數目有增加的情形，這些結果顯示血清素受體2B可能與痛覺訊息傳遞或調控有關。因為血清素可以增加酸敏感受體3的基因表現，同時在酸敏感受體3基因剔除後血清素受體2B表現量增加，所以我們推測血清素可能藉著受體2B而使酸敏感受體3基因表現增加。為了驗證這個假設，我利用聚合酶鏈反應擴增小鼠的酸敏感受體3啟動子的基因，將此基因選殖入載體用以進行啟動子活性分析。結果發現即使細胞中轉染的血清素受體2B具有活性，血清素亦不能增加酸敏感受體3啟動子的活性。但是，這並不代表在真正生物體中，血清素不能藉由血清素受體2B去影響酸敏感受體3的基因表現，其可能原因我們將於此論文中討論。 Tissue acidosis, an important feature of tissue injury or inflammation, is a dominant factor causing pain. Among those acid sensing receptors, acid sensing ion channel 3 (ASIC3) is believed to be the major receptor responsible for acid-induced pain. Surprisingly, ASIC3-deficient mice have displayed a higher sensitivity in pain sensation than wild-type mice. It is likely that other molecules compensate ASIC3-deficiency. Using microarray analysis, previous studies have found that the transcripts of serotonin receptor 2B (5-HT2B) are up-regulated in ASIC3 knockout mice. However, the role of 5-HT2B in pain sensation and its relationship with ASIC3 remain unclear. The objective of this thesis is to explore the function of 5-HT2B in pain and to understand the relationship between 5-HT2B and ASIC3. To achieve this aim, I first confirmed the results of microarray and examined 5-HT2B distribuction. 5-HT2B is expressed in all tested tissues from wild-type and ASIC3 knockout mice. The expression is enhanced two folds in dorsal root ganglion (DRG) from ASIC3 knockout mice. Using in situ hybridization, I have found that the majority of 5-HT2B is expressed in a subset of pain-relevant, small-diameter neurons, and this population increases in ASIC3 knockout DRG. Since serotonin increases ASIC3 transcripts and 5-HT2B transcription is enhanced in a loss of ASIC3 gene, it is possible that serotonin enhances ASIC3 transcription through 5-HT2B. To test this hypothesis, I have cloned ASIC3 promoter to examine the promoter activity in the presence of 5-HT2B. After serotonin stimulation, ASIC3 promoter activity is not enhanced, even though 5-HT2B is activated. The possible reasons will be discussed in the thesis.
