【作者】 李林；

【导师】 谭志建； 陶娟；

【作者基本信息】 华中科技大学 ， 皮肤病与性病学， 2010， 硕士

【摘要】 目的:探究CC型趋化因子CCL20及其受体CCR6在早期硬皮病患者皮损中的表达,研究CCL20/CCR6是否参与硬皮病的发生和发展。方法:一.免疫组织化学及荧光定量PCR分别检测早期硬皮病患者皮损中CC型趋化因子CCL20及其受体CCR6蛋白及mRNA的表达。二.采用组织块培养法分别分离硬皮病患者及正常人真皮成纤维细胞,以含10%胎牛血清DMEM培养基进行体外原代培养。通过形态学及免疫细胞化学的方法对其进行鉴定,MTT法测细胞的增殖。分别用不同浓度的Th1及Th2细胞因子进行处理,在不同的时间点分别取上清行ELISA,细胞mRNA作实时定量PCR检测。三.检测成纤维细胞产生的CCL20的趋化活性,并比较硬皮病成纤维细胞和正常成纤维细胞所产生的CCL20的趋化活性是否有差异。结果:一.CCL20及其受体CCR6蛋白及mRNA在早期硬皮病皮损中的表达增加,而且二者在皮损中的分布部位一致。二.体外原代培养的皮肤成纤维细胞可快速稳定增殖。硬皮病患者真皮成纤维细胞和正常人真皮成纤维细胞在形态学和免疫细胞化学上无明显差异。基础状态下,CCL20 mRNA及蛋白质在两种细胞中均很低,但在硬皮病患者真皮成纤维细胞表达水平较正常人真皮成纤维细胞高;Th1型细胞因子(TNF-αand IL-1β)能够随剂量和时间的增加显著提高成纤维细胞CCL20的表达水平;相反,硬皮病患者真皮成纤维细胞CCL20的表达水平则随Th2型细胞因子(IL-4)浓度和时间的增加而降低。三.重组型及成纤维细胞上清中的CCL20对PBMCs的趋化功能呈浓度依赖性。两种成纤维细胞上清的CCL20较重组型CCL20对PBMCs具有更强的趋化活性;硬皮病患者真皮成纤维细胞上清中的CCL20的趋化活性较正常皮肤成纤维细胞高;CCL20抗体能完全阻断重组型CCL20的趋化活性,但对硬皮病患者成纤维细胞上清PBMCs的趋化活性仅能阻断43.98±10.48%。结论:1. CC型趋化因子CCL20及其受体CCR6参与了硬皮病早期的发生和发展,对硬皮病皮损中单个核细胞的浸润起到重要作用。2.硬皮病患者的成纤维细胞处于持续活化状态,能够自身及在促炎症性细胞因子的刺激下合成CCL20。3.除CCL20外,成纤维细胞还可能合成其他趋化因子,共同趋化MNCs进入硬皮病皮损中。更多还原

【Abstract】 ObjectiveTo investigate the expressions of CC chemokine CCL20 and its receptor CCR6 in early scleroderma skin lesions and study whether CCL20/CCR6 pair are involved in pathogenesis of scleroderma.Methods1.The protein and mRNA expression levels of CCL20 and CCR6 in early scleroderma skin lesions were investigated by IHC and Real-time RT-PCR, respectively.2. Primary scleroderma dermal fibroblasts (SDF) and normal adult dermal fibroblasts (NDF) were isolated by tissue explants, cultured in DMEM with 10% FBS and were indentified by morphology and ICC. Cell proliferation was mesured by MTT colorimetric assay and cells were treated with gradient concentrations of Th1 or Th2 cytokines. At indicated time points following cytokine exposure, conditioned media and cells were collected for CCL20 protein and mRNA quantification, respectively.3. Biological activity of CCL20 protein in the supernatants of SDF and NDF was evaluated by chemotaxis assays. We investigated whether CCL20 from NDF and SDF had a difference in chemotactic activity.Results1. CCL20 and CCR6 were significantly up-regulated in early scleroderma lesional vs normal skins and the locations of both were totally consistent.2. Dermal fibroblasts were successfully isolated and cultured in vitro steadily and rapidly. SDF and NDF have no significant difference in morphology and ICC. Under basal conditions, the levels of CCL20 protein and mRNA were very low both in NDF and SDF, but were significantly higher in NDF than in SDF. Th1 cytokines (TNF-αand IL-1β) markedly upregulated CCL20 mRNA and protein in a time and dose-dependent manner in both NDF and SDF; On the contrary, CCL20 mRNA and protein expression in SDF decreased with the increase of IL-4 concentrations and time. 3. PBMCs migrated toward recombinant CCL20 and culture supernants of NDF and SDF in a concentration-dependent manner. Conditioned media derived from NDF and SDF both displayed stronger chemotactic activity for PBMCs than recombinant CCL20, while supernant from SDF showed a even stronger chemotactic activity than that from NDF.4. Chemotatic response to recombinant CCL20 was almost completely blocked by human CCL20/MIP-3αantibodies. However, this chemotatic activity was only inhibited by 43.98±10.48% in supernants from SDF.Conclusion1.The interaction between CC chemokine CCL20 and its receptor CCR6 are involved in the pathogenesis of scleroderma and may serve an important role in the selective recruitment of inflammatory cells in early scleroderma leisional skins.2.SDF is in a persistently activated condition and can produce CCL20 in the presence or in the absence of exogenous proinflammatory stimuli.3.Except for CCL20, some other chemokines might also participate in the recruitment of MNCs into scleroderma leisional skins.更多还原