目的:銀屑病是一種慢性炎症性皮膚疾病，特點是角質形成細胞過度增生。角質形成細胞過度增生在銀屑病家族中被證實與位於染色體6p2 1. 3 上的編碼人類白細胞抗原的基因組區有關。通過全基因組掃描，至少確定了10 個銀屑病易感基因位點(PSORS 卜的ORS10) ，而6p21 區的PSORS1 被認為是銀屑病最主要的易感位點，而HLA-C， CCHCRl (coiled coil alpha-helixrod homolog) 和CDSN(corneodesmosin) 是該位點上的重要的侯選基因。然而，這三個基因之間的連鎖不平衡很難將他們的個體效應分開。我們的目標是研究CCHCCRl 基因以及它的相關蛋白在皮膚上皮細胞增生過程中的作用。, 方法:本研究應用免疫螢光技術'免疫印跡分析和即時反轉錄聚合臨鏈反應 (Real Time RT-PCR) 三種方法比較CCHCRl 基因在銀屑病人的皮損區與正常人的皮膚細胞的表達。其次比較了CCHCRl 蛋白在喜樹鹼(拓撲異構酪I 抑制劑/凋亡誘導劑)的作用下在三種細胞(永生化的人皮膚角質形成細胞-HaCaT '人直結腸癌細胞-HT29 和人子宮頸癌細胞- HeLa) 內的表達。最後，我們應用了皮膚器官樣培養物( OTC) 研究HaCaT 細胞於皮膚角質化過程中CCHCRl 蛋白的表達。, 結果:✹HCR1 蛋白在正常皮膚和銀屑病人皮損皮膚表達模式不同，銀屑病人皮損皮膚有五種CCHCR1 蛋白染色模式，而正常人皮膚只有兩種染色模式。免疫印跡分析顯示CCHCR1 蛋白在正常皮膚含量較銀屑病人高。免役螢光顯微技術顯示角蛋白17 (K17) ，一種細胞增生的標誌性蛋白，只表達在正常皮膚基底層的角質細胞中，而在銀屑病人皮膚， K17 從基底層到顆粒層都有顯著表達，並且和CCHCR1 蛋白表達區域相同。這種現象也存在於OTC 發育過程中所有角質細胞中，而CCHCR1 蛋白表達隨OTC 的培養時間從10 天到21 天呈上升趨勢，說明CCHCR1 蛋白與細胞增殖有一定的關係。當細胞生長在培養血表面形成細胞與細胞接合全面的細胞層時， CCHCR1 的信使核糖核酸水平大幅升高，顯示CCHCR1 的信使核糖核酸表達當細胞生長速度呈負相關。CCHCR1 的信使核糖核酸水平在同步化的HaCaT 細胞G2/M 期輕度升高，而蛋白水平在G1 期達到最高。在G2/M 期，高爾基體出現崩解，而CCHCR1 蛋白和其他高爾基蛋白一樣分散到胞漿。這種現象同樣地出現在經2 州喜樹鹼刺激48小時的HeLa 和HaCaT 細胞內，免疫細胞化學染色顯示CCHCR1 和golgin-97 分散到胞漿。CCHCR1 信使核糖核酸水平在HeLa 和HaCaT 細胞都升高，而蛋白在HaCaT 細胞明顯上調。這說明CCHCR1 蛋白量的上調和細胞生長停滯有關。應用siRNA 或shRNA 抑制CCHCR1 蛋白表達後， golgin-97 分散到胞漿，但是HeLa細胞可以繼續增殖。這說明CCHCR1 蛋白可能有助於維持高爾基複合體的完整，喜樹鹼對細胞生長的抑制作用可能與CCHCR1 蛋白的上調相關。, 結論: CCHCR1 蛋白在正常皮膚表皮細胞和銀屑病人皮損區細胞胞漿內都有表達，但是正常皮膚表達較銀屑病人高，在OTC 增生後期和細胞生長緩慢或停滯期也明顯升高，說明CCHCRl 基因的上調可能抑制細胞的增生。經喜樹鹼刺激後，細胞生長受到抑制，細胞停滯在Gl 期並誘導細胞凋亡， CCHCRl 的信使核糖核酸和蛋白都上調，表明CCHCRl 與細胞的增生抑制有關。同時，它的下調也造成高爾基複合體的崩解，說明CCHCRl 蛋白可以維持高爾基複合體的結構完整性。因此， CCHCRl 不僅可以保持高爾基複合體的完整而且和細胞的增生有關。另一方面，大量證據表明長期性的高血糖會導致胰島 細胞功能紊亂。鑒於此，揭示胰島功能調節的潛在機理并闡明胰島功能与高血糖症之間的關係變得尤為重要。, Aim: Psoriasis is one of the common chronic inflammatory skin disorders characterized by keratinocyte hyperproliferation, T lymphocyte-mediated inflammation, and abnormal differentiation. Genome-wide scans have revealed that at least ten different susceptibility loci, PSORS1-PSORS10, were linked to psoriasis, among which PSORS 1 on chromosome 6p21.3 was unambiguously associated with families with psoriasis. Three strongly psoriasis-associated susceptibility alleles have been identified in PSORS1, namely HLA-C, CCHCR1 (coiled-coil alpha-helical rod protein 1 ), and CDSN ( comeodesmosin); their strong linkage disequilibrium, however, makes it very difficult to distinguish their individual genetic effects. Among them, we were interested in the CCHCR1 gene, and its possible roles with associated proteins in the process of skin epidermal cell proliferation were studied in this thesis., Methods: The cellular expressiOn of CCHCR1 protein in the epidermis was compared between human skins from healthy donors and psoriasis patients by immunofluorescence microscopy and immunoblotting analysis. Its intracellular expression was investigated in cultured human immortalized skin keratinocyte cell line (HaCaT), human colorectal cancer cell line (HT29) and human cervical carcinoma HeLa cell line, as well as in the skin keratinocyte-fibroblast organotypic culture (OTC). The responses of these cells to camptothecin (CPT), a topoisomerase I inhibitor, were compared in HaCaT cells, HT29 cells and HeLa cells. In cell experiments, CCHCR1 expression was studied by immunofluorescence microscopy, immunoblotting analysis, and real-time reverse transcriptase-polymerase chain reaction (real-time RT-PCR)., Results: The immunofluorescence staining patterns for CCHCR1 protein were different between normal and psoriasis skin biopsies. There were at least five patterns (Patterns I, III-VI) in psoriatic skins, but only two (Patterns I and II) in the normal skin, implicating that the expression of CCHCR1 protein was changed in psoriasis. Besides the different cellular location of CCHCR1 protein in normal and psoriasis skins, a higher level of CCHCR1 expression was measured in normal skin than that in psoriatic skin by immunoblotting analysis, and immunofluorescence microscopy further revealed a co-localization of CCHCR1 protein with keratin 17 (K17), a proliferation marker protein and also putative autoantigen for psoriasis, to basal keratinocytes of normal skins, and extended further fro m the basal to granular keratinocytes in the epidermis of psoriasis patients, suggesting a close association of CCHCR1 protein to cell proliferation. During epidermal development in OTC, the CCHCR1 protein was expressed in all keratinocyte layers in a time-dependent manner from day 1 0 to day 21, reaching a maximal level at day 21 when cell proliferation decreased and differentiation to the corneum became active. Furthermore, immunofluorescence for K17 remained co-localized to CCHCR1-positive cells in OTC from day 10 to day 14, but the level of K17 was very weak in the keratinocytes of suprabasallayers on day 21. In HaCaT cell culture, CCHCR1 transcription level at 100% confluence was 25 folds higher than that in subconfluence. Taken all these results together, the up-regulation of CCHCR1 was closely related to cell growth inhibition and differentiation. Immunofluorescence microscopy revealed a co-localization of CCHCR1 protein with a Golgi marker protein, golgin-97, in the compact Golgi complex at the juxtanuclear region of HaCaT cells and HeLa cells. CCHCR1 gene transcribed in all phases of the cell cycle, but was slightly higher in the G2/M phase of synchronous HaCaT cells, and its tran, Conclusion: CCHCR1 protein was expressed in the cytoplasm of epidermal keratinocytes of both normal and psoriasis skins, with a higher level detected in the normal skins. It was also found especially abundant in the keratinocytes of skin organotypic cultures during their transition from proliferation to differentiation. CCHCR1 gene transcription was increased obviously in cultured HaCaT cells at confluence. CCHCR1 was also up-regulated at both transcriptional and translational levels in response to the antiproliferative drug, camptothecin, in HaCaT cell experiments, and was accompanied by G 1 arrest and subsequent apoptosis. When CCHCR1 was knockdowned in HeLa cells, the Golgi complex disassembled, implicating a role of CCHCRl protein in the maintenance of Golgi integrity and in the control of cell proliferation., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Detailed summary in vernacular field only., Wang, Lijun., Thesis (Ph.D.)--Chinese University of Hong Kong, 2009., Includes bibliographical references (leaves 107-133)., s also in Chinese., p.i, Acknowledgements --- p.viii, List of Abbreviation --- p.ix, Chapter Chapter 1 --- General Introduction --- p.1, Chapter 1.1 --- Psoriasis --- p.1, Chapter 1.2 --- Skin --- p.9, Chapter 1.3 --- An In Vitro Model for Human Skin Equivalent --- p.10, Chapter 1.4 --- Aim of Study --- p.12, Chapter Chapter2 --- Expression of CCHCRl Protein in Psoriasis skin --- p.13, Chapter 2.1 --- Background of Skin and Psoriasis --- p.13, Chapter 2.2 --- Materials and Methods --- p.18, Chapter 2.3 --- Results --- p.21, Chapter 2.4 --- Discussion and Conclusion --- p.25, Chapter Chapter3 --- Expression of CCHCRl Protein in Skin Organotypic Culture --- p.37, Chapter 3.1 --- Background about Skin Organotypic Culture --- p.37, Chapter 3.2 --- Materials and Methods --- p.41, Chapter 3.3 --- Results --- p.45, Chapter 3.4 --- Discussion and Conclusion --- p.49, Chapter Chapter4 --- Expression of CCHCRl Protein in HaCaT Cells Treated with Camptothecin --- p.60, Chapter 4.1 --- Background --- p.60, Chapter 4.2 --- Materials and Methods --- p.61, Chapter 4.3 --- Results --- p.68, Chapter 4.4 --- Discussion and Conclusion --- p.73, Chapter Chapter 5 --- General Discussion --- p.95, References --- p.107, Publications --- p.172, http://etheses.lib.cuhk.edu.hk/servlet/list?action=abstract_tab&barcode=920151174, 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/)