【作者】 颜克香；

【导师】 郑志忠； 黄琼； 方栩；

【作者基本信息】 复旦大学 ， 皮肤性病学， 2010， 博士

【摘要】 第一部分：Foxp3+调节性T细胞在斑块型和点滴型银屑病皮损中的差异表达目的：了解寻常型银屑病皮损中Foxp3+细胞的数量与皮损严重程度及疾病分型、分期间的关系。方法：通过免疫组化单染或双染技术对41例寻常型银屑病患者和10例正常对照皮肤中Foxp3+细胞的数量及Foxp3+细胞占CD3+细胞的比例进行检测。采用PSI评分或PASI评分分别对活检部位皮损的严重程度及疾病的严重程度进行评估。结果：银屑病皮损中Foxp3+细胞主要位于真皮乳头层和网状层上方,表皮内较少,在皮损周边外观正常皮肤及正常对照皮肤中Foxp3+细胞较少或没有。银屑病皮损中Foxp3+细胞的数量与皮损的严重程度(PSI评分)呈正相关(p<0.0001),斑块型银屑病皮损中Foxp3+细胞的数量高于点滴型,进展期高于稳定期,稳定期高于消退期(p<0.05)。Foxp3+细胞占CD3+细胞的比例在真皮乳头层高于表皮层和真皮网状层(p<0.0001),且从皮损的中央到边界到周边外观正常皮肤逐渐减少。结论：Foxp3+细胞在斑块型和点滴型银屑病皮损中的差异表达提示两者可能代表银屑病皮损免疫反应过程中的不同阶段。Foxp3+细胞在真皮乳头层和皮损中央的高表达可能参与了银屑病皮损发生和维持。第二部分：Treg与Thl7细胞在斑块型和点滴型银屑病外周血中的差异表达目的：了解寻常型银屑病患者外周血Treg与Th17细胞的水平与疾病分型间的关系。方法：通过流式细胞学方法检测17例寻常型银屑病患者和12例正常对照外周血Foxp3+CD4+Treg细胞和IL-17+CD4+Th17细胞占CD4+细胞的比例。根据CD25和CD45RA的表达及CD25表达强弱的不同,分析了11例斑块型银屑病和10例正常对照aTreg、rTreg、non-Treg细胞的比例及CD4+CD25+T细胞中CD4+CD25high、CD4+CD25mid、CD4+CD25low细胞的比例。结果：寻常型银屑病患者外周血Foxp3+CD4+Treg细胞的水平与疾病严重程度(PASI评分)正相关(p<0.05)。斑块型银屑病患者外周血Foxp3+CD4+Treg细胞的水平高于点滴型(p<0.0001)；而IL-17+CD4+Thl7细胞的水平低于点滴型(p<0.0001)。斑块型银屑病患者CD4+CD25highTreg和aTreg细胞的比例显著高于正常人(p<0.001,p<0.0001),差异有统计学意义。斑块型银屑病患者CD4+CD25+T细胞的比例高于正常人(p<0.05)；但rTreg、non-Treg及CD4+CD25mid、CD4+CD25low细胞的比例在斑块型银屑病和正常人间无显著差异。结论：Treg与Thl7细胞间的失衡在斑块型和点滴型银屑病发病中起重要作用。第三部分：Treg细胞对炎症细胞因子的抑制功能在斑块型和点滴型银屑病间的差异目的：了解寻常型银屑病患者TGF-β-Foxp3+iTreg细胞对CD4+CD25-T细胞产生炎症细胞因子的抑制功能和血清中炎症细胞因子的含量与疾病分型间的关系。方法：将CD4+CD25-T细胞在有或无TGF-p刺激下培养5天,通过流式细胞学方法检测TGF-β刺激后CD4+CD25-T细胞中CD25+Foxp3+细胞的比例,培养上清和血清中炎症细胞因子TNF-α、IL-6、IL-1β、IL-17的含量通过ELISA方法检测。结果：斑块型银屑病患者CD4+CD25-T细胞受TGF-β刺激后CD25+Foxp3+细胞的比例显著高于点滴型银屑病和正常对照(p<0.0001),点滴型银屑病高于正常对照(p<0.05),差异有统计学意义。斑块型银屑病患者CD4+CD25-T细胞受TGF-β刺激后产生更多的TNF-α;点滴型银屑病患者则产生更多的IL-6和IL-1β；但正常人CD4+CD25-T细胞受TGF-p刺激后TNF-α、IL-6和IL-1β的产生均降低。点滴型银屑病患者血清中IL-17和IL-6水平高于斑块型,但TNF-α水平低于斑块型。结论：斑块型和点滴型银屑病患者TGF-β-Foxp3+iTreg细胞对CD4+CD25-T细胞产生炎症细胞因子的差异调控和血清中炎症细胞细胞因子的差异表达提示两者具有不同的免疫发病机理。更多还原

【Abstract】 PartⅠFoxp3+ regulatory T cells differentially expressed in the skin lesions of plaque versus guttate psoriasisObjective To investigate the number of Foxp3+ cell in the skin lesions of psoriasis vulgaris in correlation with the severity of skin lesions and the subtype and staging of disease.Methods The number of Foxp3+ cell and the percentage of Foxp3+ cell among CD3+ cell were examined in the skin lesion of 41 patients with psoriasis vulgaris and 10 normal skin specimens as control by immunohistochemical single or double-staining technique. The severity of skin lesions biopsied and disease was evaluated by PSI scoring and PASI scoring, respectively.Results Foxp3+ cell in psoriatic lesions was predominantly located in papillary and upper reticular layers of dermis, minimally in epidermis, and it was absent or scant in perilesional normal-appearing skin and normal skin as control. The number of Foxp3+ cell in psoriatic lesions was positively correlated with the severity of skin lesions (PSI scoring) (p＜0.0001), and it was higher in plaque psoriasis than in guttate psoriasis, and in progressive stage higher than, and higher in stable stage than in regressive stage(p<0.05). The percentage of Foxp3+ cell among CD3+ cell was higher in papillary dermis than in epidermis and upper reticular layer of dermis(p<0.0001), and it was gradually declined from the center lesion to the border and perilesional normal-appearing skin.Conclusion The differential expression of Foxp3+ cell in plaque versus guttate psoriasis implicate their different stage of immunopathogenesis. The highest expression of Foxp3+ cell in papillary dermis and center lesion might participate in the initiation and maintenance of psoriasis vulgaris.Part II Treg and Th17 cells differentially expressed in peripheral blood of plaque versus guttate psoriasisObjective To investigate the level of Treg cell and Th17 cell in peripheral blood of psoriasis vulgaris in correlation with the subtype of disease.Methods The level of Foxp3+CD4+Treg cell and IL-17+CD4+Th17 cell among CD4+ cell in peripheral blood of 17 patients with psoriasis vulgaris and 12 normal controls was measured by flow cytometry. The percentage of aTreg, rTreg, non-Treg cell and the percentage of CD4+CD25high, CD4+CD25mid, CD4+CD251ow cell among CD4+CD25+T cell in 11 plaque psoriasis and normal controls were analyzed according to the expression of CD25 and CD45RA and the difference in strength of CD25 expression.Results The level of Foxp3+CD4+Treg cell in peripheral blood of psoriasis vulgaris was positively correlated with the severity of disease (PASI scoring) (p<0.05). The level of Foxp3+CD4+Treg cell in peripheral blood of plaque psoriasis was higher than in guttate psoriasis(p<0.0001); whereas, the level of IL-17+CD4+ Th17 cell was lower than in guttate psoriasis(p<0.0001). The percentage of CD4+CD25highTreg and aTreg in plaque psoriasis was obvious higher than normal controls(p<0.001, p<0.0001). The difference was statistically significant. The percentage of CD4+CD25+ cell in plaque psoriasis was higher than in normal controls(p<0.05); however, no significant difference in the percentage of rTreg, non-Treg cell and CD4+CD25mid, CD4+CD251ow cell was found between plaque psoriasis and normal controls.Conclusion The imbalance between of Treg and Thl7 cells play an important role in the pathogenesis of plaque versus guttate psoriasis.PartⅢThe difference in inhibitory function of Treg cell to inflammatory cytokines between plaque and guttate psoriasisObjective To investigate the inhibitory function of TGF-(3-Foxp3+iTreg cell in psoriasis vulgaris to inflammatory cytokines produced by CD4+CD25-T cells and the content of inflammatory cytokines in serum in correlation with the subtype of disease.Methods CD4+CD25-T cells with or without TGF-βstimulation were cultured for 5 days. The percentage of CD25+Foxp3+ cell in CD4+CD25-T cell stimulated with TGF-βwas detected by flow cytometry. The content of cytokines such as TNF-α, IL-6, IL-1β, IL-17 and IFN-y in serum and supernatants were determined by ELISA.Results The percentage of CD25+Foxp3+ cell among CD4+CD25-T cell stimulated with TGF-βin plaque psoriasis was significant higher than in guttate psoriasis and normal controls(p<0.0001), and in guttate psoriasis was higher than in normal controls(p<0.05). The difference was statistically significant. CD4+CD25-T cells in plaque psoriasis produced more TNF-a after stimulated with TGF-β; CD4+CD25-T cells in guttate psoriasis produced more IL-6 and IL-1β; however, the level of TNF-a, IL-6 and IL-1(3 produced by CD4+CD25-T cells in normal controls was reduced after stimulated with TGF-β. The level of IL-17 and IL-6 in serum of guttate psoriasis was higher than in plaque psoriasis; however, the level of TNF-a was lower than in plaque psoriasis.Conclusion The differential expression of inflammatory cytokines in serum and regulation of TGF-β-Foxp3+iTreg cells to inflammatory cytokines produced by CD4+CD25-T cells in plaque versus guttate psoriasis implicate their different immunopathogenesis.更多还原