血清素 (serotonin, 5-HT) 及其受體在神經細胞的許多發育分化過程中扮演很重要的角色。先前的研究曾發現血清素在細胞培養中能促進大腦皮質神經細胞的存活。相較之下麩氨酸受體 (Glutamate receptor) 的亞型N-methyl-D-aspartate (NMDA) 受體在胚胎大腦皮質中表現的時間較晚。因此，NMDA受體的表現有可能會受到血清素及其受體活性之影響。本實驗探討：長期暴露於血清素受體拮抗劑，對大白鼠初級大腦皮質神經細胞中NMDA受體的表現及活性之影響。結果顯示大白鼠初級皮質神經細胞之NR1、NR2A、NR2B等NMDA受體次單元的表現，隨著培養天數而逐漸增加，於第10至20天達到高峰。長時期 (10個培養天) 暴露於高濃度的methysergide maleate (100μM) 或dihydroergocristine mesylate (10μM) 兩種5-HT1及5-HT2的非特異性拮抗劑會導致明顯的細胞毒性。低濃度methysergide maleate (1~10μM) 或dihydroergocristine mesylate (1μM) 則不會產生細胞毒性。長期培養於dihydroergocristine mesylate ( 1μM) 會降低NMDA受體次單元NR1及NR2A的表現量。而細胞長期培養於methysergide maleate (1~10μM)、 dihydroergocristine mesylate (1μM)、5-HT1a 拮抗劑pindolol (1μM) 或 5-HT2A 拮抗劑cyproheptadine hydrochloride (1μM) 會顯著降低細胞對NMDA引發的細胞毒性的敏感度。此結果顯示早期阻斷血清素受體活性可明顯抑制NMDA受體的表現，且此種抑制作用可能包含受體量與質的變化。

Serotonin (5-HT) is known to regulate many developmental processes of serotonergic neurons and their target area through its many receptors with distinct characters and functions. In developing rat brain, the axons of serotonergic neuron reach the cortex around embryonic day 16, when neurogenesis is taking place. Previously, it has been shown that serotonin can promote the survival of cortical glutamatergic neurons in culture. Since the expression of NMDA receptor in cortex is after the development of serotonergic neurons, it is possible that the expression of NMDA receptor is under the influence of the activity of 5-HT and its receptors. To test this hypothesis, we used the rat primary cortical cell culture to determine whether long-term exposure of non-selective antagonists of 5-HT1/2 receptor, the methysergide maleate and dihydroergocristine mesylate, affected the expression of the NMDA receptor subunit protein, namely NR1, NR2A and NR2B, and whether the alteration of the receptor affected the sensitivity of cultured cell to the NMDA-induced neurotoxicity. Immunoblotting assay showed that the expressions of NR1, NR2A, and NR2B increased as increase in the day in vitro (DIV), and reached the plateau around DIV10 to 20. 10-day incubation with high concentration of the methysergide maleate (100μM) or dihydroergocristine mesylate (10μM) is cytotoxic to the cortical cell culture. However, low concentration of methysergide maleate (1μM or 10μM) or dihydroergocristine mesylate (1μM) did not produce cell toxicity. Long-term exposure to dihydroergocristine mesylate (1μM) significantly decreases the expression of NR1 and NR2A subunits. LDH assay showed that long-term exposure to methysergide maleate (10μM), dihydroergocristine mesylate (1μM), and pindolol (1μM), a selective antagonist of 5-HT1A and cyproheptadine hydrochloride (1μM), a selective antagonist of 5-HT2A, reduced the sensitivity of cell to NMDA-induced cell toxicity. This result showed that long-term decreasing the activity of serotonin receptor could decrease the expression of NMDA receptor in both quantitative and qualitative manner in developing cortical neuronal cultured cell.

章節目錄

血清素受體對於發育中初級神經細胞NMDA受體表現之影響

中文摘要 III
英文摘要 V
第壹章 緒論 1
壹、 血清素(SEROTONIN) 2
貳、 血清素與血清素受體 (RECEPTORS) 在神經發育中扮演的角色 3
參、 麩氨酸受體 (GLUTAMATE RECEPTOR) 5
肆、 N-METHYL-D-ASPARTIC ACID受體 (NMDA RECEPTOR) 6
伍、 NMDA 受體次單元的分類及組成 7
陸、 NMDA 受體的功能及在神經發育中扮演的角色 8
柒、 實驗計畫目的 9
第貳章 實驗材料及方法 11
壹、 實驗材料 12
貳、 實驗方法 16
第參章 實驗結果 23
壹、 初級大腦皮質神經細胞其NMDA受體各次單元的表現 24
貳、 於體外培養的發育中初級大腦皮質神經細胞加入血清素受體拮抗劑的細胞毒性分析 24
參、 加入血清素受體拮抗劑的發育中初級大腦皮質神經細胞其NMDA受體各次單元的表現量 25
肆、 NMDA加入低濃度血清素受體拮抗劑的發育中初級大腦皮質神經細胞培養試驗 26
第肆章 實驗討論 27
附圖 31
參考文獻 43

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