【作者】 金宛宛；

【导师】 涂彩霞；

【作者基本信息】 大连医科大学 ， 皮肤病与性病学， 2010， 硕士

【摘要】 背景和目的：白癜风是一种获得性的皮肤色素异常,表现为脱色性的白斑。愈来愈多的证据表明白癜风的发病和免疫学说密切相关。免疫耐受的破坏可能在白癜风发病中起作用。CD4+CD25+T细胞(regulatory T cells,Treg)是一群独特的免疫调节细胞,具有免疫抑制作用。CD4+CD25+T细胞无论是数量上还是功能上的缺陷均可导致自身免疫疾病的发生。CD4+CD25+T细胞可以通过分泌的细胞因子来抑制淋巴细胞的活化,其中最为重要的是转化生长因子β1 (transforming growth factory-beta, TGF-β)。因此,对CD4+CD25+T细胞及其分泌的的TGF-β1研究有助于阐明白癜风的发病机制。我们前期研究发现进展期白癜风患者外周血CD4+CD25+T细胞占外周血淋巴细胞的百分比显著低于正常人,foxp3的表达低于正常人表达,支持白癜风患者CD4+CD25+T细胞存在数量不足和/或功能缺陷。为进一步探讨白癜风患者可能存在的CD4+CD25+T细胞功能缺陷,本研究检测白癜风患者血清及外周血CD4+CD25+T细胞分泌TGF-β1水平并分析其与年龄、性别、病程和皮损面积的相关性。方法：门诊寻常型白癜风患者46例,其中进展期28例,稳定期18例,及正常对照组25例。收集白癜风患者和正常对照组血清,体外分离、纯化外周血单个核细胞,采用免疫磁珠两步法分离纯化CD4+CD25+T细胞,流式细胞仪检测分离纯度。获得的CD4+CD25+T细胞体外培养,加用anti-CD3mAb和anti-CD28mAb激活,培养第5天收集细胞,采用ELISA法分别检测白癜风和正常对照组血清及CD4+CD25+T细胞培养液TGF-β1水平,并分析TGF-β1水平与年龄、性别、病程和皮损面积的相关性。结果：一、白癜风患者血清中TGF-β1水平分析：1.进展期白癜风组血清中TGF-β1的水平(1019.49±276.34pg／ml)低于稳定期白癜风组(6839.74±235.25 pg／ml)及正常对照组(7068.32±755.81 pg／ml),差异均有统计学意义(P<0.05)。稳定期白癜风组血清中TGF-β1水平与正常对照组比较,差异无统计学意义(P>0.05)。2.白癜风组和正常对照组血清中TGF-β1水平差异与皮损面积呈正相关性(r=0.337,P<0.05),与年龄、性别、病程无相关性(P>0.05)。二、体外培养白癜风患者CD4+CD25+T细胞分泌TGF-β1水平分析：1.进展期白癜风组CD4+CD25+T细胞分泌TGF-β1的水平(57.78±2.87pg／ml)低于稳定期白癜风组(71.33±5.50pg／ml)及正常对照组(71.97±4.42pg／ml),差异均有统计学意义(P<0.05)。稳定期白癜风组TGF-β1的水平与正常对照组比较,差异无统计学意义(P>0.05)。2.白癜风组和正常对照组CD4+CD25+T细胞分泌TGF-β1水平差异与皮损面积呈正相关性(r=0.34,P<0.05),与年龄、性别、病程无相关性(P>0.05)。结论：1.无论是血清中或体外培养CD4+CD25+T细胞培养液中,进展期白癜风患者TGF-β1的水平均低于正常对照组和稳定期白癜风患者,提示进展期白癜风患者可能存在CD4+CD25+T细胞功能缺陷。2.无论血清中或体外培养CD4+CD25+T细胞的培养液中,白癜风组和正常对照组TGF-β1水平差异均与皮损面积呈正相关性,与年龄、性别、病程无相关性,提示TGF-β1可能从一定程度上反应病情严重性。更多还原

【Abstract】 Background and objectives:Vitiligo is an acquired pigmentary anomaly of the skin manifested by depigmented white patches surrounded by a normal or a hyperpigmented border. More and more evidences indicated that immune theory is close to the morbidity of vitiligo. Some scholars consider that the breakdown of immunological tolerance may play an important role in vitiligo pathogenesis.The relationship between the level of CD4+CD25+regulatory T cells (CD4+CD25+T cells) expressed in human beings and autoimmune diseases has become a hot spot in the research nowadays. TGF-βis a molecle functioning in immunological regulation and immune suppression. TGF-βcontributes to the maintenance of immunological self-tolerance. Abnormal expression of CD4+CD25+regulatory T cells and related cytokines may be involved in the pathogenesis of vitiligo. Therefore, study on CD4+CD25+T cells and related cytokines may be helpful with illuminating the mechanism of immunological regulation on the molecular level.In the previous study, we examined the proportion of CD4+CD25+T cells in the peripheral lymphocytes from patients with progressing vitiligo and found that the number of CD4+CD25+T cells and the expression of Foxp3 were lower than the normal controls, suggesting that the decrease of CD4+CD25+T cells or abnormality of CD4+CD25+T cells may be correlated with the development of vitiligo. The aim of the present study is to investigate the the level of TGF-β1 from serum and CD4+CD25+T cells in patients with vitiligo and analyze its clinical significance, in order to study the role of CD4+CD25+T cells and TGF-β1 in vitiligo. The relevance with the age,sex and course of disease as well as lesion area were conducted by correlation analysis.Methods:Forty six patients with vitiligo from out-patient clinic and 25 age-matched and sex-matched healthy control subjects were included in the study. Twenty six of the patients were in active period,20 in stable phase. Lymphocytes were separated from patients with vitiligo and normal group, and serum samples were collected from vitiligo patients and healthy controls. CD4+CD25+regulatory T cells were separated from the human PBMC in two steps by magnetic cell sorting (MACS) system. CD4+T cells were negatively sorted by biotin-antibody cocktail and antibiotic microbeads, and then CD4+CD25+T cells were positively sorted by CD25 microbeads. The purity and the survival rate of the sorted cells were measured by flow cytometry, and then stimulated with anti-CD3mAb and anti-CD28mAb.The CD4+CD25+T cells on day 5 after culture was collected. The levels of TGF-P both in the serum and supernatant in the medium of cultured CD4+CD25+T cells were detected by ELISA. In order to study the role of CD4+CD25+T cells and TGF-βin vitiligo, the relations with the age, sex and course of disease as well as lesion area were conducted by correlation analysis using spss software.Results:Analysis of.Serum TGF-β1 levels:1.Serum TGF-β1 levels(1019.49±276.34pg/ml) were significantly decreased in the progressing vitiligo group compared with the control group (7068.32±755.81pg/ml)(P<0.05);Serum TGF-β1 levels(1019.49±276.34pg/ ml) were significantly decreased in the progressing vitiligo group compared with the stable vitiligo group(6839.74±235.25pg/ml)(P<0.05).No difference was detected between the stable vitiligo group(6839.74±235.25 pg/ml) and control group (7068.32±755.81pg/ml) in mean levels of serum TGF-β1 (P >0.05).2. The level of serum TGF-β1 was not correlated with the age, sex and course of vitiligo(P>0.05), while positively correlated with area of skin lesions(r=0.337, P<0.05).Analysis of. TGF-β1 levels secreted by CD4+CD25+T: 1. The levels of (57.78±2.87pg/ml) TGF-β1 secreted by CD4+CD25+T cells were decreased in the progressing vitiligo group compared with the control group (71.97±4.42pg/ml)(P<0.05).The levels of(57.78±2.87pg/ml) TGF-β1 secreted by CD4+CD25+T cells were decreased in the progressing vitiligo group compared the stable vitiligo group (71.33±5.50pg/ml) (P<0.05).The levels of (71.33±5.50) TGF-β1 secreted by CD4+CD25+T cells was no difference in the stable vitiligo group compared with the control group (71.967±4.42pg/ml) (P>0.05).2. The level of TGF-β1 secreted by CD4+CD25+T cells was not correlated with the age, sex and course of vitiligo(P>0.05),and positively correlated with area of skin lesions(r=0.34, P<0.05).Conclusions:1.TGF-β1 levels both from serum and CD4+CD25+T cells were decreased in the progressing vitiligo group compared with the control group.TGF-β1 levels both from serum and CD4+CD25+T cells were decreased in the progressing vitiligo group compared with stable vitiligo group. There was no difference between the stable vitiligo group and control group in.TGF-β1 levels both from serum and CD4+CD25+T cells The level of serum TGF-β1 was not correlated with the age, sex and course of vitiligo, while positively correlated with area of skin lesions.2. The level of TGF-β1 secreted by CD4+CD25+T cells was not correlated with the age, sex and course of vitiligo, while positively correlated with area of skin lesions.更多还原