寻常型白癜风患者血清及CD4~+CD25~+T细胞分泌TGF-β1水平检测
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。
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.
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。
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.
引文
1. Sakaguchi S, Sakaguchi N, AsanoM et al. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor a-chains(CD25). Breackdown of single mechanism of self-tolerance causes various autoimmune diseases. J Immunol.1995,155(3):1151-1164.
2. Crispin JC, Martinez A, Alcocer-Varela J. Quantification of regulatory T cells in patients with systemic lupus erythematosus. J Autoimmun.2003,21(3):273-276.
3. Liu MF, Wang CR, Fung LL, et al. Decreased CD4+CD25+T cells in peripheral blood of patients with systemic lupus erythematosus. Scand J Immunol. 2004,59(2) 198-202.
4. Sugiyama H, Gyulai R, Toichi E. Dysfunctional blood and target tissue CD4+CD25+regulatory T cells in psoriasis:mechanism underlying unrestrained pathogenice fector T cell proliferation. J Immunol.2005,174:164-173.
5. McElwee KJ, Freyschmidt-Paul P, Hoffmann R, et al. Transfer of CD8(+) cells induces localized hair loss whereas CD4(+)/CD25(-) cells promote systemic alopecia areats and CD4(+)/CD25(+) cells blockade disease onset in the C3H/HeJ mouse model. J Invest Dermatol.2005,124(5):947-957.
6. 陈小敏,杨秀丽,史维平.斑秃患者外周血CD4+CD25+Foxp3调节性T细胞及T淋巴细胞亚群的测定.中华皮肤科杂志.2008,4(11)：29-31.
7. Ehl AR, Gaus B, Tuderman LB, et al. Function and CLA expression of CD4+CD25+FOXP3+regulatory T cells in bullous pemphigoid Experimental. Dermatology.2006,16(1):13-21.
8. Dubois B, Chapat L, Goubier A, et al. Innate CD4+CD25+regulatory T cells are required for oral tolerance and inhibition of CD8+T cells mediating skin inflammation. Blood.2003,102(9):3295-3301.
9. Lee DJ, Modlin RL. Breaking tolerance-another piece added to the vitiligo puzz-le. J Invest Dermatol.2005,124:xiii-xv.
10. Verhagen J, Akdis M, Traidl-HC, et al. Absence of T-regulatory cell expression and function in atopic dermatitis skin. J Allergy Clin Immunol.2006,117 (1): 176-183.
11. Piccirillo CA, Shevach EM. Naturally-occurring CD4+CD25+immunoregulatory T cells:central players in the arena of peripheral tolerance. Semin Immunol. 2004,16(2):81-88.
12. Fehervari Z, Sakaguchi S. CD4+Tregs and immune control. J Clin Invest. 2004,114(9):1209-1217.
13. Shevach E M. Certified professionals:CD4+CD25+suppressor T cells.J Exp Med. 2001,193(11):F41-F45.
14. Shimizu J, Yamazaki S, Takahashi T, et al. Stimulation of CD25+CD4+regulatory T cells through GITR breaks immunological self-tolerance. Nat Immunol.2002,3 (2):135-142.
15. Walsh PT, Taylor DK, Turka I A. Tregs and transplantation tolerance. J Clin Invest.2004,114(10):1398-1403.
16. Faria AM, Weiner HL. Oral tolerance. Immunol Rev.2005,206:232-259.
17. Jiang H, Chess L. An integrated view of suppressor T cell subsets in immunoregulation. J Clin Invest.2004,114(9):1198-1208.
18. Dennler S, Itoh S, Vivien D, et al. Direct binding of Smad3 and Smad4 to critical TGF-β-inducible elements in the promoter of human plasminogen activator inhibitor-type 1 gene. The EMBO Journal.1998,17(11):3091-3100.
19. Petritsch C, Beug, et al TGF-P inhibits p70 S6 kinase via protein phosphatase 2A to induce G1 arrest. Genes and Development.2004,14(9):3093-3101.
20. Ostroukhova M, Qi ZB, et al. Treg-mediated immunosuppression involves activation of the Notch-HES1 axis by membrane-bound TGF-β. J Clin Invest. 2006,116 (4):996-1004.
21. Thompson C, Powrie F. Regulatory T cells. Current Opinion in Pharmacology. 2004,4(4):408-414.
22. Asano M, Toda M, Sakaguchi N, et al. Autoimmune disease as a consequence of developmental abnormality of a T cell subpopulation. J Exp Med.1996,184 (2):387-396.
23. Ruth YL, Aftab AA, Lian ZX ct al. Regulatory T cells:development function and role in autoimmunity. Autoimmunity.2005,4(6):351-363.
24.李泓馨,王华,高天..寻常型进展期白癜风患者外周血CD4+CD25+调节性T细胞检测.临床皮肤科杂志.2008,37(11)：714-716.
25.吴燕虹,杨慧兰,李薇,等.白癜风患者CD4+CD25+调节性T细胞水平检测.广东医学.2008,29(3)：481-482.
26.李延慧,侯晓彬,肖漓,等.CD4+CD25+调节性T细胞与Foxp3表达在白癜风发病中的作用.中国美容医学.2009,18(6)：819-822.
27. Alanko T, Saksela O. Transforming growth factor beta 1 induces apoptosis in normal melanocytes but not in nevus cells grown in type I collagen gel. Invest Dermatol.2000,115:286-291.
28. Basak PY, Adiloglu AK,et al.The role of helper and regulatory T Cells in the pathogenesis of vitiligo. J Am Acad Dermatol.2009,60(2):256-260.
29. Moretti S, Spallanzani A, Amato L, et al. New insights into the pathogenesis of vitiligo:imbalance of epidermal cytokines at sites of lesions. Pigment Cell Res. 2002,15(2)87-92.
30.徐前喜,杜娟,张建中,等.白癜风患者转化生长因子β1蛋白和mRNA的表达.中华皮肤科杂志.2003,36(10)：595.
1. 林元玮,南国荣.皮肤性病诊治与康复[M].北京：人民卫生出版社.1998.274.
2. 谢忠,季素珍,沈丽玉等.白癜风患者血清中抗黑素细胞自身抗体与临床发病关系.中华皮肤科杂志.1995,28(4)：232.
3. Gunduz K, Ozturk G, Terzioglu E, et al. T cell subpopulations and IL-2R in vitiligo. J Dermatol.2005,124(1):144-150.
4. 高晓明.CD4+CD25+调节性T细胞研究的意义.中华微生物和免疫学杂志.2002,22(2)：121-122.
5. Westerhof W, d'Ischia M. Vitiligo puzzle:the pieces fall in place. Pigment Cell Res.2007,20(5):345-359.
6. Groux H, Bigler M, Vries JE, et al. Interleukin 10 induces a long-term antigen specific anergic state in human CD4+T cells. J Exp Med.1996,184(1):19-29.
7. Shevach EA. Regulatory T cells in autoimmunity. Annu Rev Immunol 2000,18:423.
8. Seddon BD. Mason The third function of the thymus. Immunol Today. 2000,21:95.
9. Detlef D, Heidi P, Susanne B, et al. Ex vivo isolation and characterization of CD4+CD25+T cells with regulatory properties from human blood. J Exp Med. 2001,193(11):1303-1310.
10. Thornton AM, Shevach EM. CD4+CD25+immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukins-2 production. J Exp Med.1998,188:287.
11. Cederbom L, Hall H. CD4+CD25+regulatory T cells down regulate costimulatory molecules on antigen-presenting cells. Eur J Immunol.2000,30:1538-1540.
12.高晓明,鲁琰.CD4+CD25+调节性T细胞的研究现状及展望.细胞与分子免疫学杂志.2002,18(3)：201-202.
13. Lee DJ, Modlin RL Breaking tolerance-another piece added to the vitiligo puzzle. J Invest Dermatol 2005,124:ⅹⅲ-ⅹⅴ.
14.钟翠屏.CD4+CD25+调节性T细胞及其功能.解剖学杂志.2008,31(5)：605-607.
15. Thompson C, Powrie F Regulatory T cells. Current Opinion in Pharmacology. 2004,4(4):408-414.
16.中国中西医结合学会皮肤性病专业委员会色素病学组.白癜风临床分型及疗效标准(2003年修订稿).中华皮肤科杂志.2004,37(7)：440.
17. Maloy KJ, Salaun L, Cahil R, et al. CD4+CD25+T(R)cells suppress innate immune pathology through cytokine-dependent mechanisms. J Exp Med. 2003,197(1):111-119.
18. Masaki H, Cherry IK, Masanori N, et al. IL-10 is required for regulatory.
19. T cells to mediate tolerance to alloantigen in vivo. J Immunol. 2001,166(9):3789-3796.
20. Yamagiwa S, Gray J, Hashimoto S, et al. A role for TGF-βin the generation and expansion of CD4+CD25+regulatory T cells from human peripheral blood. J Immnunol.2001,166(12):728-729.
21. Kukreja A, Cost G, Marker J, et al. Multiple immuno-regulatory defects in type 1 diabetes. J Clin Invest.2002,109(1):131-140.
22. Cao D, van Vollenhoven R. Klareskog L, et al. CD25brightCD4+regulatory T cells en riched in inflamed joints of patients with chromic rheumatic disease. Arthri-tis Res Ther.2004,6(4):335-346.
23. Sugiyama H, Gyulai R, Toichi E, et al. Dysfunctional blood and target tissue CD4+CD25high regulatory T cells in psoriasis:mechanism underlying unrestrained pathogenic effector T cell proliferation. J Immunol.2005,174:164-173.
24. Crispin JC, Martinez A, Alcocer-Varela J. Quantification of regulatory T cells in patients with systemic lupus erythematosus J Autoimmun 2003,21(3):273-276.
25. Danese S, Rutella S. The Janus face of CD4+CD25+regulatory T cells in cancer and autoimmunity. Curr Med Chem.2007,14(6):649-666.
26. Ruth YL, Aftab AA, Lian ZX ct al. Regulatory T cells:development function and role in autoimmunity. Autoimmunity.2005,4(6):351-363.
27.李泓馨,王华,高天.寻常型进展期白癜风患者外周血CD4+CD25+调节性T细胞检测.临床皮肤科杂志.2008,37(11)：714-716.
28.吴燕虹,杨慧兰,李薇,等.白癜风患者CD4+CD25+调节性T细胞水平检测.广东医学.2008,29(3)：481-482.
29.李延慧,侯晓彬,肖漓,等.CD4+CD25+调节性T细胞与Foxp3表达在白癜风发病中的作用.中国美容医学.2009,18(6)：819-822.
30. Basak PY, Adiloglu AK, Ceyhan AM, et al. The role of helper and regulatory T Cells in the pathogenesis of vitiligo. J Am Acad Dermatol.2009,60(2):256-260.
31. Thompson C, Powrie F. Regulatory T cells. Current Opinion in Pharmacology. 2004,4(4):408-414.
32. K.Zhang, X.Li, G.Yin, et al. Functional characterization of CD4+CD25+ regulatory T cells differentiated in vitro from bone marrow-derived haematopoietic cells of psoriasis patients with a family history of the disorder. British Journal of Dermatology.2008,158(2):298-305
2. Crispin JC, Martinez A, Alcocer-Varela J. Quantification of regulatory T cells in patients with systemic lupus erythematosus. J Autoimmun.2003,21(3):273-276.
3. Liu MF, Wang CR, Fung LL, et al. Decreased CD4+CD25+T cells in peripheral blood of patients with systemic lupus erythematosus. Scand J Immunol. 2004,59(2) 198-202.
4. Sugiyama H, Gyulai R, Toichi E. Dysfunctional blood and target tissue CD4+CD25+regulatory T cells in psoriasis:mechanism underlying unrestrained pathogenice fector T cell proliferation. J Immunol.2005,174:164-173.
5. McElwee KJ, Freyschmidt-Paul P, Hoffmann R, et al. Transfer of CD8(+) cells induces localized hair loss whereas CD4(+)/CD25(-) cells promote systemic alopecia areats and CD4(+)/CD25(+) cells blockade disease onset in the C3H/HeJ mouse model. J Invest Dermatol.2005,124(5):947-957.
6. 陈小敏,杨秀丽,史维平.斑秃患者外周血CD4+CD25+Foxp3调节性T细胞及T淋巴细胞亚群的测定.中华皮肤科杂志.2008,4(11)：29-31.
7. Ehl AR, Gaus B, Tuderman LB, et al. Function and CLA expression of CD4+CD25+FOXP3+regulatory T cells in bullous pemphigoid Experimental. Dermatology.2006,16(1):13-21.
8. Dubois B, Chapat L, Goubier A, et al. Innate CD4+CD25+regulatory T cells are required for oral tolerance and inhibition of CD8+T cells mediating skin inflammation. Blood.2003,102(9):3295-3301.
9. Lee DJ, Modlin RL. Breaking tolerance-another piece added to the vitiligo puzz-le. J Invest Dermatol.2005,124:xiii-xv.
10. Verhagen J, Akdis M, Traidl-HC, et al. Absence of T-regulatory cell expression and function in atopic dermatitis skin. J Allergy Clin Immunol.2006,117 (1): 176-183.
11. Piccirillo CA, Shevach EM. Naturally-occurring CD4+CD25+immunoregulatory T cells:central players in the arena of peripheral tolerance. Semin Immunol. 2004,16(2):81-88.
12. Fehervari Z, Sakaguchi S. CD4+Tregs and immune control. J Clin Invest. 2004,114(9):1209-1217.
13. Shevach E M. Certified professionals:CD4+CD25+suppressor T cells.J Exp Med. 2001,193(11):F41-F45.
14. Shimizu J, Yamazaki S, Takahashi T, et al. Stimulation of CD25+CD4+regulatory T cells through GITR breaks immunological self-tolerance. Nat Immunol.2002,3 (2):135-142.
15. Walsh PT, Taylor DK, Turka I A. Tregs and transplantation tolerance. J Clin Invest.2004,114(10):1398-1403.
16. Faria AM, Weiner HL. Oral tolerance. Immunol Rev.2005,206:232-259.
17. Jiang H, Chess L. An integrated view of suppressor T cell subsets in immunoregulation. J Clin Invest.2004,114(9):1198-1208.
18. Dennler S, Itoh S, Vivien D, et al. Direct binding of Smad3 and Smad4 to critical TGF-β-inducible elements in the promoter of human plasminogen activator inhibitor-type 1 gene. The EMBO Journal.1998,17(11):3091-3100.
19. Petritsch C, Beug, et al TGF-P inhibits p70 S6 kinase via protein phosphatase 2A to induce G1 arrest. Genes and Development.2004,14(9):3093-3101.
20. Ostroukhova M, Qi ZB, et al. Treg-mediated immunosuppression involves activation of the Notch-HES1 axis by membrane-bound TGF-β. J Clin Invest. 2006,116 (4):996-1004.
21. Thompson C, Powrie F. Regulatory T cells. Current Opinion in Pharmacology. 2004,4(4):408-414.
22. Asano M, Toda M, Sakaguchi N, et al. Autoimmune disease as a consequence of developmental abnormality of a T cell subpopulation. J Exp Med.1996,184 (2):387-396.
23. Ruth YL, Aftab AA, Lian ZX ct al. Regulatory T cells:development function and role in autoimmunity. Autoimmunity.2005,4(6):351-363.
24.李泓馨,王华,高天..寻常型进展期白癜风患者外周血CD4+CD25+调节性T细胞检测.临床皮肤科杂志.2008,37(11)：714-716.
25.吴燕虹,杨慧兰,李薇,等.白癜风患者CD4+CD25+调节性T细胞水平检测.广东医学.2008,29(3)：481-482.
26.李延慧,侯晓彬,肖漓,等.CD4+CD25+调节性T细胞与Foxp3表达在白癜风发病中的作用.中国美容医学.2009,18(6)：819-822.
27. Alanko T, Saksela O. Transforming growth factor beta 1 induces apoptosis in normal melanocytes but not in nevus cells grown in type I collagen gel. Invest Dermatol.2000,115:286-291.
28. Basak PY, Adiloglu AK,et al.The role of helper and regulatory T Cells in the pathogenesis of vitiligo. J Am Acad Dermatol.2009,60(2):256-260.
29. Moretti S, Spallanzani A, Amato L, et al. New insights into the pathogenesis of vitiligo:imbalance of epidermal cytokines at sites of lesions. Pigment Cell Res. 2002,15(2)87-92.
30.徐前喜,杜娟,张建中,等.白癜风患者转化生长因子β1蛋白和mRNA的表达.中华皮肤科杂志.2003,36(10)：595.
1. 林元玮,南国荣.皮肤性病诊治与康复[M].北京：人民卫生出版社.1998.274.
2. 谢忠,季素珍,沈丽玉等.白癜风患者血清中抗黑素细胞自身抗体与临床发病关系.中华皮肤科杂志.1995,28(4)：232.
3. Gunduz K, Ozturk G, Terzioglu E, et al. T cell subpopulations and IL-2R in vitiligo. J Dermatol.2005,124(1):144-150.
4. 高晓明.CD4+CD25+调节性T细胞研究的意义.中华微生物和免疫学杂志.2002,22(2)：121-122.
5. Westerhof W, d'Ischia M. Vitiligo puzzle:the pieces fall in place. Pigment Cell Res.2007,20(5):345-359.
6. Groux H, Bigler M, Vries JE, et al. Interleukin 10 induces a long-term antigen specific anergic state in human CD4+T cells. J Exp Med.1996,184(1):19-29.
7. Shevach EA. Regulatory T cells in autoimmunity. Annu Rev Immunol 2000,18:423.
8. Seddon BD. Mason The third function of the thymus. Immunol Today. 2000,21:95.
9. Detlef D, Heidi P, Susanne B, et al. Ex vivo isolation and characterization of CD4+CD25+T cells with regulatory properties from human blood. J Exp Med. 2001,193(11):1303-1310.
10. Thornton AM, Shevach EM. CD4+CD25+immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukins-2 production. J Exp Med.1998,188:287.
11. Cederbom L, Hall H. CD4+CD25+regulatory T cells down regulate costimulatory molecules on antigen-presenting cells. Eur J Immunol.2000,30:1538-1540.
12.高晓明,鲁琰.CD4+CD25+调节性T细胞的研究现状及展望.细胞与分子免疫学杂志.2002,18(3)：201-202.
13. Lee DJ, Modlin RL Breaking tolerance-another piece added to the vitiligo puzzle. J Invest Dermatol 2005,124:ⅹⅲ-ⅹⅴ.
14.钟翠屏.CD4+CD25+调节性T细胞及其功能.解剖学杂志.2008,31(5)：605-607.
15. Thompson C, Powrie F Regulatory T cells. Current Opinion in Pharmacology. 2004,4(4):408-414.
16.中国中西医结合学会皮肤性病专业委员会色素病学组.白癜风临床分型及疗效标准(2003年修订稿).中华皮肤科杂志.2004,37(7)：440.
17. Maloy KJ, Salaun L, Cahil R, et al. CD4+CD25+T(R)cells suppress innate immune pathology through cytokine-dependent mechanisms. J Exp Med. 2003,197(1):111-119.
18. Masaki H, Cherry IK, Masanori N, et al. IL-10 is required for regulatory.
19. T cells to mediate tolerance to alloantigen in vivo. J Immunol. 2001,166(9):3789-3796.
20. Yamagiwa S, Gray J, Hashimoto S, et al. A role for TGF-βin the generation and expansion of CD4+CD25+regulatory T cells from human peripheral blood. J Immnunol.2001,166(12):728-729.
21. Kukreja A, Cost G, Marker J, et al. Multiple immuno-regulatory defects in type 1 diabetes. J Clin Invest.2002,109(1):131-140.
22. Cao D, van Vollenhoven R. Klareskog L, et al. CD25brightCD4+regulatory T cells en riched in inflamed joints of patients with chromic rheumatic disease. Arthri-tis Res Ther.2004,6(4):335-346.
23. Sugiyama H, Gyulai R, Toichi E, et al. Dysfunctional blood and target tissue CD4+CD25high regulatory T cells in psoriasis:mechanism underlying unrestrained pathogenic effector T cell proliferation. J Immunol.2005,174:164-173.
24. Crispin JC, Martinez A, Alcocer-Varela J. Quantification of regulatory T cells in patients with systemic lupus erythematosus J Autoimmun 2003,21(3):273-276.
25. Danese S, Rutella S. The Janus face of CD4+CD25+regulatory T cells in cancer and autoimmunity. Curr Med Chem.2007,14(6):649-666.
26. Ruth YL, Aftab AA, Lian ZX ct al. Regulatory T cells:development function and role in autoimmunity. Autoimmunity.2005,4(6):351-363.
27.李泓馨,王华,高天.寻常型进展期白癜风患者外周血CD4+CD25+调节性T细胞检测.临床皮肤科杂志.2008,37(11)：714-716.
28.吴燕虹,杨慧兰,李薇,等.白癜风患者CD4+CD25+调节性T细胞水平检测.广东医学.2008,29(3)：481-482.
29.李延慧,侯晓彬,肖漓,等.CD4+CD25+调节性T细胞与Foxp3表达在白癜风发病中的作用.中国美容医学.2009,18(6)：819-822.
30. Basak PY, Adiloglu AK, Ceyhan AM, et al. The role of helper and regulatory T Cells in the pathogenesis of vitiligo. J Am Acad Dermatol.2009,60(2):256-260.
31. Thompson C, Powrie F. Regulatory T cells. Current Opinion in Pharmacology. 2004,4(4):408-414.
32. K.Zhang, X.Li, G.Yin, et al. Functional characterization of CD4+CD25+ regulatory T cells differentiated in vitro from bone marrow-derived haematopoietic cells of psoriasis patients with a family history of the disorder. British Journal of Dermatology.2008,158(2):298-305