N-甲基-D-天門冬胺酸鹽受器活化對視前區性別差異性神經核的基因調控

Gene Regulation By NMDA Receptor Activation In The SDN-POA During Sexual Development

邵佩琳(Shao Pei-Lin)

指導教授 ： 許勤 教授

高雄醫學大學/醫學院/醫學研究所/碩士(2003年)

摘要

摘要
雄鼠視前區性別差異性神經核大於雌鼠約七倍。造成此核區差異的原因為初生期間雄鼠神經元凋亡數目比雌鼠少。本實驗室最近的研究結果指出雄鼠NMDA受器表現量較多在保護神經元免於凋亡扮演重要的角色。NMDA受器活化導致大量鈣離子進入細胞並間接活化轉錄因子如NFκB及CREB，這些轉錄因子調控凋亡有關的基因。本論文主要釐清生理性活化NMDA受器可能參與保護神經元免於凋亡的訊息傳遞路徑。以suppression subtractive hybridization (SSH) 及RT-PCR 證實將NMDA受器阻斷之後表現受影響的基因；以Western blot analysis比較正常雌、雄鼠及MK-801處理雌、雄鼠之間Bcl-2蛋白質表現量；以electro-mobility shift assay (EMSA)分析NFκB及CREB的DNA結合活性。結果顯示：（1）SSH後篩選出NMDA受器阻斷之後表現被抑制的基因包括促神經生長的基因（neurotrophic genes）如RBM3, alpha-tubulin及凋亡相關基因（apoptosis-related genes）如Bcl-2, cytochrome oxidase subunit II, cytochrome oxidase subunit III；（2）RT-PCR結果亦證實上述基因在MK-801處理雄鼠表現量少於正常雄鼠；（3）雄鼠Bcl-2 mRNA及蛋白質表現量在MK-801處理之後皆顯著減少；（4）正常雄鼠NFκB的DNA結合活性顯著大於雌鼠、MK-801處理雄鼠及MK-801處理雌鼠；（5）CREB的DNA結合活性正常雌、雄鼠及MK-801處理雌、雄鼠之間並無顯著差異。這些結果顯示性別發育過程NMDA受器活化可能調控基因參與神經元的生長及/或抗凋亡；且NFκB活化並turn on它的目標基因Bcl-2可能是保護神經細胞免於凋亡重要的訊息傳遞路徑之一。其他基因參與NMDA受器調控的性別發育過程的角色須進一步釐清。

關鍵字

N-甲基-D-天門冬胺酸鹽受器 ； 視前區性別差異性神經核

並列摘要

Abstract
The sexual dimorphic nucleus of the POA (SDN-POA) of male rats exhibits about a seven-fold greater nuclear volume than females. This difference in the nuclear volume has been attribute to the less neonatal neuronal apoptosis in male rats than that in females. Recently, we provided evidence that the predominant expression of NMDA receptor in male rats plays an important role in preventing neurons from apoptosis in the SDN-POA during sexual development. Activation of NMDA receptor causes calcium influx and may subsequently activate some transcriptional factors such as NFκB or CREB, which may regulate apoptosis-related genes. Therefore, the present study is designed to find out the possible signaling pathways, which may be involved in preventing neurons from apoptosis after physiological activation of NMDA receptor. Genes responsive to the blockage of the NMDA receptor by antagonist (MK-801) were screened after suppression subtractive hybridization (SSH) and then confirmed by RT-PCR. The protein expression of the bcl-2 was further compared among intact male, MK-801 treated male, intact female and MK-801 treated female rats by Western blot analysis. The DNA binding activities of NFκB and CREB were evaluated by electro-mobility shift assay (EMSA). The results showed that (1) differential screen after SSH finds out that some neurotrophic genes (RBM3, alpha-tubulin) as well as apoptosis-related genes (Bcl-2, cytochrome oxidase subunit II, cytochrome oxidase subunit III) were downregulated by blocking NMDA receptor. (2) The RT-PCR products of aforementioned genes in MK-801 treated male was significantly less than that in intact male. (3) Especially, the expressions of Bcl-2 mRNA including Bcl-2 protein of male rats were significantly suppressed by MK-801 treatment. (4) The binding activity of NFκB was significantly higher in male rat than that in females and the binding activity of NFκB in male rat was significantly diminished by blocking NMDA receptor with MK-801. (5) No significant difference of CREB binding activity was observed among male, MK-801 treated male, female and MK-801 treated female groups. These results suggested that genes regulated by NMDA receptor activation might participate in the neuronal growth and/or anti-apoptosis during sexual development and supported an important signaling pathway of NFκB activation and its target gene, Bcl-2, in preventing neurons in SDN-POA from apoptosis. The role of other genes in participating NMDA receptor-mediated sexual development needs further investigation.

並列關鍵字

NMDAR：N-methyl-D-aspartate receptor ； SDN-POA：sexual dimorphic nucleus of preoptic area

參考文獻

參考文獻

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