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研究生：	莊媛茹 Chuang, Yuan-Yu
論文名稱：	人類TIAM2基因在發育時期血清素神經纖維的作用探討 The role of human TIAM2S gene in the developing of serotonergic fibers
指導教授：	朱俊憲 Chu, Chun-Hsien
學位類別：	碩士 Master
系所名稱：	醫學院 - 分子醫學研究所 Institute of Molecular Medicine
論文出版年：	2019
畢業學年度：	108
語文別：	英文
論文頁數：	55
中文關鍵詞：	血清素纖維、TIAM2 、早期生長發育
外文關鍵詞：	serotonergic fibers, TIAM2 gene, postnatal development
相關次數：	點閱：100 下載：0
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T細胞淋巴瘤侵襲和轉移2（TIAM2S）蛋白在健康的非腫瘤性腦組織中呈現高表達量。在先前的研究中，全基因轉錄組(whole-transcriptome)和基因功能富集分析(gene set enrichment analysis)結果顯示，TIAM2S-knockdown（TIAM2S-KD）與腦結構發育和分化、血清素相關信號傳導以及代表神經行為發展的疾病標誌物有密切相關。由於血清素(serotonin, 5-HT)對於5-羥色胺能神經元(serotonergic neurons)的末端發育至關重要。因此我們假設TIAM2S可以調節出生後，5-羥色胺能纖維(serotonergic fibers)的生長以及成年後的5-羥色胺的表現量。首先，透過重建3D圖像分析對血清素進行免疫熒光（IF）染色，於出生後第14天和第28天過表達人類TIAM2S蛋白（TIAM2S-TG小鼠）的WT和TG小鼠中測量八個的大腦區域（putamen, globus pallidus, primary somatosensory cortex layer IV, hippocampus, basolateral amygdala, medial prefrontal cortex, substantial nigra, and dorsal lateral geniculate nucleus）的血清素神經纖維的數量和總長度。實驗結果顯示，在出生後第14天，WT和TIAM2S-TG小鼠在大部分腦區中，小鼠的血清素纖維數量和總長度相似。有趣的是，TIAMS-TG小鼠在出生後第28天顯示出血清素纖維的數量和長度增加，特別是在basolateral amygdala、dorsal lateral geniculate、hippocampus及substantial nigra之大腦區域。此外，同時運用HPLC測量成年之WT和TIAM2S-TG小鼠hippocampus中的血清素濃度。實驗數據顯示，與WT小鼠（291.6 pg / mg）相比，TIAM2S-TG小鼠hippocampus的血清素濃度（353.9 pg / mg）約高21％。另外，TIAM2S-KD NT2/D1細胞中的血清素比shRNA_Leu對照細胞中的血清素低約50％（圖3G，P <0.01，t = 4.51，Student's t-test）。
儘管TIAM2S-TG小鼠在發育和生長過程中，大腦外觀正常，但經實驗結果進一步顯示，當TIAM2S過表達，其調節出生後血清素神經纖維(serotonergic fibers)的末端發育進而改變血清素分布走向，並提高了成年時期血清素的表現量。然而，人類TIAM2S對於血清素纖維的調控機制更值得進一步研究。簡言之，本研究顯示人類TIAM2S可作為新發現的調節子，增強出生後血清素神經纖維和血清素的增長。

The short form of the T-cell lymphoma invasion and metastasis 2 (TIAM2S) protein is abundant in healthy, non-neoplastic brain tissue. Previously, a whole-transcriptome and enrichment analysis with total RNA sequencing revealed that TIAM2S-knockdown (TIAM2S-KD) is strongly associated with the cellular processes related to the structural development and differentiation of the brain, serotonin-related signaling, and the markers of diseases representing neurobehavioral developmental disorders. Since the serotonin signal is critical for the terminal field development of serotonergic neurons, we determined whether TIAMS can regulate the postnatal stages of serotonergic fiber growth as well as the subsequent serotonin levels in adulthood. First, immunofluorescence (IF) staining for serotonin and an analysis of reconstructed 3D images was used to measure the number and total length of serotonergic fibers in eight region-matched brain areas (putamen, globus pallidus, primary somatosensory cortex layer IV, hippocampus, basolateral amygdala, medial prefrontal cortex, substantial nigra, and dorsal lateral geniculate nucleus) in WT and TG mice overexpressing human TIAM2S protein (TIAM2S-TG mice) at postnatal days 14 and 28. Our data showed that at postnatal day 14, the WT and TIAM2S-TG mice had a similar number and total length of serotonergic fibers among these brain regions. Interestingly, TIAMS-TG mice displayed increased number and length of serotonergic fibers at postnatal day 28, especially in the basolateral amygdala, dorsal lateral geniculate nucleus, hippocampus, and substantial nigra brain regions. Second,
we applied HPLC to carefully measure the serotonin levels in the hippocampus of WT and TIAM2S-TG mice at young adulthood. Our data revealed that the TIAM2S-TG mice had a ~21% higher serotonin concentrations in the hippocampus (353.9 pg/mg) compared with the WT mice (291.6 pg/mg). Furthermore, the serotonin level in the TIAM2S-KD NT2/D1 cells was ~50% lower than that in the shRNA_Leu control cells. Although the TIAM2S-TG mice had normal brain appearance during development and growth, our results further revealed that TIAM2S overexpression altered the serotonergic projection through modulation of the terminal field development of serotonergic neurons at postnatal stages in order to the elevation of serotonin level in adulthood. Nevertheless, determining the regulatory mechanisms related to the development of serotonergic fibers by human TIAM2S is of interest for further investigation. In conclusion, the present study demonstrates that human TIAM2S acts as a novel regulator to enhance postnatal growth of serotonergic fibers and serotonin levels.

中文摘要	I
Abstract	III
誌  謝	V
Table of Contents	1
Introduction	4
1 Human T-cell lymphoma invasion and metastasis 2 (TIAM2)	4
2 Serotonin system in CNS	4
2.1 Serotonergic transmission system	5
2.2 Serotonergic neuron/fiber	5
3 The function of TIAM subtypes in the neurons of human brains	6
3.1 TIAM1 (T-cell lymphoma invasion and metastasis 1)	6
3.2 Stef (Sif- and Tiam1-like exchange factor)	7
4 The function of TIAM2 in the neurons of the human brain	7
5 Objective of this study	8
6 The aims of this study	8
Materials and Methods	9
1 Cell culture	9
2 CRISPR interfere system (CRISPRi)	9
2.1 sgRNA design and CRISPRi all-in-one vector	9
2.2 Lentiviral delivery and limit dilution	9
3 Sample collection	10
3.1 Total RNA extraction	10
3.2 Total protein extraction	10
4 Serotonin ELISA	11
4.1 Acylation of Samples and Controls	11
4.2 Coating samples into ELISA detective plate	11
4.3 Serotonin detection using a photometer	12
5 Animal models	13
6 Western blot and Antibodies	13
7 Immunofluorescence staining and 3D-reconstruction imaging	14
8 Statistical Analysis	14
Results	15
1 Changes in serotonergic fiber projection during the development of the post-natal brain in TG mouse models	15
1.1 Effect of TIAMS overexpression on he serotonergic fiber projection in the mouse brain region at the P14 stage	15
1.2 TIAM2S overexpression alters phenomenon and process of serotonergic fibers in the P28 stage	16
2 TIAM2S involves in regulation of postnatal growth of serotonergic fiber projection	17
3 A novel role of TIAM2S in regulating serotonin level	17
Discussion	18
1 Establishment of the TIAM2S-KD cells using the CRISPRi system	18
2 TIAM2S, serotonin and serotonin fibers	19
Conclusion	22
Figures	23
Figure 1. The brain regions of serotonergic fibers measured in this study.	23
Figure 2. Serotonergic fiber projection in the P14 TG mouse models	32
Figure 3. Serotonergic fibers projection in P28 age TG mouse model	41
Figure 4. Changes in the projection of serotonin fiber in the development of the brain nerve system	43
Figure 5. Serotonin level in the TIAM2S mouse brain	44
Figure 6. Both TIAM2 and serotonin were decreased in the knockdown stable clone	45
Supplemental Figures	46
Figure S1. Establishment of TIAM2 knockdown stable clone using the CRISPRi system	48
References	49
Appendix Tables	55
Appendix Tables 1. sgRNA used to knockdown TIAM2S in the CRISPRi system	55



                                    

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