臺灣博碩士論文加值系統

躁鬱症又稱為雙向情緒型障礙 (Bipolar affective disorder)，是一種常見的複雜型多基因遺傳疾病，患者具有在狂躁及憂鬱兩者相互循環的嚴重情緒障礙，週期性的情緒轉換也嚴重降低患者及其家人的生活品質。在人口比例中約有1~3%患有躁鬱症。過去我們實驗室研究發現指出血清素(人體內主要的神經傳導物質之一)系統基因上的多型性標誌分布和躁鬱症有關連性存在。針對這些血清素系統基因的序列變異的功能性分析，也證實序列變異的確會造成蛋白質功能或表現量異常。另外，在實驗室初步的細胞實驗中也顯示，當神經細胞在抗憂鬱劑(SSRIs)及情緒穩定劑(Lithum)的處理下，血清素含量會迅速增加，證明血清素傳遞系統參與在藥物治療躁鬱症的生理機轉。有趣的是我們發現在血清素含量迅速增加後，細胞會立刻做出反應來調控血清素在細胞內的平衡。這些研究結果皆顯示血清素傳遞系統無法正常調控血清素反應路徑可能與躁鬱症的致病機轉有關。因此本研究的目的主要是觀察在血清素傳遞路徑受到擾動的神經細胞內，血清素系統基因會如何做動態調控以維持細胞內血清素的恆定，藉以來幫助我們瞭解血清素傳遞路徑失常在躁鬱症病理成因上扮演的角色。在本研究中，我們利用NT2D1細胞株成功建立了分化的血清素分泌神經細胞(NT2N)。藉由神經細胞模型來監控細胞內血清素傳遞及恆定情形，並運用外加血清素的方式，擾動分化的神經細胞內血清素的傳遞系統，以模擬躁鬱症患者在狂躁情緒有過量血清素的生理環境。在外加血清素的處理下，我們分析了血清素處理後不同時間點的細胞外分泌血清素濃度、血清素系統基因的訊息核醣核酸(mRNAs)及蛋白質表現量。結果顯示，相較於未進行任何處理的神經細胞，外加血清素的神經細胞組中，血清素的含量大體上增加了六倍，但處理後各個時間點之間的血清素濃度卻沒有明顯差異。過量的血清素環境中，血清素合成酵素(Tryptophan hydroxylase；TPHs)和血清素受體1A (Serotonin receptor；HTR1A)的訊息核醣核酸表現量並沒有改變，然而其中第二血清合成酵素II(TPH2)的蛋白質表現量卻隨著時間有顯著的下降。除此之外，血清素轉運子(Serotonin transporet；SLC6A4)、血清素代謝酵素(Monoamine oxidase；MAOA)及其他血清素受體的訊息核醣核酸和蛋白質表現量在外加血清素的處理下，明顯地與未處理的神經細胞也有所不同。藉由這些實驗結果，我們確立了當血清素傳遞途徑受到過量血清素的干擾時，神經細胞動態調控血清素系統基因來維持細胞內血清素的恆定的網路。這些資訊將幫助我們對躁鬱症的病理成因提出更深層的看法。

Bipolar affective disorder (BPD) is a complex disease which is characterized by recurrent episodes of mania and depression. It affects about 1~3% of the populations worldwide with decreasing the quality of life. Our previous works have demonstrated the sequence variants of serotonergic genes which impair proteins’ function are strongly associated with BPD. The preliminary study using SSRI and lithium treated SH-SY5Y cells suggested that cell needs to maintain its serotonin homeostasis, but the underlying mechanism remains unclear. These results suggested that disruption of serotonin homeostasis leading to abnormal serotonergic transmission may play an important role in BPD etiology. To dissect the regulation of serotonin transmission, current study aims to investigate the dynamic changes of the expression of serotonergic genes in response to the disturbed serotonin transmission in the cells. We have successfully differentiated NT2D1 cells into serotonergic neuronal NT2N cells. The differentiated neuron model was used to monitor the homeostasis and transmission of serotonin in the cells. The terminal differentiated NT2N cells were treated with serotonin solution to mimic the disturbed serotonin transmission of excess serotonin in BPD patients. The extracellular serotonin concentration, mRNA and protein expression of several serotonergic genes were measured. We found the overall serotonin amount increased about six fold in treated cells, but no difference was observed over the time. The mRNA expression of both TPH1, TPH2 and HTR1A were unchanged under the treatment while the TPH2 protein level revealed significantly decreased through the time period of treatment. And the mRNA and protein level of SLC6A4, MAOA and several receptors in treated cells are obviously different from untreated cells. Results from this study demonstrated the network of dynamic regulation of serotonergic genes maintains serotonin homeostasis under the condition of excess amount of serotonin. Results from this study help us to understand how cell maintains serotonin homeostasis, and provide a base for future investigation of BPD etiology

摘 要 I
Abstract III
誌 謝 V
Table of contents VII
List of figures X
List of table XI

1. Introduction 1
1.1. Bipolar affective disorder 1
1.2. Serotonergic system and BPD 2
1.2.1. Genetics studies of serotonergic system and BPD 3
1.2.2. Clinical observations of BPD under drug treatment 3
1.3. Serotonergic transmission system 4
1.4. Serotonergic system genes 5
1.4.1. Tryptophan hydroxylase (TPH) 5
1.4.2. Serotonin transporter (SLC6A4) 6
1.4.3. Monoamine Oxidase A (MAOA) 7
1.4.4. Serotonin receptors (HTRs) 7
1.5. Preliminary study of serotonin homeostasis 9
1.6. Objective of this study 10
2. Materials and methods 11
2.1. Cell culture 11
2.2. Serotonergic neuron model setup 11
2.2.1. Matrigel coating and cell seeding 11
2.2.2. Neuron induction 11
2.2.3. NT2N cells characterization 12
2.3. Serotonin addition 12
2.4. Sample collection 13
2.4.1. Culture medium collection 13
2.4.2. Total RNA extraction 13
2.4.3. Total protein extraction 15
2.5. Serotonin ELISA 15
2.6. Reverse Transcription-PCR 17
2.7. Real-time quantitative PCR (Real-time qPCR) 17
2.7.1. Materials 17
2.7.2. Quantitative RT-PCR procedure 18
2.8. Antibodies 19
2.9. Western blot 19
2.9.1. Protein concentration determination 19
2.9.2. Protein sample preparation 20
2.9.3. Gel electrophoresis 20
2.9.4. Wet transfer 20
2.9.5. Blocking 21
2.9.6. Hybridization 21
2.9.7. Detection 22
2.10. Statistical analysis 22
3. Results 23
3.1. Establish terminal differentiated serotonergic neuron model 23
3.1.1. Morphological changes under differentiation 23
3.1.2. Differentiated NT2N cells express neuronal markers and serotonergic genes 23
3.1.3. Differentiated NT2N cells increase expression of extracellular serotonin 24
3.2. Excess extracellular serotonin amounts after serotonin addition 24
3.3. Alteration of serotonergic genes mRNA level under serotonin addition 25
3.4. Alteration of the levels of serotonergic proteins under serotonin addition 27
4. Discussions 28
4.1. Induction of terminal differentiated human neuron model 28
4.2. Serotonin homeostasis through fluctuated control of serotonergic system 29
4.3. Disturbed serotonergic neurons under excess serotonin 31
4.4. Responses of serotonergic genes under irregular serotonin transmission 32
4.4.1. Tryptophan hydroxylase (TPHs) 32
4.4.2. Serotonin transporter (SLC6A4) 33
4.4.3. Serotonin degrading enzyme (MAOA) 34
4.4.4. Serotonin auto-receptors (HTR1A and HTR1B) 35
4.4.5. Serotonin receptors (HTR6 and HTR7) 37
5. Conclusion 39
6. References 56

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