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標題: | 探討IL-15對於表皮層之先天性免疫反應調控 Regulation of innate immune response in the epidermis by IL-15 |
作者: | Tsung-Lin Lee 李宗霖 |
指導教授: | 顧家綺(chia-chi ku) |
關鍵字: | 第十五號細胞激素,表皮細胞,皮膚發炎,先天性免疫反應, IL-15,Epidermis,Skin inflammation,innate immune response, |
出版年 : | 2013 |
學位: | 碩士 |
摘要: | IL-15是一個多功能的細胞激素,除了是維持自然殺手細胞發育與記憶型CD8+ T細胞數恆定的生長因子,在人類的發炎性疾病與自體免疫相關疾病中,也被證實是一種具有促發炎反應功能的細胞激素。中研院基因突變鼠動物模式核心實驗室(MMPCF)利用ENU的致突變機制所產生的191品系突變鼠(簡稱P191)在IL-15基因上的第七外顯子具有的點突變容易導致生成IL-15選擇性剪接異構體(本論文中稱為IL-15_ASE7)。過去在HSV-1疱疹病毒的小鼠動物實驗模式中,我們發現P191小鼠的皮膚比B6小鼠容易產生較大的疱疹傷口,且皮膚的修復時間延遲,真皮層中表現Gr-1與F4/80表面抗原的白血球浸潤細胞數量也較B6小鼠少,此實驗結果顯示出皮膚中表現IL-15_ASE7可能會影響皮膚控制HSV-1病毒感染的抗病毒免疫反應與受損皮膚的修復。
本研究論文發現,P191小鼠皮膚中的IL-15與IL-15_ASE7 mRNA表現量相當,但是在不改變總表現量的狀態下,骨骼肌與脾臟中的IL-15_ASE7大約比IL-15多出兩倍。本研究藉由機械性刺激的方式比較刮擦刺激後P191小鼠皮膚與野生型B6和IL-15基因缺失鼠(Il-15-/-)皮膚在先天性免疫反應方面的差異,以探討高量IL-15_ASE7表現在皮膚組織的可能影響。組織切片的病理分析結果顯示P191小鼠的表皮細胞增生程度(p=0.03)以及真皮層增厚現象(p=0.02)均較B6小鼠低,特別是在刮擦處真皮層的嗜中性白血球浸潤顯著地比B6小鼠減少(p<0.01**)。藉由DNA微陣列分析B6與P191刮擦皮膚的全基因組轉錄圖譜,顯示氧化磷酸化與穀胱甘肽代謝有關的基因群在B6與P191小鼠中有差異表現。定量PCR的實驗結果證實Uqcrb基因與抗氧化Sod-1基因表現量在刮擦刺激48小時之後的P191小鼠皮膚分別明顯地下降2倍(p<0.01**)與3倍(p<0.01**)。此外,表皮細胞分化基因如Krt,Ivl以及Lor在P191小鼠中表現量皆較高實驗結果顯示P191小鼠皮膚在受損刺激下表皮細胞的活化程度不若B6小鼠皮膚顯著。雖然B6 的表皮細胞所產生的發炎細胞激素包括G-CSF,KC與MCP-1經過刮擦後顯著上升,甚至在Il-15-/-鼠中更明顯,然而在P191小鼠表皮細胞所產生的這些發炎細胞激素不論在轉錄與轉譯層次都比B6 與Il-15-/-下降。將P191小鼠的皮膚移植到B6宿主上,經由刮擦所刺激的嗜中性白血球浸潤也同樣明顯較B6 皮膚減少。顯示出在IL-15_ASE7的表現下,可能會影響表皮細胞受到刺激活化後的狀態,進而影響隨後的發炎反應進行。IL-15_ASE7如何調控皮膚發炎,其詳細的分子機制需要更多實驗進行佐證。 Interleukin-IL-15 (IL-15) is a pleiotropic cytokine. Not only IL-15 is known as a growth factor for the development of natural killer cells and the homeostasis of memory CD8+ T cells, it is also demonstrated as a pro-inflammatory cytokine in several human inflammatory disorder and autoimmune diseases. An ENU-mutagenized pedigree 191 (P191) generated at the Mouse Mutagenesis Program Core Facility (MMPCF) was found bearing a mutation in the exon 7 of IL-15 mRNA and thus enhanced the generation of alternatively spliced IL-15 mRNA isoform, IL-15_ASE7. Using HSV-1 zosteriform mouse model, our laboratory has previously found that HSV-1 created more severe skin lesions and delayed skin healing in P191 compared with B6 mice. Reduced dermal infiltrating cells such as Gr-1+ and F4/80+ granulocytes were also observed in P191 lesional skin, suggesting expression of IL-15_ASE7 in P191 might modulate innate immunity in skin after viral infection. In this study, I demonstrated that comparable levels of canonical IL-15 and IL-15_ASE7 mRNA were expressed in skin but about two-fold increase in the transcription of IL-15_ASE7 mRNA in the skeletal muscles and the spleen of P191 mice by TaqMan q-PCR analysis. The effects of elevated IL-15_ASE7 in skin on innate immune responses were further investigated by mechanically stimulating flank skin of P191 compared with wild type B6 and Il-15-/- mice. Histological and immunohistochemical analysis of skin sections demonstrated that proliferation of epidermal keratinocytes (p=0.03) and the dermal thickness (p=0.02) were reduced in P191 compared with B6 mice. Dermal cellular infiltration especially in the recruitment of neutrophils in P191 lesional skin was also reduced (p<0.01**) compared with in B6 abraded skin. Genome-wide transcriptional profiling of B6 and P191 abraded skin by DNA microarray showed that genes involved in oxidative phosphorylation and glutathione metabolism were distinguishably expressed between B6 and P191 abraded skin. Particularly, transcriptional levels of Uqcrb and anti-oxidant Sod-1 gene were significantly reduced by 2-fold (p<0.01**) and 3-fold (p<0.01**) in P191 skin at 48 hours after abrasion. Additionally, expression of genes for keratinocyte differentiation, such as Krt10, Ivl, and Lor were higher in P191 compared with B6 skin. Results from these experiments suggested that proliferation and activation of the epidermal keratinocytes were alleviated in P191 mice in response to abrasion stimulation. While inflammatory cytokines produced by the epidermal keratinocytes including G-CSF, KC, MCP-1 were significantly induced by abrasion in B6 and even higher in Il-15-/- mice, they were all decreased in P191 abraded skin both at transcriptional and translational levels. Reduction of neutrophils recruitment to P191 abraded skin was also observed when the skin was grafted on the wild type host. These experiments have suggested that expression of IL-15_ASE7 in skin might have function on the homeostasis of epidermal keratinocytes and subsequently in modulate cutaneous inflammation. More experiments will be performed to clarify the detailed mechanisms by which IL-15_ASE7 regulates the process. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/17272 |
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