背根神經節（DRG）上的初級感覺神經元通常負責感覺從環境中有害的刺激，但新出現的證據表明，它亦負責對危險的感覺。Toll-樣受體-4（TLR4）通常見於小膠質細胞，它是負責識別病原體相關分子模式（PAMPs）或損傷相關分子模式（DAMPs）並誘發炎症。奇怪的是，儘管TLR4在中樞神經系統通常見於神經膠質細胞，在DRG發現的TLR4僅見於初級感覺神經元，但從未見於周邊的衛星膠質細胞（SGC）。而重要的是，在感覺神經節中激活TLR4是會導致神經病理性疼痛的，但我們仍然未知道初級感覺神經元上的TLR4是否導致疼痛的唯一來源。本研究旨在探討在DRG細胞的TLR4信號傳導的分子和細胞機理，並探討在DRG的神經元和膠質細胞上TLR4活動的差異，在生物學上有甚麼意義。 / 為了研究在DRG神經元和膠質細胞的相互作用，我們首先在一個既定的混合DRG細胞培養模型上研究了谷氨酰胺合成酶（ GS ）的表達模式。GS是一種只會在SGC上表達的特異性酶，並於神經元和神經膠質細胞之間的谷氨酰胺 - 谷氨酸循環產生相互作用。在典型的DRG細胞培養，神經元通過擴散因子促進了GS在神經膠質細胞上的表達，然而，GS的表達亦受到TLR4激動劑，即脂多醣（LPS），的抑制。這表明DRG神經元和神經膠質細胞的關係受到TLR4介導的炎症之影響。在混合DRG細胞中，我們對TLR4-免疫反應（IR）進行了鑑定，發現TLR4最主要的是表達在神經元細胞的表面。另外，LPS（ 1微克/毫升，2小時）會刺激混合DRG細胞，通過在DRG細胞中MyD88依賴性信令，誘導環加氧酶-2（COX -2），白細胞介素-1β（ IL-1β）和腫瘤壞死因子-α（TNFα）的轉錄。此外，在DRG細胞， LPS（ 1微克/毫升， 24小時）亦會觸發依賴COX-2 的前列腺素E2（PGE₂）和的前列環素（PGI₂）的產生。但在LPS刺激後，我們發現DRG神經元和神經膠質細胞都對 COX-2-IR呈陽性反應。這證明DRG神經膠質細胞對TLR4誘發的神經炎症也擔任一定的角色。 / 為了純粹研究神經膠質細胞有沒有任何TLR4活性，我們把神經元從混合DRG細胞中除去，從而把神經膠質細胞純化。出乎意料的是，在純化後，大約80的神經膠質細胞對TLR4 -IR呈陽性反應。而且，時間和濃度依賴性的研究表明，純化後的神經膠質細胞對LPS刺激的COX-2表達反應在有效性和效率上比混合DRG細胞的顯著更高。明顯地，神經元對神經膠質細胞的TLR4活性有抑制作用。我們並且發現，神經元的抑制作用是透過由細胞與細胞之間的接觸介導，而不是由擴散因子介導。 / 重要的是， LPS也能誘導純化後的神經膠質細胞去產生依賴COX-2活性的前列腺素E2（PGE₂）。反過來， PGE₂能區別地調節依賴TLR4的炎症基因轉錄，說明在DRG 由TLR4介導的神經炎症是受多重複雜的機理控制。然而有趣的是，從受熱休克性損害的感覺神經元所收集的培養基可以激活純化膠質細胞，並通過對TLR4局部依賴性的方式，誘導COX-2的轉錄。此外，我們利用斑馬魚作為疼痛行為反應的模型，發現COXs的活性與瞬時受體電位通道亞家族V1（ TRPV1）有密切關係。斑馬魚幼蟲的疼痛行為反應是一個適合於篩選新型鎮痛化合物的體內模型。 / 總括來說，透過細胞與細胞之間的接觸和擴散因子，感覺神經元可以控制神經膠質細胞的表型。我們的研究確定感覺神經元是在DRG中表達TLR4的主要細胞類型，但當神經元施加的抑制被削弱，SGC可以成為完全勝任TLR4信息傳遞的細胞。因此我們推測TLR4的活性在DRG中被嚴格調控，以防止不必要的神經炎症發生。至於未來，我們認為在DRG中的TLR4/COX-2/PGE₂信號通路可以成為研究方的新型鎮痛化合物的方向。而轉基因斑馬魚則可用作篩選新型鎮痛化合物的工具。 / Primary sensory neurons of the dorsal root ganglia (DRG) are classically responsible for the detection of physiological stimuli from the environment, but emerging evidences suggests that they are also involved in the sensation of danger. Toll-like receptor 4 (TLR4) is commonly found on microglia for the recognition of pathogen- or damage- associated molecular patterns (PAMPs or DAMPs) and to the activation of TLR4 leads to inflammation. Curiously, while commonly found in glial cells in central nervous system, TLR4 expression was only found in primary sensory neurons but not the satellite glial cells (SGCs) in the DRG. Importantly, activation of TLR4 in sensory ganglia mediates neuropathic pain, but it remains unknown whether neurons are the only source of TLR4 activity. The present study aims to study the cellular and molecular mechanism(s) of TLR4 signalings and explore the biological significance of differential cellular TLR4 activity in the DRG. / To investigate neuron-glia interactions in the DRG, the expression of glutamine synthetase (GS), a SGC-specific enzyme in the glutamine-glutamate shuttle between neuron and glia, was studied in an established model of mixed DRG cells culture. In typical mixed DRG cell cultures, neurons promoted the GS expression in glial cells through diffusible factors. However, GS expression was negatively regulated by theTLR4 agonist, lipopolysaccharide (LPS), indicative of a change in neuron-glia relationships by TLR4 mediated inflammation. In mixed DRG cells, cell surface TLR4-immunoreactivity (-ir) was predominantly identified on the neurons. LPS (1 μg/mL, 2 h) stimulation induced cyclooxygenases-2 (COX-2), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNFα) transcription through MyD88-dependent signalings in DRG cells. Furthermore, LPS (1 μg/mL, 24 h) triggered COX-2-dependent production of prostaglandin E₂ (PGE₂) and prostacyclin (PGI₂) in mixed DRG cells. / To study the TLR4 activity of glial cells, glial cell cultures were purified by removing neurons from mixed DRG cell culture. Unexpectedly, approximately 80% of purified glial cells become TLR4-ir positive. Moreover, a time- and concentration-dependent study showed that the efficacy and efficiency of purified glial cells to express COX-2 in response to LPS was significantly higher than that of mixed DRG cells. We found that neuron inhibited glial cells through cell-cell contact, but not by diffusible factors. Importantly, LPS also induced COX-2 dependent PGE₂ production in purified glial cells. In turn, PGE₂ can differentially modulate TLR4-dependent gene transcription, suggestive of a complex regulation of TLR4-mediated inflammation in the DRG. Intriguingly, conditioned media from heat-shocked damaged sensory neurons activated purified glial cells to induce COX-2-transcription through a partially TLR4-dependent mechanism. Using zebrafish as a model of nocifensive behavior, we found that the activity of COXs was closely associated with the transient receptor potential channel subfamily V1 (TRPV1), and the nocifensive behavior of zebrafish larvae is suitable for in vivo screening of novel analgesic compounds. / To conclude, sensory neurons regulate the phenotypes of DRG glial cells through cell-cell contact and diffusible factors. Here, sensory neurons are confirmed to be the predominant cell type expressing TLR4 in the DRG, but SGCs become fully competent for TLR4 signalings when the neuronal inhibitions are diminished. We therefore hypothesize that TLR4 activity is tightly regulated in the DRG to prevent unwanted neuroinflammation. Future studies with genetically modified zebrafish can be used for the screening of novel analgesic compound targeting the TLR4/COX-2/PGE₂ signaling pathway. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Tse, Kai Hei. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 190-222). / Abstracts also in Chinese.
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_1077648 |
| Date | January 2014 |
| Contributors | Tse, Kai Hei (author.), Rudd, John Anthony (thesis advisor.), Chinese University of Hong Kong Graduate School. Division of Biomedical Sciences, (degree granting institution.) |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography, text |
| Format | electronic resource, electronic resource, remote, 1 online resource (xi, 222 leaves) : illustrations (some color), computer, online resource |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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