臺灣博碩士論文加值系統

介白質-33(Interleukin-33, IL-33)也稱作IL-1F11，屬於IL-1超家族中的一員，可作為細胞激素，當組織受到損害時細胞核中的pro-IL-33可經過蛋白酶修飾後送到細胞外與其受體ST-2、IL-1rap的結合，誘導T細胞之極化反應，進而使T細胞轉變為第二型輔助T細胞(Th2)來調控發炎反應的產生。Pro-IL-33具有核定位序列可以進入細胞核中，但IL-33在細胞核中的功能尚未釐清。在本研究中也發現IL-33主要大量表現在大鼠C6腦膠質瘤細胞與大鼠膠質前驅細胞(GPCs)之細胞核中。利用DNA染色質纖維染色發現到IL-33與DNA結合。另外透過核蛋白免疫共沉澱的方式證明IL-33在膠質瘤細胞中不與NF-B相互作用。以上結果表明IL-33可能主要儲存在腦膠質瘤細胞以及膠質前驅細胞的細胞核。此外，在本文研究中發現小鼠大腦白質區的寡突膠細胞也會大量表現IL-33。透過TEM分析，發現與WT小鼠相比，IL-33基因剃除小鼠(IL33-KO)在大腦胼胝體區的髓鞘結構鬆散且較薄。有趣的是，在動物行為測試(高架十字迷宮與開放場域試驗)的結果可以看到IL33-KO小鼠較不畏懼且顯示較低的焦慮程度。利用西方墨點法證實與WT小鼠相比，OLGs成熟所需的蛋白質MBP、PLP在IL33-KO的小鼠表現量都較低。另外，在體外培養之大鼠OPCs的IL-33抑制後也觀察到與寡突膠細胞分化相關基因(Olig2、 Myrf、 Id4、 Hes-5、Sirt2、 Tspan2)以及與成熟相關基因(MBP、PLP)表現下調。更進一步，分別利用GC、MBP、CNPase抗體以免疫螢光染色的方式觀察細胞型態並發現IL33-KD組別的分化受到明顯的抑制，綜合以上結果，證實IL-33在寡突膠細胞發育及分化的過程是不可或缺的。另外，IL-33基因的缺失使寡突膠細胞成熟受阻以及髓鞘形成的缺陷或許是導致不正常的動物行為結果的原因。

Interleukin-33 (IL-33) is a member of IL1 family and acts as an extracellular cytokine after conversion from pro-IL-33. IL-33 is an “alarmin” to induce T helper 2 (Th2) immune responses in immune cells via interaction with its receptor ST2 and IL-1 receptor accessory protein. The pro-IL-33 protein contains a nuclear localization sequence (NLS), an N-terminal homeodomain-like helix-turn-helix (HTH) motif, and a chromatin-binding domain. Although the pro-IL33 can translocate from the cytoplasm into the nucleus, the function of the nuclear IL-33 in the cells remains unclear. In this study, we showed that IL-33 was enriched in the nuclear fractions of rat C6 glioma cells and rat glial progenitor cells (GPCs). Through the chromatin fiber analysis, IL-33 binding to chromatin was observed. The results from the experiments by co-immunoprecipitation with anti-IL-33 and anti- NF-B indicated that nuclear IL-33 did not interact with NF-B in glioma cells. These observations reveal that IL-33 might be stored mainly in the nucleus of glioma and progenitor cells. Furthermore, the in vivo study indicated that IL-33 was highly expressed in oligodendrocytes (OLGs) at the corpus callosum (CC) of the CNS. By Transmission Electron Microscopy (TEM) analysis we found that the myelin structure in the CC region of mice with IL-33 deficiency (IL33-KO) was disrupted compared to those observed in WT mice. Interestingly, the results from animal behaviors tests (i.e. elevated plus maze test and open field test) showed that IL33-KO mice displayed a fearless behavior, suggesting that the degree of anxiety was reduced in mice after IL33-KO. Oligodendrocytes (OLGs) derived from oligodendrocyte precursor cells (OPCs) prepared from the brains of IL33-KO mice expressed lower levels of OLG myelin basic protein (MBP) and myelin proteolipid protein (PLP) that OLGs generated from WT mice did. The further in vitro study using lentivirus-mediated shRNA delivery against IL-33 expression (IL33-KD) also indicated that OLG differentiation-related genes (i.e. Olig2, Myrf, Id4, Hes5, Sirt2, Tspan2) and OLG maturation proteins (i.e. MBP and PLP) were downregulated after IL33-KD. Moreover, the morphological examination of OLGs by immunofluorescence using anti-GC, anti-CNPase and anti-MBP antibodies revealed that IL33-KD suppressed OLG differentiation and maturation. Overall, our findings demonstrate that IL-33 is required for the proper development of OLG differentiation. IL-33 gene deficiency can disrupt OLG maturation and interfere myelin compaction, which might lead to the behavior change in mice.

目錄
中文摘要 I
英文延伸摘要 II
致謝 VI
目錄 1
圖目錄 5
縮寫表 7
前言 9
一、中樞神經系統膠細胞 9
二、寡突膠細胞的發育與成熟 10
三、寡突膠質細胞與中樞神經系統疾病 11
四、介白素33 12
五、IL-33與中樞神經系統疾病 13
材料與方法 16
一、實驗材料 16
1、細胞培養材料與試劑 16
2、化學藥品 16
3、抗體 17
4、引子 17
5、病毒載體與試劑組 18
二、實驗方法 19
1、細胞實驗 19
1-1、大鼠腦膠質瘤細胞培養(Rat C6 glioma cell line) 19
1-2、寡突膠質前驅細胞培養 (Preparation of OPCs) 19
1-3、膠質前驅細胞培養 (Preparation of GPCs) 20
1-4、慢病毒載體轉殖(Lentiviral Vector Transduction) 20
1-5、核蛋白萃取 (Nuclear protein extraction) 21
1-6、西方墨點法 (Western Blot Analysis) 21
1-7、染色體纖維分析 (Chromatin fibers) 22
1-8、核蛋白免疫共沉澱 (Co-Immunoprecipitation of nuclear protein) 22
1-9、細胞免疫螢光染色 (Immunofluorescence) 22
1-10、核酸即時定量分析 (Quantitative Real-Time Polymerase Chain Reaction；Q-PCR) 23
1-11、細胞增殖實驗 (MTT cell proliferation assay) 24
2、動物實驗 25
2-1、動物組織處理 25
2-2、組織切片 26
2-3、組織免疫螢光染色 (Immunofluorescence) 26
2-4、穿透式電子顯微分析 (Transmission Electron Microscopy；TEM) 26
2-5、動物行為測試 (Animal behavior tests) 27
3、統計分析 (Statistical analysis) 28
實驗結果 29
一、IL-33大量表現於細胞核內 29
二、IL-33表現在胼胝體的寡突膠細胞 30
三、抑制IL-33表現對OLGs的影響 30
四、IL-33表現下降對OLGs分化前期的影響 31
五、IL-33缺失對小鼠OLGs的影響 31
六、電子顯微影像觀察白質區髓鞘微結構 32
七、IL-33缺失的小鼠進行動物行為的分析 33
討論 35
一、IL-33在膠質瘤細胞中的影響 35
二、IL-33在寡突膠質細胞核中功能角色 36
三、IL-33下調影響寡突膠細胞的成熟 37
結論 39
參考文獻 40


圖目錄
Figure 1. High nuclear level of IL-33 in C6-1 cells and C6-2 with IL-33 gene overexpression. 45
Figure 2. IL-33 immunostaining to examine its interaction with chromatin fiber and NF-B. 46
Figure 3. Nuclear expression of IL-33 in CC1+-OLGs located at rodent corpus callosum. 47
Figure 4. Cell viability of OPCs with IL-33 knockdown. 48
Figure 5. Effect of IL-33 knockdown on gene expression in OLGs. 49
Figure 6. Down regulation of IL-33 expression reduced the cell process formation and OLGs maturation. 50
Figure 7. Expression of OLGs myelin-related genes were downregulated in IL33-KD OPCs. 51
Figure 8. IL-33 gene deficiency reduces the cell process formation and OLGs maturation. 52
Figure 9. TEM imaging analysis indicates interrupted myelin structure in the corpus callosum of IL-33 knockout mice. 53
Figure 10. EPM shows the decreased in anxiety-like behavior in IL33-KO mice. 54
Figure 11. Declined anxiety-like behavior in IL33-KO mice. 55
Figure 12. No difference in recognition memory capability between WT and IL33-KO mice. 56

參考文獻
Ali S, Mohs A, Thomas M, Klare J, Ross R, Schmitz ML, Martin MU. 2011. The dual function cytokine IL-33 interacts with the transcription factor NF-kappaB to dampen NF-kappaB-stimulated gene transcription. J Immunol 187:1609-16.
Allan D, Fairlie-Clarke KJ, Elliott C, Schuh C, Barnett SC, Lassmann H, Linnington C, Jiang HR. 2016. Role of IL-33 and ST2 signalling pathway in multiple sclerosis: expression by oligodendrocytes and inhibition of myelination in central nervous system. Acta Neuropathol Commun 4:75.
Barrette B, Nave KA, Edgar JM. 2013. Molecular triggers of neuroinflammation in mouse models of demyelinating diseases. Biol Chem 394:1571-81.
Bradl M, Lassmann H. 2010. Oligodendrocytes: biology and pathology. Acta Neuropathol 119:37-53.
Bramow S, Frischer JM, Lassmann H, Koch-Henriksen N, Lucchinetti CF, Sorensen PS, Laursen H. 2010. Demyelination versus remyelination in progressive multiple sclerosis. Brain 133:2983-98.
Bujalka H, Koenning M, Jackson S, Perreau VM, Pope B, Hay CM, Mitew S, Hill AF, Lu QR, Wegner M and others. 2013. MYRF is a membrane-associated transcription factor that autoproteolytically cleaves to directly activate myelin genes. PLoS Biol 11:e1001625.
Cao K, Liao X, Lu J, Yao S, Wu F, Zhu X, Shi D, Wen S, Liu L, Zhou H. 2018. IL-33/ST2 plays a critical role in endothelial cell activation and microglia-mediated neuroinflammation modulation. J Neuroinflammation 15:136.
Carlock C, Wu J, Shim J, Moreno-Gonzalez I, Pitcher MR, Hicks J, Suzuki A, Iwata J, Quevado J, Lou Y. 2017. Interleukin33 deficiency causes tau abnormality and neurodegeneration with Alzheimer-like symptoms in aged mice. Transl Psychiatry 7:e1191.
Carroll WM, Jennings AR. 1994. Early recruitment of oligodendrocyte precursors in CNS demyelination. Brain 117 ( Pt 3):563-78.
Chari DM. 2007. Remyelination in multiple sclerosis. Int Rev Neurobiol 79:589-620.
Chen WY, Hong J, Gannon J, Kakkar R, Lee RT. 2015. Myocardial pressure overload induces systemic inflammation through endothelial cell IL-33. Proc Natl Acad Sci U S A 112:7249-54.
Choi YS, Park JA, Kim J, Rho SS, Park H, Kim YM, Kwon YG. 2012. Nuclear IL-33 is a transcriptional regulator of NF-kappaB p65 and induces endothelial cell activation. Biochem Biophys Res Commun 421:305-11.
Czepiel M, Boddeke E, Copray S. 2015. Human oligodendrocytes in remyelination research. Glia 63:513-30.
Dawson MR, Polito A, Levine JM, Reynolds R. 2003. NG2-expressing glial progenitor cells: an abundant and widespread population of cycling cells in the adult rat CNS. Mol Cell Neurosci 24:476-88.
Dohi E, Choi EY, Rose IVL, Murata AS, Chow S, Niwa M, Kano SI. 2017. Behavioral Changes in Mice Lacking Interleukin-33. eNeuro 4.
Edgar N, Sibille E. 2012. A putative functional role for oligodendrocytes in mood regulation. Transl Psychiatry 2:e109.
El Waly B, Macchi M, Cayre M, Durbec P. 2014. Oligodendrogenesis in the normal and pathological central nervous system. Front Neurosci 8:145.
Fang KM, Yang CS, Lin TC, Chan TC, Tzeng SF. 2014. Induced interleukin-33 expression enhances the tumorigenic activity of rat glioma cells. Neuro Oncol 16:552-66.
Foster SL, Talbot S, Woolf CJ. 2015. CNS injury: IL-33 sounds the alarm. Immunity 42:403-5.
Fu AK, Hung KW, Yuen MY, Zhou X, Mak DS, Chan IC, Cheung TH, Zhang B, Fu WY, Liew FY and others. 2016. IL-33 ameliorates Alzheimer's disease-like pathology and cognitive decline. Proc Natl Acad Sci U S A 113:E2705-13.
Gadani SP, Walsh JT, Smirnov I, Zheng J, Kipnis J. 2015. The glia-derived alarmin IL-33 orchestrates the immune response and promotes recovery following CNS injury. Neuron 85:703-9.
Gautier HO, Evans KA, Volbracht K, James R, Sitnikov S, Lundgaard I, James F, Lao-Peregrin C, Reynolds R, Franklin RJ and others. 2015. Neuronal activity regulates remyelination via glutamate signalling to oligodendrocyte progenitors. Nat Commun 6:8518.
Herrera J, Yang H, Zhang SC, Proschel C, Tresco P, Duncan ID, Luskin M, Mayer-Proschel M. 2001. Embryonic-derived glial-restricted precursor cells (GRP cells) can differentiate into astrocytes and oligodendrocytes in vivo. Exp Neurol 171:11-21.
Hill RA, Li AM, Grutzendler J. 2018. Lifelong cortical myelin plasticity and age-related degeneration in the live mammalian brain. Nat Neurosci 21:683-695.
Hines JH, Ravanelli AM, Schwindt R, Scott EK, Appel B. 2015. Neuronal activity biases axon selection for myelination in vivo. Nat Neurosci 18:683-9.
Jakoby P, Schmidt E, Ruminot I, Gutierrez R, Barros LF, Deitmer JW. 2014. Higher transport and metabolism of glucose in astrocytes compared with neurons: a multiphoton study of hippocampal and cerebellar tissue slices. Cereb Cortex 24:222-31.
Jensen MB, Poulsen FR, Finsen B. 2000. Axonal sprouting regulates myelin basic protein gene expression in denervated mouse hippocampus. Int J Dev Neurosci 18:221-35.
Karin M, Cao Y, Greten FR, Li ZW. 2002. NF-kappaB in cancer: from innocent bystander to major culprit. Nat Rev Cancer 2:301-10.
Kondo T, Raff M. 2000. The Id4 HLH protein and the timing of oligodendrocyte differentiation. EMBO J 19:1998-2007.
Liew FY, Girard JP, Turnquist HR. 2016. Interleukin-33 in health and disease. Nat Rev Immunol 16:676-689.
Liu A, Li J, Marin-Husstege M, Kageyama R, Fan Y, Gelinas C, Casaccia-Bonnefil P. 2006. A molecular insight of Hes5-dependent inhibition of myelin gene expression: old partners and new players. EMBO J 25:4833-42.
Liu Q, Turnquist HR. 2013. Implications for Interleukin-33 in solid organ transplantation. Cytokine 62:183-94.
Madouri F, Guillou N, Fauconnier L, Marchiol T, Rouxel N, Chenuet P, Ledru A, Apetoh L, Ghiringhelli F, Chamaillard M and others. 2015. Caspase-1 activation by NLRP3 inflammasome dampens IL-33-dependent house dust mite-induced allergic lung inflammation. J Mol Cell Biol 7:351-65.
Miller RH. 2002. Regulation of oligodendrocyte development in the vertebrate CNS. Prog Neurobiol 67:451-67.
Murakami-Satsutani N, Ito T, Nakanishi T, Inagaki N, Tanaka A, Vien PT, Kibata K, Inaba M, Nomura S. 2014. IL-33 promotes the induction and maintenance of Th2 immune responses by enhancing the function of OX40 ligand. Allergol Int 63:443-55.
Peterson LK, Fujinami RS. 2007. Inflammation, demyelination, neurodegeneration and neuroprotection in the pathogenesis of multiple sclerosis. J Neuroimmunol 184:37-44.
Pinto SM, Nirujogi RS, Rojas PL, Patil AH, Manda SS, Subbannayya Y, Roa JC, Chatterjee A, Prasad TS, Pandey A. 2015. Quantitative phosphoproteomic analysis of IL-33-mediated signaling. Proteomics 15:532-44.
Podbielska M, Levery SB, Hogan EL. 2011. The structural and functional role of myelin fast-migrating cerebrosides: pathological importance in multiple sclerosis. Clin Lipidol 6:159-179.
Ransohoff RM, Engelhardt B. 2012. The anatomical and cellular basis of immune surveillance in the central nervous system. Nat Rev Immunol 12:623-35.
Revishchin AV, Korochkin LI, Okhotin VE, Pavlova GV. 2008. Neural stem cells in the mammalian brain. Int Rev Cytol 265:55-109.
Shipman L. 2017. Allergy: Neonatal IL-33 drives allergy. Nat Rev Immunol 17:80-81.
Simons M, Trajkovic K. 2006. Neuron-glia communication in the control of oligodendrocyte function and myelin biogenesis. J Cell Sci 119:4381-9.
Thangaraj MP, Furber KL, Gan JK, Ji S, Sobchishin L, Doucette JR, Nazarali AJ. 2017. RNA-binding Protein Quaking Stabilizes Sirt2 mRNA during Oligodendroglial Differentiation. J Biol Chem 292:5166-5182.
Theoharides TC, Petra AI, Taracanova A, Panagiotidou S, Conti P. 2015. Targeting IL-33 in autoimmunity and inflammation. J Pharmacol Exp Ther 354:24-31.
Tkachev D, Mimmack ML, Ryan MM, Wayland M, Freeman T, Jones PB, Starkey M, Webster MJ, Yolken RH, Bahn S. 2003. Oligodendrocyte dysfunction in schizophrenia and bipolar disorder. Lancet 362:798-805.
Vainchtein ID, Chin G, Cho FS, Kelley KW, Miller JG, Chien EC, Liddelow SA, Nguyen PT, Nakao-Inoue H, Dorman LC and others. 2018. Astrocyte-derived interleukin-33 promotes microglial synapse engulfment and neural circuit development. Science 359:1269-1273.
Wang CY, Deneen B, Tzeng SF. 2017a. MicroRNA-212 inhibits oligodendrocytes during maturation by down-regulation of differentiation-associated gene expression. J Neurochem 143:112-125.
Wang CY, Sun YT, Fang KM, Ho CH, Yang CS, Tzeng SF. 2018. Function of B-Cell CLL/Lymphoma 11B in Glial Progenitor Proliferation and Oligodendrocyte Maturation. Front Mol Neurosci 11:4.
Wang H, Moyano AL, Ma Z, Deng Y, Lin Y, Zhao C, Zhang L, Jiang M, He X, Ma Z and others. 2017b. miR-219 Cooperates with miR-338 in Myelination and Promotes Myelin Repair in the CNS. Dev Cell 40:566-582 e5.
Yaseen IH, Monk PN, Partridge LJ. 2017. Tspan2: a tetraspanin protein involved in oligodendrogenesis and cancer metastasis. Biochem Soc Trans 45:465-475.
Zuchero JB, Barres BA. 2013. Intrinsic and extrinsic control of oligodendrocyte development. Curr Opin Neurobiol 23:914-20.