臺灣博碩士論文加值系統

介白質-15 (interlukine-15, IL-15) 是細胞激素的一種。除了參與重要的免疫反應之外，同時也涉及了宿主體內其他的生理反應，諸如:骨骼肌的代謝亦或是角質細胞的增生。基於作用的多效性以及分佈的廣泛性，IL-15生物活性的調控就顯得相當的重要。近幾年來，科學界在自然情況下發現人體內的許多細胞激素選擇性剪接異構體，並且藉用體外實驗的方式觀察到這些異構蛋白對於原型細胞激素的生物活性扮演了抑制調控的角色。然而，這些細胞激素剪接異構體的免疫生物活性尚未在體內實驗裡得到進一步的証實。本論文利用由國家型基因突變鼠核心實驗室以ENU誘導突變所建立的突變鼠品系191(P191)，經過人類疱疹一型病毒(Human Herpes Simplex Virus-1，HSV-1)的感染模式，希望能進一步地了解IL-15選擇性剪接異構體對原型IL-15調控下的免疫機制的影響。
　　 我們將小鼠皮膚經過HSV-1感染後，藉由組織切片染色的方法比較突變鼠與正常鼠對病毒感染的反應結果發現:P191小鼠比野生型小鼠(C57BL/6，B6)能更快地產生病毒感染所引起的典型皮層細胞的病理變化並且顯示較嚴重程度的細胞破壞。經由免疫組織化學染色的結果得知這些皮膚組織的破壞程度和病毒蛋白表現的豐富程度相關。此外，從免疫組織螢光染色的結果觀察到，B6小鼠經過HSV-1 病毒感染後雖然能在皮膚組織誘發表現IL-15及IL-15Rα，但是這些因子在P191小鼠的皮膚表現卻相對地降低。雖然小鼠的皮膚經過病毒的感染會發生皮層細胞的壞死，正常鼠卻能在感染後七天內開始出現皮膚修復的現象，同時伴隨表現Gr-1抗原的細胞的浸潤與聚集。不過，這些表現Gr1抗原的細胞卻無法在病毒感染後各個時間點所收集的P191小鼠皮膚中觀察到，同時P191小鼠需要經過較長的時間才能修復受到病毒感染而破壞的皮膚組織。雖然不論是正常鼠或是突變鼠的皮膚在病毒感染後都能觀察到CD3+ T 細胞的浸潤，它們對於二種品系的小鼠在控制病毒感染的作用與角色還需要進一步地研究與探討。總之，本實驗結果發現，在基因轉錄水平上表現較高程度IL-15異構體的P191小鼠經過HSV-1的表皮感染後，不但比正常鼠容易產生病毒的複製並且造成較嚴重的組織破壞，對於控制病毒感染與進行組織修復的時間與機智也與正常鼠有許多的差異。究竟這些差異如何由IL-15異構體轉錄本所造成以及該異構體轉錄本是否能在體內轉譯為蛋白質後並且進一步地影響原型IL-15的生物活性,或是同時能影響其它多項控制疱疹病毒感染的免疫機制，仍有待未來更進一步的實驗證明與探討。

Interlukine-15 (IL-15) is one of important cytokines and exhibits a high degree of pleiotropy, controlling a wide range of functions including immune responses, metabolism of skeletal muscle and proliferation of keratinocytes. However, very little is known about the mechanisms by which the expression and bioactivity of IL-15 are controlled. Whereas several forms of IL-15 alternative splice variants are identified in normal mice, their roles in the control of IL-15 expression remain unclear. In this study, we used an ENU mutant mouse model generated by the MMPCF (Mouse Mutagenesis Program Core Facility) to investigate how the immune responses against virus infection are affected when the host predominantly expresses IL-15 splice variants.
Normal C57BL/6 (B6) and mutant pedigree 191 (P191) were infected with Human Herpes Simplex Virus-1 (HSV-1) via epicutanous route. Skin samples with lesional areas were collected and sectioned for further analysis. Results from H & E stain showed that HSV-1 induced typical pathological changes in skin tissues of both types of mice including cell ballooning, vesicular degeneration and epidermal cytolysis. However, these changes appeared earlier and more severe in P191 than in wild type B6 skin. Expression of HSV-1 proteins was also more abundant in P191 than in B6 skin by immunohistochemical stain. Whereas expressions of IL-15 and IL-15R were induced in the subdermal area of B6 skin after HSV-1 infection, they were much reduced in P191 lesional skin. In addition, the time for the skin to be healed after HSV-1 infection was longer for P191 than B6 mice. While significant numbers of Gr-1+ cells were detected on day 5 and peaked at day 10 in the dermis of infected B6 skin, they were nearly detected at all times in P191 skin after HSV-1 infection. Although CD3+ T cells were all detected in B6 and P191 skin after HSV-1 infection, their roles in the control of virus infection remain clarified. In summary, results from our study have shown that infection of P191 mice with HSV-1 resulted in altered immune responses as compared with wild type B6 mice. The differences at least but not limited include the susceptibility to HSV-1 infection, time for skin healing and recruitment of immune cells to lesional areas. How does the elevated level of IL-15 splice variants in P191 mice contribute to these deviations is not clear. More experiments will be done to understand whether IL-15 splice variants are expressed as variant proteins to antagonize prototype IL-15 and/or acquire novel functions in regulating host immune responses.

誌謝 1
中文摘要 2
英文摘要 4
第一章 緒論 6
第一節 細胞激素與免疫反應 6
1. 調控細胞激素表達的機制 7
2. IL-2 cytokine family異構體的生物性特色 7
IL-2 cytokine family 7
IL-2 cytokine family與選擇性剪接異構體 8
3. 細胞激素異構體的應用發展性 10
第二節 動物模式在生物學研究上的重要性 10
1. ENU的致突變機轉 11
2. 從表型篩選到基因探索 13
3. ENU突變鼠在免疫學研究上的應用 13
Pedigree 191 (P191) 14
4. P191 基因突變鼠免疫偏差(Immune Deviation)之探討 16
第三節 人類單純性疱疹一型病毒(Human Herpes Simplex Virus-1，HSV-1)的感染與免疫反應 16
1. HSV-1的特性與致病機轉 17
2. 宿主的防禦機制 18
2.1. 補體連鎖反應的活化 18
2.2. 免疫細胞與細胞激素的相互作用 19
3. 利用HSV-1病毒感染的模式探討P191小鼠的免疫偏差 20
第二章 材料與方法 22
第三章 實驗結果 34
1. 利用RT-PCR的方式確認Pedigree 191(P191)所表現的IL-15選擇性剪接異構體 34
2. 人類疱疹一型病毒感染小鼠上皮組織之感染動物模式的建立 35
3. 人類疱疹一型病毒感染小鼠上皮組織之病理現象 35
4. 人類疱疹一型病毒感染後，小鼠皮膚組織內病毒蛋白的偵測 36
5. 人類疱疹一型病毒感染皮膚後誘導的免疫反應 37
5.1. IL-15 與 IL-15Rα 37
5.2. Gr1+浸潤細胞 38
5.3. CD3+浸潤細胞 38
6. 總結 39
第四章 討論 41
1. 比較人類疱疹一型病毒感染野生型 B6 與 P191小鼠後皮膚組織病理變化差異之原因 41
2. 人類疱疹一型病毒的感染引誘皮膚組織CD3+ T 細胞的浸潤 42
3. HSV-1的清除和Gr-1+ 細胞的浸潤 43
4. IL-15的表達與Neutrophils的浸潤在抗HSV-1感染上的相關性 44
第五章 圖表 47
第六章 參考資料 60

圖表目次
1. Detection of IL-15 mRNA splice variant in p191 by RT-PCR 47
2. Development of HSV-1 zosteriform model in B6 mice 49
3. HSV-1 induced cytopathological changes in mouse skin by H&E stain 50
4. Demonstration of expressions of HSV-1 proteins in skin by immunohistochemical staining 52
5. Expressions of IL-15 and IL-15 Rα in mouse skin after HSV-1 infection by immunofluorescence analysis 54
6. Detection of Gr-1 expressing cells within the cellular infiltration in HSV-1 infected skin by confocal microscopy 56
7. Detection of CD3 expressing cells within the cellular infiltration in HSV-1 infected skin by confocal microscopy 58

Ahmad, A., Ahmad, R., Toma, E., Morisset, R., and Menezes, J. (2000). Impaired induction of IL-15 in response to herpes simplex virus type 1 infection in peripheral blood mononuclear cells of HIV-infected patients. AIDS 14, 744-746.
Alves, N.L., Arosa, F.A., and van Lier, R.A. (2007). Common gamma chain cytokines: dissidence in the details. Immunol Lett 108, 113-120.
Atamas, S.P., Choi, J., Yurovsky, V.V., and White, B. (1996). An alternative splice variant of human IL-4, IL-4 delta 2, inhibits IL-4-stimulated T cell proliferation. J Immunol 156, 435-441.
Balling, R. (2001). ENU mutagenesis: analyzing gene function in mice. Annu Rev Genomics Hum Genet 2, 463-492.
Bouchard, A., Ratthe, C., and Girard, D. (2004). Interleukin-15 delays human neutrophil apoptosis by intracellular events and not via extracellular factors: role of Mcl-1 and decreased activity of caspase-3 and caspase-8. J Leukoc Biol 75, 893-900.
Carson, W.E., Giri, J.G., Lindemann, M.J., Linett, M.L., Ahdieh, M., Paxton, R., Anderson, D., Eisenmann, J., Grabstein, K., and Caligiuri, M.A. (1994). Interleukin (IL) 15 is a novel cytokine that activates human natural killer cells via components of the IL-2 receptor. J Exp Med 180, 1395-1403.
Cassatella, M.A. (1995). The production of cytokines by polymorphonuclear neutrophils. Immunol Today 16, 21-26.
Cunningham, A.L., Turner, R.R., Miller, A.C., Para, M.F., and Merigan, T.C. (1985). Evolution of recurrent herpes simplex lesions. An immunohistologic study. J Clin Invest 75, 226-233.
DiSanto, J.P., Muller, W., Guy-Grand, D., Fischer, A., and Rajewsky, K. (1995). Lymphoid development in mice with a targeted deletion of the interleukin 2 receptor gamma chain. Proc Natl Acad Sci U S A 92, 377-381.
Flaherty, L., Messer, A., Russell, L.B., and Rinchik, E.M. (1992). Chlorambucil-induced mutations in mice recovered in homozygotes. Proc Natl Acad Sci U S A 89, 2859-2863.
Futatsugi, A., Kuwajima, G., and Mikoshiba, K. (1995). Tissue-specific and developmentally regulated alternative splicing in mouse skeletal muscle ryanodine receptor mRNA. Biochem J 305 ( Pt 2), 373-378.
Han, G.W., Iwatsuki, K., Inoue, M., Matsui, T., Nishibu, A., Akiba, H., and Kaneko, F. (1999). Interleukin-15 is not a constitutive cytokine in the epidermis, but is inducible in culture or inflammatory conditions. Acta Derm Venereol 79, 37-40.
Henkel, G., and Brown, M.A. (1994). PU.1 and GATA: components of a mast cell-specific interleukin 4 intronic enhancer. Proc Natl Acad Sci U S A 91, 7737-7741.
Justice, M.J., Carpenter, D.A., Favor, J., Neuhauser-Klaus, A., Hrabe de Angelis, M., Soewarto, D., Moser, A., Cordes, S., Miller, D., Chapman, V., et al. (2000). Effects of ENU dosage on mouse strains. Mamm Genome 11, 484-488.
Kennedy, M.K., Glaccum, M., Brown, S.N., Butz, E.A., Viney, J.L., Embers, M., Matsuki, N., Charrier, K., Sedger, L., Willis, C.R., et al. (2000). Reversible defects in natural killer and memory CD8 T cell lineages in interleukin 15-deficient mice. J Exp Med 191, 771-780.
Kishimoto, T., Taga, T., and Akira, S. (1994). Cytokine signal transduction. Cell 76, 253-262.
Loser, K., Mehling, A., Apelt, J., Stander, S., Andres, P.G., Reinecker, H.C., Eing, B.R., Skryabin, B.V., Varga, G., Schwarz, T., and Beissert, S. (2004). Enhanced contact hypersensitivity and antiviral immune responses in vivo by keratinocyte-targeted overexpression of IL-15. Eur J Immunol 34, 2022-2031.
Malek, T.R., Yu, A., Vincek, V., Scibelli, P., and Kong, L. (2002). CD4 regulatory T cells prevent lethal autoimmunity in IL-2Rbeta-deficient mice. Implications for the nonredundant function of IL-2. Immunity 17, 167-178.
McInnes, I.B., and Gracie, J.A. (2004). Interleukin-15: a new cytokine target for the treatment of inflammatory diseases. Curr Opin Pharmacol 4, 392-397.
Noveroske, J.K., Weber, J.S., and Justice, M.J. (2000). The mutagenic action of N-ethyl-N-nitrosourea in the mouse. Mamm Genome 11, 478-483.
Peschon, J.J., Morrissey, P.J., Grabstein, K.H., Ramsdell, F.J., Maraskovsky, E., Gliniak, B.C., Park, L.S., Ziegler, S.F., Williams, D.E., Ware, C.B., et al. (1994). Early lymphocyte expansion is severely impaired in interleukin 7 receptor-deficient mice. J Exp Med 180, 1955-1960.
Popp, R.A., Bailiff, E.G., Skow, L.C., Johnson, F.M., and Lewis, S.E. (1983). Analysis of a mouse alpha-globin gene mutation induced by ethylnitrosourea. Genetics 105, 157-167.
Ratthe, C., and Girard, D. (2004). Interleukin-15 enhances human neutrophil phagocytosis by a Syk-dependent mechanism: importance of the IL-15Ralpha chain. J Leukoc Biol 76, 162-168.
Russell, L.B., Hunsicker, P.R., Cacheiro, N.L., Bangham, J.W., Russell, W.L., and Shelby, M.D. (1989). Chlorambucil effectively induces deletion mutations in mouse germ cells. Proc Natl Acad Sci U S A 86, 3704-3708.
Russell, W.L., Hunsicker, P.R., Raymer, G.D., Steele, M.H., Stelzner, K.F., and Thompson, H.M. (1982). Dose--response curve for ethylnitrosourea-induced specific-locus mutations in mouse spermatogonia. Proc Natl Acad Sci U S A 79, 3589-3591.
Russell, W.L., Kelly, E.M., Hunsicker, P.R., Bangham, J.W., Maddux, S.C., and Phipps, E.L. (1979). Specific-locus test shows ethylnitrosourea to be the most potent mutagen in the mouse. Proc Natl Acad Sci U S A 76, 5818-5819.
Tagaya, Y., Kurys, G., Thies, T.A., Losi, J.M., Azimi, N., Hanover, J.A., Bamford, R.N., and Waldmann, T.A. (1997). Generation of secretable and nonsecretable interleukin 15 isoforms through alternate usage of signal peptides. Proc Natl Acad Sci U S A 94, 14444-14449.
Tan, X., and Lefrancois, L. (2006). Novel IL-15 isoforms generated by alternative splicing are expressed in the intestinal epithelium. Genes Immun 7, 407-416.
Todd, M.J., Viitanen, P.V., and Lorimer, G.H. (1993). Hydrolysis of adenosine 5''-triphosphate by Escherichia coli GroEL: effects of GroES and potassium ion. Biochemistry 32, 8560-8567.
Tsytsikov, V.N., Yurovsky, V.V., Atamas, S.P., Alms, W.J., and White, B. (1996). Identification and characterization of two alternative splice variants of human interleukin-2. J Biol Chem 271, 23055-23060.
Tumpey, T.M., Chen, S.H., Oakes, J.E., and Lausch, R.N. (1996). Neutrophil-mediated suppression of virus replication after herpes simplex virus type 1 infection of the murine cornea. J Virol 70, 898-904.
van Lint, A., Ayers, M., Brooks, A.G., Coles, R.M., Heath, W.R., and Carbone, F.R. (2004). Herpes simplex virus-specific CD8+ T cells can clear established lytic infections from skin and nerves and can partially limit the early spread of virus after cutaneous inoculation. J Immunol 172, 392-397.