SLE患者T细胞TCR/CD3复合物介导的[Ca++]i反应与患者临床特征相关性的研究

为研究SLE患者原发的T细胞功能异常与TCR/CD3复合物介导生物化学信号转导异常相关,及与其临床特征的相关性。用尼龙柱分离肝素化的静脉血得到T细胞悬液,用CD3单抗与羊抗鼠二抗IgG相交联刺激T细胞,粘附细胞仪连续观察10min,观察T细胞犤Ca++犦i的变化,并分析其与患者的临床特征相关性。发现正常人和SLET细胞的[Ca++]i反应的基准值相似(P=0.105),SLE患者T细胞高峰值和平台值均明显高于正常对照(P<0.001,P<0.001),且与患者临床特征无关。     

抗CD3单抗介导的系统性红斑狼疮患者T细胞内钙离子反应与临床特征的相关性

本实验研究了抗CD3单抗刺激SLE患者T细胞TCR/CD3复合物后的[Ca⁺⁺]i反应,及其与患者临床特征相关性。     

间质干细胞对SLE患者外周血T细胞的免疫抑制作用

目的 探讨体外骨髓间质干细胞(MSC)对SLE患者外周血T淋巴细胞(T细胞)的免疫调节作用及可能机制.方法 分离培养健康人骨髓MSC并鉴定其纯度,分离SLE患者外周血T细胞,在植物血凝素(PHA)刺激下,用不同比例的MSC与T细胞作用.分为T细胞、T细胞+MSC_1(T:MSC_1=1:0.02)、T细胞+MSC_2(T:MSC_2=1:0.2)3组.采用MTF法检测T细胞增殖,流式细胞仪检测T细胞CD152~+和CD28~+的表达,实时PCR测定IFN-γ和IL-6 mRNA的表达水平.结果 MSC可明显抑制SLE患者T细胞增殖.T细胞增殖在T细胞组A值为0.765±0.036,T细胞+MSC_1组为0.484±0.032,T细胞+MSC_2组为0.308±0.025,与对照组相比,A值均明显降低(P＜0.05),T细胞+MSC_1组和T细胞+MSC_2组之间差异也有统计学意义(P＜0.05).T细胞组CD28~+细胞百分比为(74.73±3.74)%,T细胞+MSC_1组为(60.39±3.92)%,T细胞+MSC_2组为(45.05±3.46)%,与对照组相比均明显降低(P＜0.05),T细胞+MSC_1组和T细胞+MSC_2组之间差异也有统计学意义(P＜0.05).MSC对T细胞CD152+表达无显著影响.MSC明显抑制T细胞IFN-γ、IL-6 mRNA表达(P＜0.05),并且随着MSC数昔的增加,抑制作用增强.结论 MSC对SLE患者T细胞有免疫抑制作用,可能与抑制T细胞增殖、抑制T细胞CD28~+表达和抑制T细胞IFN-γ、IL-6 mRNA表达有关.     

CD4+CD25+T细胞和NK细胞与SLE病情活动相关性的研究

目的: 了解CD4+CD25+T细胞和NK细胞与SLE病情活动的相关性.方法: 用流式细胞仪检测17例SLE患者治疗前后和11例正常人外周血中CD3+T(总T)、CD4+T、CD8+T、NK细胞、CD4+CD25+T、CD4+CD25-T细胞.结果: SLE活动期CD8+T细胞百分率升高(P<0.05),导致CD4+/CD8+比例降低(P<0.05),CD4+CD25+T、NK细胞百分率明显降低(P<0.01),而CD3+T、CD4+T细胞、CD4+CD25-T细胞百分率无明显改变(P>0.05);治疗后患者SLEDAI评分下降而NK细胞和CD4+CD25+T细胞百分率均明显上升(均P<0.01);SLEDAI评分与CD4+CD25+T细胞百分率有相关性(P<0.05).结论: SLE存在CD4+T、CD8+T细胞亚群分布和活化的异常;CD4+CD25+T细胞数量明显降低,且量的变化与疾病活动相关;NK细胞数量虽然也明显降低,治疗后也上升,但量的变化与疾病活动无明显相关.     

系统性红斑狼疮患者CD4~+ CD25~+调节性T细胞及其细胞因子的改变

目的探讨系统性红斑狼疮(SLE)患者CD4+CD25+调节性T细胞(Tr)及其细胞因子的改变。方法用流式细胞仪直接免疫荧光法检测SLE患者外周血CD4+CD25+T细胞的百分率,FoxP3+分子表达,细胞因子IL-10,TGF-β1的表达。结果 SLE患者活动期组、稳定期组、健康对照组CD4+CD25+T细胞占CD4+T细胞的百分率分别是(3.57±1.45)%,(5.14±1.63)%,(6.03±1.96)%。活动期患者明显低于稳定期患者和健康对照组,差异均有统计学意义(P均<0.05)。CD4+CD25+FoxP3+T细胞、CD4+CD25-FoxP3+T细胞占CD4+T细胞的百分率,活动期患者明显高于稳定期患者和健康对照组,差异均有统计学意义(P<0.05)。CD4+CD25+FoxP3+T细胞与疾病的活动指数(DAI)正相关(r=0.659,P<0.05);而CD4+CD25-FoxP3+T细胞与DAI无统计学相关性(r=0.184,P>0.05)。活动期患者CD4+CD25+IL-10+细胞明显高于稳定期患者和健康对照组,差异均有统计学意义(P均<0.05)。然而CD4+CD25-TGF-β+细胞在活动期患者、稳定期患者和健康对照组之间比较差异无统计学意义(P均>0.05)。结论 SLE患者外周血中Tr细胞数量减少,分泌的IL-10增加,TGF-β减少,可能导致细胞免疫抑制功能减弱,自身免疫耐受平衡被打破,出现激活的自身反应性T细胞增多;此外,T淋巴细胞激活增多,还可辅助B细胞产生更多相关抗体,表现出多种临床症状。     

慢性荨麻疹患者外周血CD4+CD25+调节性T细胞的表达

目的:探索调节性T细胞(regulatory T cells,Tregs)在慢性荨麻疹(CU)发病中的作用.方法:采用流式细胞术分别检测CU患者和健康人外周血CD3+、CD4+、CD8+、CD4+CD25+T细胞的表达水平.结果:与健康对照组比较,CU患者外周血CD3+T细胞比例无明显变化,CD4+T细胞增高(P ＜ 0.05),CD4+CD25+T细胞表达水平显著增高(P ＜ 0.01),CD8+T细胞数量降低(P ＜ 0.05),CD4+/ CD8+比值明显升高(P ＜ 0.05).自身血清皮肤试验(ASST)阳性组与阴性组的CU患者CD4+CD25+Tregs 细胞的数量无统计学差异.结论:CU患者外周血存在Treg细胞及其他T淋巴细胞亚群的数量异常和分化失衡,这一异常在CU发病中的作用值得进一步研究.     

系统性红斑狼疮T细胞的TCR／CD3信号转导

SLE是一种病因未明,累及多系统的自身免疫性疾病,从过去20年的研究中,我们认识到SLE的细胞免疫失调是一个复杂的多细胞系的功能紊乱,T细胞被认为是SLE发病机理之关键.Dayal等[1]提出,原发的T细胞异常,信号转导的生化途径失调,导致基因调节或表达的异常.使占主导地位的Tho细胞因子分泌方式转变为Th2方式,Tho细胞可产生Th1CK(如:IL-2、INF-γ)和Th2CK(如IL4、5、10),缺陷性T细胞的分泌细胞因子的转变,影响了B细胞克隆的免疫调节,导致多克隆B细胞的活化和无限制的致病自身抗体的产生.本文就SLE中TCR/CD3复合体介导的信号转导途径的异常作一简要的阐述.     

系统性红斑狼疮患者T细胞亚群与T淋巴细胞CD2免疫分子功能研究

系统性红斑狼疮(SLE)与T细胞受体介导的信号传导异常有关。红细胞具有天然免疫特性，又具有适应性免疫的特性，有识别、黏附、杀伤抗原，清除循环免疫复合物的能力．参与机体免疫调控。红细胞CD58、CD59与T细胞的CD2分子密切相关，二者互为天然配体，其相互作用是红细胞调控细胞因子产生的主要分子基础。我们通过对SLE患者红细胞的天然免疫研究，探讨T淋巴细胞CD2及T细胞亚群CD4^ 、CD8^ 以及CD4^ ／CD8^ 的比值在SLE患者中的意义，进一步探讨SLE的发病机制。     

特应性皮炎患者外周血CD4+CD25+调节性T细胞的检测

目的 探讨CD4+CD25+调节性T细胞（CD4+CD25+ Treg）在特应性皮炎（AD）发病中的作用机制及临床意义。方法 流式细胞仪分析AD患者外周血中CD4+CD25+ Treg细胞数量,实时荧光定量PCR检测外周血单核细胞（PBMC）中Foxp3 mRNA水平,ELISA检测血清中IL-2、IL-4、IL-10、IFN-γ水平。结果 AD患者外周血中CD4+CD25+ Treg细胞占CD3+ T细胞及CD4+ T细胞的比例均明显低于正常人对照组（t′ = 3.775、4.533,P值均 ＜ 0.01）;外周血中CD4+CD25+ Treg细胞占CD3+ T细胞比例在AD患者急性期明显低于慢性期（t = 2.217,P < 0.05）,而在急性期与亚急性期、亚急性期与慢性期之间差异均无统计学意义（t = 1.558、0.49,P值均 ＞ 0.05）。AD患者PBMC中Foxp3 mRNA的水平低于正常人对照组（z = -2.368,P ＜ 0.05）;其外周血中CD4+CD25+ Treg细胞与血清中IL-2和IL-10成正相关（r = 0.512、0.494,P值均 ＜ 0.05）,与IL-4和IFN-γ的相关性无统计学意义（r = -0.110、-0.237,P值均 ＞ 0.05）。结论 在AD患者中,外周血中CD4+CD25+ Treg细胞数量及Foxp3 mRNA水平均下降,从而可能减少对Th2细胞增生及其细胞因子分泌的抑制,使Th2占优势,参与AD的发病。     

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

目的 研究斑秃患者外周血中CD4+CD25+调节性T细胞数量及CD4+、CD8+ T淋巴细胞亚群数量与斑秃疾病严重程度的关系。方法 对斑秃进行病情分组，以流式细胞仪检测17例重度、15例局限型斑秃患者和25例正常人对照者外周血中有功能活性的CD4+CD25+调节性T细胞（即CD4+CD25+ Foxp 3 T 细胞）在CD4+ T淋巴细胞中的比率，CD4+和CD8+占T淋巴细胞的比率。 结果 重度斑秃患者外周血中有功能活性的CD4+CD25+ Foxp 3 T细胞占CD4+ T细胞比率为0.54% ± 0.31%，显著低于正常人对照组（3.21% ± 0.76%）及局限型斑秃患者（2.71% ± 0.37%，P ＜ 0.001）；与正常人对照组比较，差异无统计学意义（P ＞ 0.05）。重度斑秃患者的CD4+占T淋巴细胞的比率为32.61% ± 3.48%，显著低于正常人对照组（43.0% ± 3.63%，P ＜ 0.001），而CD8+占T淋巴细胞的比率为40.96% ± 8.54%，显著高于正常人对照组（25.23% ± 2.14%，P ＜ 0.001）。局限型斑秃患者的此两项指标分别为41.25% ± 4.27%和26.6% ± 2.28%，与对照组差异无统计学意义（P ＞ 0.05）。重度斑秃患者的CD8+占T淋巴细胞的比率与CD4+CD25+ Foxp 3调节性 T 细胞占CD4+ T细胞的比率有负相关关系（r ＝ －0.94，P ＜ 0.001）。结论 重度斑秃可能与外周血中CD4+CD25+ T细胞数量的减少和功能活性的降低有关。     

Keratinocytes cultured from patients with Hailey-Hailey disease and Darier disease display distinct patterns of calcium regulation

BACKGROUND: Hailey-Hailey disease (HHD) (OMIM 16960) and Darier disease (DD) (OMIM 124200) are dominantly inherited acantholytic skin diseases, respectively, caused by mutations in the genes encoding the Golgi secretory pathway Ca2+-ATPase (SPCA1, ATP2C1) and the sarco/endoplasmic reticulum Ca2+-ATPase type 2 (SERCA2, ATP2A2) genes. OBJECTIVES: To investigate calcium regulation in keratinocytes cultured from patients with HHD and DD by measuring intracellular calcium resting levels and the cellular responses to ATP and thapsigargin. METHODS: The study was carried out using keratinocyte cultures established from four patients with HHD and four with DD. Calcium concentrations were measured with fluorescence ratio imaging using fura-2 loading. RESULTS: Control and HHD keratinocytes displayed approximately the same Ca2+ levels in resting phase, while DD keratinocytes showed elevated Ca2+ levels. Application of ATP caused less pronounced elevation of intracellular calcium concentration ([Ca2+]i) in both HHD and DD keratinocytes than in control cells. HHD keratinocytes did not lower their [Ca2+]i as efficiently as control keratinocytes after treatment with thapsigargin. In addition, DD keratinocytes were practically incapable of lowering their [Ca2+]i after treatment with thapsigargin. CONCLUSIONS: The results demonstrate that the defects in SPCA1 and SERCA2 calcium ATPases result in distinct patterns of calcium metabolism. This is also supported by the different clinical features of the diseases.     

Influence of ion channel blockers on proliferation and free intracellular Ca2+ concentration of human keratinocytes.

The proliferation of in vitro cultivated keratinocytes from the HaCaT cell line while under the influence of different ion channel blockers, i.e. calcium channel blockers tetraethylammonium (TEA) and the nonspecific anion channel blocker 4',4'-diisothiocyanato-stilbene-2-2' disulfonic acid (DIDS) was investigated. There were changes in proliferation of the keratinocytes brought on by TEA and DIDS. It can be assumed that the effects of TEA on proliferated keratinocytes lead to a temporary arrest of cells in the G(1)-phase of the cell cycle. The change in the extracellular K+ concentration ([K+]e) had no influence on the proliferation of keratinocytes. During the entire course of investigations, the adding of DIDS leads to an increase in proliferation. Proliferation of keratinocytes is known to be greatly dependent on intracellular Ca2+ content ([Ca2+]i). The blocking of ion channels with TEA or DIDS does not lead to a change in the free [Ca2+]i. Also a change in the [K+]e does not lead to a displacement of the [Ca2+]i of keratinocytes.     

Adenylate cyclase induces intracellular calcium increase in single human epidermal keratinocytes measured by fluorescence microscopy using Fura 2-AM

Intracellular calcium ([Ca2+]i) is an important second messenger of extracellular signals to induce various cellular responses. Extracellular and intracellular Ca2+ are considered to be important for cellular differentiation and proliferation of epidermal keratinocytes. Several mechanisms which increase [Ca2+]i have been demonstrated in various tissues, but in epidermal keratinocytes these mechanisms are poorly understood. In epidermal keratinocytes the adenylate cyclase-cyclic AMP response is thought to regulate cell proliferation and differentiation. However, the series of reactions which follow the cyclic AMP response remain unknown. Beta-adrenergic agonists increase [Ca2+]i in cultured epidermal keratinocytes, and we have therefore studied whether stimulation of keratinocyte adenylate cyclase could induce [Ca2+]i increase, by using fluorescence microscopy with Fura 2-AM. Adenosine and histamine, which are known to be keratinocyte adenylate cyclase receptor agonists, induced transient [Ca2+]i increase, as did epinephrine. In addition, forskolin, a direct adenylate cyclase activator, and dibutyryl-cyclic AMP also induced an increase in [Ca2+]i. In a calcium-free medium epinephrine, adenosine, histamine and dibutyryl-cyclic AMP induced an increase in [Ca2+]i. These results suggest that cyclic AMP in human epidermal keratinocytes regulates [Ca2+]i, which is released from intracellular stores.     

Calcium release activated calcium entry in a human skin derived cell line (HaCaT)

Using isolated cells from a spontaneously immortalized human keratinocyte cell line (HaCaT) loaded with Fura-2 the regulation of intracellular Ca2+ concentration ([Ca2+]i) was studied. The continuous presence of ATP induced a biphasic response in high external Ca2+. The first component reflected the release of calcium from intracellular stores since it was present after the removal of external calcium with ethylene-glycol-bis-N,N,N',N'-tetraacetic acid (EGTA). The second phase of [Ca2+]i increase was not detectable in the absence of external calcium and raising the extracellular [Ca2+] increased the rate of rise in [Ca2+]i suggesting that it was influenced by the external environment. Furthermore, after adenosine triphosphate (ATP) had emptied the intracellular store in a calcium-free milieu, the elevation of external Ca2+ induced a secondary increase in [Ca2+]i that did not require the presence of ATP. Depleting the intracellular calcium store with a Ca-ATP-ase inhibitor (cyclopiasonic acid, 10 microM) also induced calcium entry. The depletion induced calcium entry was inhibited by econazole (100 microM). These findings indicate the presence of a calcium release activated calcium influx pathway in HaCaT keratinocytes.     

淋巴细胞活化在系统性红斑狼疮发病中的意义

目的 :　探讨系统性红斑狼疮 (SLE)患者外周血淋巴细胞HLA -DR以及植物凝集素 (PHA)刺激下的CD3+ CD4 + 、CD3+ CD8+ 、CD19+ 淋巴细胞的早期活化标志CD6 9的表达。方法 :　应用三色荧光标记流式细胞术检测活动期和非活动期SLE患者外周血CD3+ 、CD3+ CD4 + 、CD3+ CD8+ 淋巴细胞晚期活化标志HLA -DR以及在PHA刺激 4h后CD3+ CD4 + 、CD3+ CD8+ 、CD19+ 淋巴细胞的早期活化标志CD6 9的表达。结果 :　①PHA刺激 4小时后 ,活动期SLECD3+ CD4 + 、CD3+ CD8+ 细胞CD6 9的表达明显低于正常对照组 (P <0 .0 5 ) ;而非活动期SLE与正常对照组之间CD6 9表达无显著性差异 (P >0 .0 5 )。②PHA刺激前SLE活动组CD19+ 细胞表达CD6 9较正常对照组和SLE非活动期明显升高 (P <0 .0 5 ) ,PHA刺激 4小时后 ,活动期SLECD19+ 细胞表达CD6 9低于正常对照组和非活动期SLE ,存在显著性差异 (P <0 .0 5 ) ,而非活动期SLE与正常对照组之间CD19+ CD6 9+ 表达无明显差异 (P >0 .0 5 )。③活动期SLECD3+ HLA -DR+细胞明显高于正常对照组 (P <0 .0 1)和非活动期SLE(P <0 .0 1) ,CD3+ CD4 + HLA -DR+ 细胞、CD3+ CD8+HLA -DR+ 细胞明显高于正常对照组 (P <0 .0 5 ) ,而非活动期SLE与正常对照组之间 ,HLA -DR表达无显著性差异 (P >0 .0 5     

系统性红斑狼疮患者外周血中T细胞CD4CD25的表达

目的了解CD4,CD25在系统性红斑狼疮患者外周血T细胞中的表达水平及其临床意义。方法用流式细胞术检测30例正常人和30例活动期系统性红斑狼疮(SLE)患者外周血中T细胞CD4,CD25的表达水平,根据CD25表达荧光强度>100者为T细胞CD4+CD25+bright,并与SLE活动指数评分(SLEDAI)进行相关分析。结果SLE患者外周血T细胞CD4+CD25+表达率为(8.82±2.98)%,显著低于对照组(12.24±5.71)%(P<0.05);患者组T细胞CD4+CD25bright表达率为(1.56±0.76)%,低于对照组(2.23±1.22)%(P<0.05),差异有统计学意义;SLE患者外周血T细胞CD4+CD25+、T细胞CD4+CD25bright表达率与SLEDAI评分两者之间无相关性;r分别为-0.156,-0.153,P均>0.05)。结论SLE患者外周血中T细胞CD4+CD25+减少,其可能在发病机制中起一定的作用,低水平T细胞CD4+CD25bright在SLE发病中的确切作用机制有待进一步阐明。     

T淋巴细胞亚群失衡与SLE中医辨证分型的关系

目的观察不同中医证型SLE患者外周血CD4~+T,CD8~+T,CD8~+CD28~+T,CD8~+CD28~-T,CD3~+CD4~+IFN-r~+T和CD3~+CD4~+IL-4~+T细胞百分率。方法 SLE患者中医辨证分为气血两燔型、肝热血瘀型、气阴两虚血瘀型和脾肾阳虚型4型,流式细胞技术检测外周静脉血中CD4~+T,CD8~+T,CD8~+CD28~+T,CD8~+CD28~-T,CD3~+CD4~+IFN-r~+T和CD3~+CD4~+IL-4~+T细胞百分率,并与正常组比较。结果不同证型SLE患者外周血CD4~+T表达均下降(P均<0.01),气阴两虚血瘀型CD8~+CD28~+T表达降低、CD8~+CD28~+T/CD8~+CD28~-T比值降低、CD8~+CD28~-T表达升高,脾肾阳虚型CD3~+CD4~+IFN-r~+T表达显著高于其他各组,其CD3~+CD4~+IL-4~+T表达也显著高于正常组及气血两燔型(P均<0.05)。结论 SLE患者存在T淋巴细胞数量及功能失衡,中医辨证分型与微观指标有一定的关系。     

The circulating lymphocyte profiles in patients with discoid lupus erythematosus and systemic lupus erythematosus suggest a pathogenetic relationship

BACKGROUND: Discoid lupus erythematosus (DLE) and systemic lupus erythematosus (SLE) are chronic inflammatory diseases of unknown aetiology; the relationship of DLE with SLE has been a subject of debate for many years. OBJECTIVES; To find evidence for systemic immune activation in DLE by analysis of the immunophenotypic profiles of circulating lymphocytes, and to compare these changes with those in patients with SLE. METHODS: The immunophenotypic profile of peripheral blood lymphocyte subsets from 23 DLE patients without clinical or laboratory evidence of systemic disease, 25 SLE patients and 38 healthy donors was characterized by two-colour immunofluorescence flow cytometry analysis. None of the patients was receiving corticosteroid or immunosuppressive treatment. RESULTS: Patients with DLE had increased numbers of circulating HLA-DR+ CD3+ T cells and HLA-DR+ CD4+ T cells, indicating systemic T-cell activation, and an expansion of CD5+ CD19+ B cells. Decreased numbers of T-cell subsets expressing the differentiation markers CD11b and CD16/56, and of CD16/56+ natural killer cells were also found. In SLE, the changes were similar but more pronounced. In addition, a profound CD4+ T-cell lymphopenia and an increase of HLA-DR+ CD8+ T cells were found only in SLE. CONCLUSIONS: Our data provide evidence for systemic activation of the cellular immune system in patients with purely cutaneous DLE. Similarities in the lymphocyte immunophenotypic profiles in patients with DLE compared with SLE suggest that there are common immunopathological processes in these two conditions.     

CD70 mRNA及其蛋白在SLE患者T淋巴细胞中的表达

目的通过研究SLE患者T淋巴细胞CD70mRNA及其蛋白的表达,探讨CD70在SLE发病机理中的作用。方法用定量RT-PCR方法测定15例活动期、15例非活动期SLE患者和15例正常人对照组外周血T淋巴细胞CD70mRNA转录水平。用流式细胞仪检测各组的CD70+CD4+T淋巴细胞阳性率。结果活动期、非活动期SLE患者及正常对照组T淋巴细胞CD70mRNA转录水平分别为(0.82±0.12),(0.73±0.11)和(0.45±0.09),活动期、非活动期SLE患者明显高于正常对照组,差异有统计学意义(P<0.01),且活动期显著高于非活动期SLE患者,差异有统计学意义(P<0.01)。活动期、非活动期SLE患者及正常人外周血CD4+CD70+T淋巴细胞阳性率分别为(80.30±11.04)%,(66.80±3.98)%和(12.48±3.45)%,活动期、非活动期SLE患者明显高于正常对照组,差异有统计学意义(P<0.01),且活动期显著高于非活动期SLE患者,差异有统计学意义(P<0.01)。结论 CD70的过度表达在SLE的发生发展中起着重要的作用,并且CD70过度表达可以作为SLE疾病活动的一个指标。