跳到主要內容

臺灣博碩士論文加值系統

(18.204.48.64) 您好!臺灣時間:2021/08/03 12:05
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

我願授權國圖
: 
twitterline
研究生:盧威仁
研究生(外文):Wei-JenLu
論文名稱:TIAM2S調節小膠質細胞的免疫啟動進而增強神經發炎與神經損傷
論文名稱(外文):TIAM2S exaggerates neuron-inflammation and neural damage through microglial priming
指導教授:孫孝芳孫孝芳引用關係朱俊憲
指導教授(外文):H.sunny SunChun-Hsien Chu
學位類別:碩士
校院名稱:國立成功大學
系所名稱:分子醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2019
畢業學年度:107
語文別:英文
論文頁數:42
中文關鍵詞:T細胞淋巴癌侵略和轉移基因小膠質細胞啟動神經發炎
外文關鍵詞:TIAM genemicroglia primingneuron-inflammation
相關次數:
  • 被引用被引用:0
  • 點閱點閱:29
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
人類T細胞淋巴癌侵略和轉移蛋白(T-cell lymphoma invasion and metastasis; TIAM)隸屬於鳥糞嘌呤核甘酸交換因子(guanine nucleotide exchange factor; GEF)的家族成員。已有文獻指出,其家族成員TIAM1和TIAM2透過其GEF activity能影響周邊免疫細胞的功能,例如:免疫細胞的趨化性(immune cell chemotaxis)。而TIAM1亦被發現在胚胎發育早期對神經發育有重要的影響。在我們先前的研究中指出,短片段的TIAM2 (short form of TIAM2; TIAM2S)具有增強神經可塑性(brain plasticity)的功能。然而TIAM2S是否具有調節中樞系統的神經免疫能力仍未有報導。因此在本研究中,我們探討TIAM2S能否能調節大腦當中的常駐免疫細胞小膠質細胞的免疫特性以及相關機制。首先確認人類小膠質細胞株(human microglia colony 3 cells; HMC3 cells)能表達TIAM2S。我們使用TIAM2S基因轉殖鼠(TIAM2S-TG mice),我們發現到過量表現TIAM2S會造成小鼠大腦中的小膠質細胞突觸長度和其分支增加,並且增強免疫啟動標記(immune priming marker) CD68的表現量。接著我們培養野生型(WT)及TIAM2S過量表現型(TIAM2S-TG)的混合膠質細胞(mixed-glia cells (80% astroglia + 20% microglia)),亦發現到大量表現TIAM2S能改變小膠質細胞的外觀型態,顯示小膠質細胞外觀的改變是由自分泌作用,並非由神經細胞所影響。另外,若將此TIAM2S過量表現型的培養液與神經細胞共同培養,並不會傷害神經細胞的生長。為瞭解小膠質細胞免疫啟動是如何發生,我們利用細胞因子陣列測試(cytokine array tests)測定細胞培養液裡發炎因子表現的表現量。我們發現到,在TIAM2S-TG mixed-glia cells的培養液裡,intercellular adhesion molecule 1 (ICAM-1; CD54)表現量有著顯著的增加。並在基因轉殖鼠的腦中,亦觀察到增加的ICAM-1表現量。而直接處理ICAM-1亦會促使HMC3 cells改變其細胞型態。最後,我們探討由TIAM2S所調節之免疫啟動的小膠質細胞是否在發炎刺激下,有著較野生株細胞產生更強的發炎因子與更易於對神經造成損傷。結果顯示TIAM2S-TG mixed-glia cells在LPS刺激下,小膠質細胞更加活化並且產生更多發炎因子例如C-X-C motif chemokine ligand 12 (CXCL12)、 CXCL13、interleukin 6 (IL-6)、 CXCL2和chemokine C-C motif ligand 1 (CCL1)及Tumor necrosis factor alpha (TNF-α)。若將其細胞培養液與神經細胞共同培養,亦導致促凋亡的cleaved caspase-3表現上升且神經細胞間網絡出現受損的現象。綜上所述,本研究說明TIAM2S能經由調控小膠質細胞的免疫啟動進而影響神經發炎與神經細胞存活的全新功能。
Human T-cell lymphoma invasion and metastasis 2 short form (TIAM2S), which belongs to the TIAM guanine nucleotide exchange factor (GEF) subfamily, functions as a novel neuroplasticity-related protein in neurons. However, the role of TIAM2S in microglia, which are resident macrophages in the brain, remains unknown. Some reports demonstrate that TIAM 1 and 2 affect the chemotaxis of peripheral immune cells via their GEF activity. In this study, we hypothesize that TIAM2S can directly regulate the immune properties of microglia. First, our data showed that TIAM2S protein exists in human microglia colony 3 cells (HMC3 cells) under normal conditions. TIAM2S-TG mice display an alternation of microglial cellular morphology and an increased in expression of CD68, which is an immune priming marker. Microglia in the mixed-glia cells from TIAM2-TG mice also show morphological changes under normal conditions, indicating that neurons don’t participate in TIAM2S-mediated microglia priming. Incubated with the cellular media of TIAM2S-TG mixed-glia cells had no effects on neurons viability. To further determine which soluble factors in cellular media from TIAM2S-TG cells trigger microglia priming, we found that intercellular adhesion molecule 1 (ICAM-1; CD54) was significantly increased at the supernatant level of TIAM2S-TG mixed-glia cells and the hippocampal region of TIAM2S-TG mice by using cytokine arrays and IF staining, respectively. Furthermore, we determined that microglia primed by TIAM2S are more sensitive to inflammatory stimulation, leading to greater neural damage. Our data showed that under an LPS challenge, TIAM2S-primed microglia became overactive, which enhanced the production of inflammatory factors such as C-X-C motif chemokine ligand 12 (CXCL12), CXCL13, interleukin 6 (IL-6), CXCL2 and chemokine C-C motif ligand 1 (CCL1), and tumor necrosis factor alpha (TNF-α) compared to that in LPS-treated WT cells. Importantly, WT neurons incubated with the media of the LPS-treated TIAM2S-TG mixed-glial cells exhibited increased levels of cleaved caspase-3 and neural disconnections compared to neurons incubated with the media from LPS-treated WT mixed-glial cells. Taken together, our data suggested that TIAM2S plays a critical role in regulation of neuroinflammation-mediated neuronal viability through microglia priming.
摘要 i
Abstract iii
誌謝 v
Introduction 1
1. T-cell lymphoma invasion and metastasis (TIAM) family 1
1.1 T-Cell Lymphoma Invasion and Metastasis-1 (TIAM1) 1
1.2 SIF and Tiam1- like exchange factor (Stef) 3
1.3 Human T-Cell Lymphoma Invasion and Metastasis-2 (TIAM2) 3
2. Microglia 4
2.1 Microglial priming 5
3. Objective of this study 6
Materials and methods 8
1. Mice 8
2. Brain cryostat section 8
3. Immunofluorescence staining 9
4. Microglia morphological analysis 9
5. HMC3 and Primary neuron-enriched and mixed-glia and cultures 10
6. Protein extraction and western blot 11
7. Cytokine analysis 11
8. Antibody and Reagents 12
9. Statistical analysis 12
Results 13
1. Mice overexpressing TIAM2S display priming phenotype of microglia 13
2. TIAM2S-primed microglia fail to damage neurons under normal conditions ……………………………………………………………………………..14
3. Increased secretory CD54 may contribute to TIAM2S-mediated microglial priming ……………………………………………………………………………..14
4. TIAM2S-primed microglia enhance LPS-induced neuron-inflammation and neurons damage 15
Discussion 17
References 22
Figures 30
Figure 1. TIAM2S protein expresses in human microglial cells. 30
Figure 2. Overexpression of TIAM2S changes microglial morphology and increases the CD68 marker 33
Figure 3. Soluble factors from the TIAM2S-overexpressd cells affect microglial activation, but not neuron viability. 36
Figure 4. CD54 plays a role in TIAM2S-mediated microglial activation 39
Figure 5. TIAM2S-primed microglia enhance LPS-induced neuroninflammation and neuron damage 42
Ambrosini, E., Remoli, M. E., Giacomini, E., Rosicarelli, B., Serafini, B., Lande, R., . . . Coccia, E. M. (2005). Astrocytes produce dendritic cell-attracting chemokines in vitro and in multiple sclerosis lesions. Journal of Neuropathology and experimental Neurology, 64(8), 706-715.
Bilbo, S. D. (2010). Early-life infection is a vulnerability factor for aging-related glial alterations and cognitive decline. Neurobiology of Learning and Memory, 94(1), 57-64.
Boespflug, N. D., Kumar, S., McAlees, J. W., Phelan, J. D., Grimes, H. L., Hoebe, K., . . . Karp, C. L. (2014). ATF3 is a novel regulator of mouse neutrophil migration. Blood, 123(13), 2084-2093.
Boissier, P., & Huynh-Do, U. (2014). The guanine nucleotide exchange factor Tiam1: a Janus-faced molecule in cellular signaling. Cell Signal, 26(3), 483-491.
Carter, B. J., Anklesaria, P., Choi, S., & Engelhardt, F. (2009). Redox Modifier Genes and Pathways in Amyotrophic Lateral Sclerosis. ANTIOXIDANTS & REDOX SIGNALING, 11(7), 1569-1586.
Chapman, K. Z., Ge, R., Monni, E., Tatarishvili, J., Ahlenius, H., Arvidsson, A., . . . Kokaia, Z. (2015). Inflammation without neuronal death triggers striatal neurogenesis comparable to stroke. Neurobiology of Disease, 83, 1-15.
Chen, J. S., Su, I. J., Leu, Y. W., Young, K. J., & Sun, H. (2012). Expression of T-cell lymphoma invasion and metastasis 2 (TIAM2) promotes proliferation and invasion of liver cancer. International Journal of Cancer, 130, 1302-1313.
Chen, S. H., Oyarzabal, E. A., & Hong, J.-S. (2013). Preparation of Rodent Primary Cultures for Neuron–Glia, Mixed Glia, Enriched Microglia, and Reconstituted Cultures with Microglia. Methods in Molecular Biology, 1041, 231-240.
Cheon, M. S., Kim, S. H., Yaspo, M.-L., Blasi, F., Aoki, Y., Melen, K., & Lubec, G. (2003). Protein levels of genes encoded on chromosome 21 in fetal Down syndrome brain: Challenging the gene dosage effect hypothesis. Amino Acids, 24, 111-117.
Chiu, C. Y., Leng, S., Martin, K. A., Kim, E., Gorman, S., & Duhl, D. M. (1999). Cloning and Characterization of T-Cell Lymphoma Invasion and Metastasis 2 (TIAM2), a Novel Guanine Nucleotide Exchange Factor Related to TIAM1. Genomics, 61(1), 66-73.
Combrinck, M. I., Perry, V. H., & Cunningham, C. (2002). Peripheral infection evokes exaggerated sickness behaviour in pre-clinical murine prion disease. Neuroscience, 112, 7-11.
Crews, F. T., & Vetreno, R. P. (2016). Mechanisms of neuroimmune gene induction in alcoholism. Psychopharmacology, 233, 1543-1557.
Cunningham, C. (2013). Microglia and neurodegeneration: the role of systemic inflammation. Glia, 61(1), 71-90.
Cunningham, C., Wilcockson, D. C., Campion, S., Lunnon, K., & Perry, V. H. (2005). Central and Systemic Endotoxin Challenges Exacerbate the Local Inflammatory Response and Increase Neuronal Death during Chronic Neurodegeneration. Neurobiology of Disease, 25(40), 9275-9284.
Ding, Y., Chen, B., Wang, S., Zhao, L., Chen, J., Ding, Y., . . . Luo, R. (2009). Overexpression of Tiam1 in hepatocellular carcinomas predicts poor prognosis of HCC patients. International Journal of Cancer, 124(3), 653-658.
Dunckley, T., Huentelman, M. J., Craig, D. W., Pearson, J. V., Szelinger, S., Joshipura, K., . . . Stephan, D. A. (2007). Whole-Genome Analysis of Sporadic Amyotrophic Lateral Sclerosis. The new england journal of medicine, 357(8).
Ehler, E., van Leeuwen, F., Collard, J. G., & Salinas, P. C. (1997). Expression of Tiam-1in the Developing Brain Suggests a Role for the Tiam-1–Rac Signaling Pathway in Cell Migration and Neurite Outgrowth. Molecular and Cellular Neuroscience, 9, 1-12.
Espagnolle, N., Balguerie, A., Arnaud, E., Sensebé, L., & Varin, A. (2017). CD54-Mediated Interaction with Pro-inflammatory Macrophages Increases the Immunosuppressive Function of Human Mesenchymal Stromal Cells. Stem Cell Reports, 8(4), 961-976.
Gerard, A., van der Kammen, R. A., Janssen, H., Ellenbroek, S. I., & Collard, J. G. (2009). The Rac activator Tiam1 controls efficient T-cell trafficking and route of transendothelial migration. Blood, 113(24), 6138-6147. doi:10.1182/blood-2008-07-167668
Ginhoux, F., Greter, M., Leboeuf, M., Nandi, S., See, P., Gokhan, S., . . . Merad, M. (2010). Fate mapping analysis reveals that adult microglia derive from primitive macrophages. Science, 330(6005), 841-845.
Glass, C. K., & Natoli, G. (2015). Molecular control of activation and priming in macrophages. Nature Immunology, 17(1), 26-33.
Godbout, J. P., Chen, J., Abraham, J., Richwine, A. F., Berg, B. M., Kelly, K. W., & Johnson, R. W. (2005). Exaggerated neuroinflammation and sickness behavior in aged mice following activation of the peripheral innate immune system. Fed. Am. Soc. Exp. Biol., 19, 1329-1331.
Grönholm, M., Jahan, F., Marchesan, S., Karvonen, U., Aatonen, M., Narumanchi, S., & Gahmberg, C. (2011). TCR-Induced Activation of LFA-1 Involves Signaling through Tiam1. The Journal of Immunology, 187(7), 3613-3619.
Griffin, W. S., Stanley, L. C., Ling, C., White, L., Macleod, V., Perrot, L. J., . . . Aroaz, C. (1989). Brain interleukin 1 and S-100 immunoreactivity are elevated in Down syndrome and Alzheimer disease Proceedings of the National Academy of Sciences of the United States America, 86, 7611-7615.
Habets, G. G. M., van der Kämmen, R. A., Jenkins, N. A., Gilbert, D. J., Copeland, N. G., Hagemeijer, A., & Collard, J. G. (1995). The invasion-inducing TlAM 1 gene maps to human chromosome band 21 q22 and mouse Chromosome 16 Cytogenet Cell Genet, 70, 48-51.
Habets, G. G. M., van der Kämmen, R. A., Stam, J. C., Micheals, F., & Collard, J. G. (1995). Sequence of the human invasion-inducing TIAM1 gene, its conservation in evolution and its expression in tumor cell lines of different tissue origin. Oncogene, 10(7), 1371-1376.
Henry, C. J., Huang, Y., Wynne, A. M., & Godbout, J. P. (2009). Peripheral lipopolysaccharide (LPS) challenge promotes microglial hyperactivity in aged mice that is associated with exaggerated induction of both pro-inflammatory IL-1β and anti-inflammatory IL-10 cytokines. Brain, Behavior, and Immunity, 23(3), 309-317.
Huber, A. K., & Irani, D. N. (2015). Targeting CXCL13 During Neuroinflammation. Advance in Neuroimmune Biology, 6(1), 1-8.
Kawauchi, T., Chihama, K., Nabeshima, Y., & Hoshino, M. (2003). The in vivo roles of STEF/Tiam1, Rac1 and JNK in cortical neuronal migration. EMBO J., 22(16), 4190-4201.
Kothur, K., Wienholt, L., Brilot, L., & Dale, R. C. (2016). CSF cytokines/chemokines as biomarkers in neuroinflammatory CNS disorders: A systematic review. Cytokine, 77, 227-237.
Kunda, P., Paglini, G., Quiroga, S., Kosik, K., & Cáceres, A. (2001). Evidence for the Involvement of Tiam1 in Axon Formation. The Journal of Neuroscience, 21(7), 2361-2372.
Kurdi, A. T., Bassil, R., Olah, M., Wu, C., Xiao, S., Taga, M., . . . Elyaman, W. (2016). Tiam1/Rac1 complex controls Il17a transcription and autoimmunity. Nature Communications, 7(13048).
Li, M., & Ransohoff, R. M. (2008). Multiple roles of chemokine CXCL12 in the central nervous system: A migration from immunology to neurobiology. Progress in Neurobiology, 84(2), 116-131.
Loane, D. J., Kumar, A., Stoica, B. A., Cabatbat, R., & Faden, A. I. (2014). Progressive Neurodegeneration After Experimental Brain Trauma: Association With Chronic Microglial Activation Journal of Neuropathology & Experimental Neurology, 73(1), 14-29.
Lott, I. T., Head, E., Doran, E., & Busciglio, J. (2006). Beta-Amyloid, Oxidative Stress and Down Syndrome. Current Alzheimer Research, 3(5), 521-528.
Luo, H. R. (2014). A dual regulator of neutrophil recruitment. Blood, 123(13), 1983-1985.
Martin, E., Boucher, C., Fontaine, B., & Delarasse, C. (2017). Distinct inflammatory phenotypes of microglia and monocyte‐derived macrophages in Alzheimer's disease models: effects of aging and amyloid pathology. Aging Cell, 16(1), 27-38.
Mertens, A. E., Roovers, R. C., & Collard, J. G. (2003). Regulation of Tiam1-Rac signalling. FEBS Letters, 546(11-16).
Miklossy, J., Doudet, D. D., Schwab, C., Yu, S., McGeer, E. G., & McGeer, P. L. (2006). Role of ICAM-1 in persisting inflammation in Parkinson disease and MPTP monkeys. Experimental Neurology, 197(2), 275-283.
Minard, M. E., Kim, L. S., Price, J. E., & Gallick, G. E. (2004). The role of the guanine nucleotide exchange factor Tiam1 in cellular migration, invasion, adhesion and tumor progression. Breast Cancer Research Treat, 84(1), 21-32.
Nimmerjahn, A., Kirchhoff, F., & Helmchen, F. (2005). Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo. Science, 308(5726), 1314-1318.
Nishimura, T., Yamaguchi, T., Kato, K., Yoshizawa, M., Nabeshima, Y., Ohno, S., . . . Kaibuchi, K. (2005). PAR-6-PAR-3 mediates Cdc42-induced Rac activation through the Rac GEFs STEF/Tiam1. Nature Cell Biology, 7(3), 270-277.
Püntener, U., Booth, S. G., Perry, V. H., & Teeling, J. L. (2012). Long-term impact of systemic bacterial infection on the cerebral vasculature and microglia. Journal of Neuroinflammation, 9(146).
Perry, V. H. (2010). Contribution of systemic inflammation to chronic neurodegeneration. Acta Neuropathologica, 120, 277-286.
Qi, Y., Huang, B., Yu, L., Wang, Q., Lan, G., & Zhang, Q. (2009). Prognostic Value of Tiam1 and Rac1 Overexpression in Nasopharyngeal Carcinoma. ournal for Oto-rhino-laryngology and its Related Specialties, 71(3), 163-171.
Ramos, T. N., Bullard, D. C., & Barnum, S. R. (2014). ICAM-1: Isoforms and Phenotypes. The Journal of Immunology, 192(10), 4469-4474.
Rossman, K. L., Der, C. J., & Sondek, J. (2005). GEF means go: turning on RHO GTPases with guanine nucleotide-exchange factors. Nature Reviews Molecular Cell Biology, 6, 167-180.
Sawant, K. V., Xu, R., Cox, R., Hawkins, H., Sbrana, E., Kolli, D., & Garofalo, R. P. (2015). Chemokine CXCL1-Mediated Neutrophil Trafficking in the Lung: Role of CXCR2 Activation. Journal of Innate Immunity, 7, 647-658.
Schafer, D. P., & Stevens, B. (2013). Phagocytic glial cells: sculpting synaptic circuits in the developing nervous system. Current Opinion in Neurobiology, 23(6), 1034-1040.
Shimoji, M., Pagan, F., Healton, E. B., & Mocchetti, I. (2009). CXCR4 and CXCL12 Expression is Increased in the Nigro-Striatal System of Parkinson’s Disease. Neurotoxicity Research, 16(3), 318-328.
Ślusarczyk, J., Trojan, E., Glombik, K., Budziszewska, B., Kubera, M., Lasoń, W., . . . Basta-Kaim, A. (2015). Prenatal stress is a vulnerability factor for altered morphology and biological activity of microglia cells. Frontiers in Cellular Neuroscience, 9(82).
Terawaki, S., Kitano, K., Mori, T., Zhai, Y., Higuchi, Y., Itoh, N., . . . Hakoshima, T. (2010). The PHCCEx domain of Tiam1/2 is a novel protein‐ and membrane‐binding module. EMBO J., 29(1), 236-250.
Van Seventer, G. A., Shimizu, Y., Horgan, K. J., & Shaw, S. (1990). The LFA-1 ligand ICAM-1 provides an important costimulatory signal for T cell receptor-mediated activation of resting T cells. The Journal of Immunology, 144(12), 4579-4586.
Wachholz, S., Eßlinger, M., Plümper, J., Manitz, M. P., Juckel, G., & Friebe, A. (2016). Microglia activation is associated with IFN-α induced depressive-like behavior. Brain, Behavior, and Immunity, 55, 105-103.
Wilcock, D. M., & Griffin, S. T. (2013). Down’s syndrome, neuroinflammation, and Alzheimer neuropathogenesis. Journal of Neuroinflammation, 10, 864.
Wright, H. L., Thomas, H. B., Moots, R. J., & Edwards, S. W. (2013). RNA-Seq Reveals Activation of Both Common and Cytokine-Specific Pathways following Neutrophil Priming. PLoS One, 8(3), e58598.
Yang, L., Wang, M., Guo, Y. Y., Sun, T., Li, Y. J., Yang, Q., . . . Wu, Y. M. (2016). Systemic inflammation induces anxiety disorder through CXCL12/CXCR4 pathway. Brain, Behavior, and Immunity, 56, 352-362.
Yoo, S., Kim, Y., Lee, H., Park, S., & Park, S. (2012). A Gene Trap Knockout of the Tiam-1 Protein Results in Malformation of the Early Embryonic Brain. Molecules and Cells, 34, 103-108.
Zaldua, N., Gastineau, M., Hoshino, M., Lezoualc'h, F., & Zugaza, J. L. (2007). Epac signaling pathway involves STEF, a guanine nucleotide exchange factor for Rac, to regulate APP processing. FEBS Letters, 581(30), 5814-5818.
Zhang, K., Tian, L., Liu, L., Feng, Y., Dong, Y. B., Li, B., . . . Chen, Y. H. (2013). CXCL1 Contributes to β-Amyloid-Induced Transendothelial Migration of Monocytes in Alzheimer’s Disease. PLoS One, 8(8), e72744.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
無相關論文
 
無相關期刊
 
無相關點閱論文