HBV变异患者外周血CD4^＋CD25^＋调节性T细胞变化及其临床意义

目的研究乙型肝炎病毒(HBV)变异患者外周血CD4 CD25 调节性T细胞(Treg细胞)及肝组织CD8 T细胞的关系,并探讨其临床意义。方法慢性乙型肝炎患者64例。HBV变异者27例,无变异者37例;急性乙型肝炎患者16例。各组间年龄、性别无明显差异。25例患者进行肝脏穿刺活检,免疫组化方法检测肝组织CD8 T细胞分布;流式细胞技术测定外周血Treg细胞变化;荧光定量PCR测定血清HBVDNA水平,PCR-RFLP测定前C区G1896A、P区YMDD变异。结果急性乙型肝炎患者、HBV变异伴肝病进展患者外周血Treg细胞比例明显低于HBV变异肝病无明显变化者(3.00±1.33,2.57±0.83vs4.32±0.96,P<0.01),后者与HBV无变异者没有明显差异(4.32±0.96vs4.77±2.11,P>0.05)。外周血Treg细胞与HBVDNA水平呈正相关(r=0.411,P<0.01);免疫组化显示HBV变异伴肝病进展患者肝组织CD8 T明显增多,急性乙型肝炎患者肝组织汇管区较多CD8 T浸润。结论Treg比例降低与HBV变异肝病进展可能有关,可作为HBV变异后临床转归的预测指标。初步显示,肝组织CD8 T浸润与外周血Treg细胞呈负相关,提示HBV变异后肝脏病进展的免疫损伤机制。     

The effect of Echinococcus granulosus on spleen cells and TGF‐β expression in the peripheral blood of BALB/c mice

Cystic echinococcosis (CE) caused by the cestode Echinococcus granulosus (E. granulosus) is a zoonotic parasitic disease. The effective immune evasion mechanisms of E. granulosus allow it to parasitize its hosts. However, the status of the innate and adaptive immune cells and their contributions to E. granulosus progression remain poorly understood. In this study, we aimed to determine the impact of E. granulosus infection on T cells, NK cell responses and TGF‐β expression during the early infection phase in BALB/c mice. In E. granulosus infections, there was an increasing tendency in the percentage of CD4⁺CD25⁺ T cells and CD4⁺Foxp3⁺ T cells and peripheral blood TGF‐β levels and relative expression of the Foxp3 gene. Moreover, there were a decreasing tendency in the percentage of NK cells and NK cell cytotoxicity and the expression of NKG2D on NK cells. The TGF‐β1/Smad pathway was activated by E. granulosus in mice. Above results can be reversed by the inhibitor SB‐525334 (potent activin receptor‐like kinase 5 inhibitor). These results suggest that the TGF‐β/Smad pathway plays an important role in changes of T‐cell or NK cell responses. These results may contribute to revealing the preliminary molecular mechanisms in establishing hydatid infection.     

不同病因肝衰竭患者外周血单个核细胞HLA-DR基因水平及血Th17和CD4+CD25+Treg细胞百分比的变化

目的探讨不同病因所致肝衰竭患者外周血单个核细胞（PBMCs）HLA-DR mRNA及Th17和CD4⁺CD25⁺Treg细胞水平的变化及其意义。方法本研究纳入肝衰竭患者50例,其中乙型肝炎肝衰竭15例,药物性肝损伤12例,酒精性肝病13例,自身免疫性肝炎10例;慢性乙型肝炎患者17例和正常人10例。采用PCR法检测PBMCs中HLA-DR mRNA水平,使用流式细胞仪检测CD4⁺CD25⁺Treg和Th17细胞百分比。结果乙型肝炎肝衰竭患者HLA-DR mRNA水平为（134.5±15.2）,显著高于药物性肝损伤组的（17.9±1.2）、酒精性肝病组的（19.6±2.0）和自身免疫性肝炎组的[（11.2±1.2）,P<0.05];不同病因肝衰竭患者Th17和CD4⁺CD25⁺Treg细胞百分比[分别为（4.4±0.6）%和（3.9±0.6）%左右]的差异无统计学意义（P>0.05）,但与慢性乙型肝炎[分别为（3.7±0.2）%和（6.1±0.4）%和正常人（2.1±0.7）%和（7.0±0.9）%比,均有显著性差异（P<0.05）;对不同病因肝衰竭患者进行动态观察发现,19例死亡患者CD4⁺CD25⁺Treg细胞百分比呈持续下降,直至死亡,而31例生存患者则逐渐恢复至接近正常水平。结论外周血单个核细胞HLA-DR mRNA水平及Th17和CD4⁺CD25⁺Treg细胞百分比的变化与肝衰竭患者的病情密切相关,可作为判断肝衰竭严重程度及预后的指标。     

外周血CD4+CD25+调节性T细胞在Graves病发病中的作用

测定不同阶段Graves病患者外周血中淋巴细胞亚群含量及CD4+ CD25+调节性T细胞功能.结果显示CD4+ CD25+调节性T细胞免疫功能减弱导致CD4+T细胞增殖失控可能是Graves病发病的重要原因,药物治愈患者其免疫功能难以完全恢复正常导致Grares病容易复发.     

慢性丙型肝炎患者外周血单个核细胞HCV RNA检测及其与机体免疫功能的相关性研究

目的观察慢性丙型肝炎患者外周血单个核细胞(PBMC)HCVRNA含量及其对T淋巴细胞亚群的影响,以探讨HCV感染者PBMC中HCVRNA水平及其与机体免疫功能的关系。方法采用荧光定量PCR(FQPCR)技术对128例丙型肝炎患者血清、外周血单个核细胞的HCVRNA含量进行了检测,同时检测CD3+、CD4+、CD8+、CD4+/CD8+。结果PBMC内HCVRNA阳性组与HCVRNA阴性组比较,前者CD3+、CD4+水平降低、CD8+水平增高,CD4+/CD8+比值下降大于后者,差异有显著性(P<0.05)。结论丙型肝炎病毒侵染PBMC后可加重患者的细胞免疫功能紊乱。     

系统性红斑狼疮患者外周血CD4+CD25highFoxp3+调节性T细胞数量和Foxp3mRNA的表达水平与疾病活动性的相关性

目的 研究系统性红斑狼疮(SEE)患者CD4+CD25highFoxp3+调节性T细胞的数量及其功能基因Foxp3 mRNA的表达水平与SLE疾病活动性和肾脏损伤的相关性.方法 采用四色流式细胞术以Foxp3-异硫氰酸荧光素(FITC )/CD25-藻红蛋白/CD4-多甲藻叶绿素蛋白(PerCP)/CD3-藻蓝蛋白7抗体组合检测40名健康对照者及42例SLE患者外周血CD4+CD25highFoxp3+调节性T细胞的数量,实时荧光定量聚合酶链反应(PCR)检测特异性转录因子Foxp3 mRNA的表达水平,并分析其与SLE患者疾病活动指数(SLEDAI)、补体C3及血清抗双链DNA(dsDNA)抗体的关系.统计学方法采用t检验和Spearman相关分析.结果 活动期SLE患者外周血CD4+CD25highFoxp3+调节性T细胞数量显著低于健康对照组[(4±3)％与(7±4)％,P＜0.05],稳定期与健康对照组差异无统计学意义(P＞0.05);活动期SLE患者外周血CD4+CD25highFoxp3+调节性T细胞数量及CD4+CD25highFoxp3+调节性T细胞/CD4+比值显著低于稳定期患者[(4±3)％,(9±6)％与(5±4)％,(10±6)％,P均＜0.05];活动期SLE患者外周血Foxp3 mRNA的表达水平明显低于稳定期和对照组(P＜0.01,P＜0.05);SLE患者并发肾病组外周血CD4+CD25highFoxp3+调节性T细胞数量及CD4+CD25highFoxp3+调节性T细胞/CD4+比值显著低于SLE非肾病组(P＜0.05).相关分析显示,SLE患者外周血CD4+CD25highFoxp3+调节性T细胞数量与SLEDAI呈负相关(r=-0.5782,P＜0.05);CD4+CD25highFoxp3+调节性T细胞/CD4+比值与SLEDAI呈负相关(r=-0.4913,P＜0.05),与补体C3呈正相关(r=0.3687,P＜0.05);SLE患者外周血CD4+CD25highFoxp3+调节性T细胞数量与Foxp3 mRNA的表达水平呈正相关(r=0.6142,P＜0.0l).结论 SLE患者外周血CD4+CD25highFoxp3+调节性T细胞和Foxp3 mRNA的变化可能是导致SLE疾病发生和发展的关键因素之一,与疾病的活动性有密切关系.     

Phenotypical analysis and cytokine release of liver-infiltrating and peripheral blood T lymphocytes from patients with chronic hepatitis of different etiology

Cytokines released by infiltrating T cells may contribute to the hepatic injury in chronic hepatitis. Therefore, we characterized peripheral blood- and liver-infiltrating T cells from patients with chronic hepatitis of different etiology and determined the T cell phenotypes and the cytokine release. Liver tissue and peripheral blood-derived T cells from patients with autoimmune hepatitis and primary biliary cirrhosis predominantly expressed CD4-molecules and the α- and β-chains of the T cell receptor (TCR). In chronic viral hepatitis B and C, liver- and blood-derived T cells were preferentially CD8+ T cells expressing the αβ TCR. Mitogenic stimulation with irradiated Daudi lymphoma cells and phytohemagglutinin led to a strong release of interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α) and interleukin-2 (IL-2) by T cells in patients with chronic hepatitis and in healthy controls. T cells from patients with primary biliary cirrhosis and some patients with autoimmune hepatitis showed a significantly higher secretion of interleukin-4 (IL-4) and interleukin-10 (IL-10) than T cells from patients with chronic viral hepatitis or healthy controls. Histologic inflammatory activity did not correlate with the amount of cytokines released after mitogenic activation. In conclusion, liver tissue and peripheral blood T cells of patients with autoimmune hepatitis and primary biliary cirrhosis were dominated by CD4+ TCR αβ+ T helper/inducer cells, whereas in chronic viral hepatitis an enrichment of CD8+ TCR αβ+ cytotoxic/suppressor T cells was observed. In addition, analysis of the cytokine release showed that T cells in autoimmune and chronic viral liver diseases secreted high amounts of IFN-γ and TNF-α, cytokines predominantly secreted by T_hl-like cells. The secretion of the T_h2 cytokines. IL-4 and IL-10, however, was increased in autoimmune hepatitis and primary biliary cirrhosis. These data show that in autoimmune and chronic viral liver diseases different functional T cell subsets are activated.     

先天性心脏病患者免疫因子Th17/Treg失衡与肺动脉高压的关系

目的:探讨患者外周血中Th17细胞、CD4+D25+FoxP3+调节性T细胞(Treg)的变化及意义。方法:75例先天性心脏病(CHD)合并肺动脉高压(PAH)患者,分为轻度肺动脉高压组(28例)、中度肺动脉高压组(32例)及重度肺动脉高压组(15例),20例健康体检者作为对照组。采用流式细胞分析法检测外周血中Th17细胞、Treg细胞占CD4+T细胞的比例,分析Th17细胞与Treg细胞的比例与肺动脉压的相关性。结果:Th17/CD4+T细胞比例、Treg/CD4+T细胞的比例和Th17/Treg细胞比值,两组间差异具有统计学意义(P<0.05),随着肺mPAP的增加,Th17/CD4+T细胞比例明显升高;Treg/CD4+T细胞的比例明显下降;Th17/Treg细胞比值显著升高。mPAP与Th17/Treg比值呈显著正相关(r=0.95,P<0.05)。结论:先天性心脏病合并肺动脉高压患者外周血中存在Th17/Treg失衡,且与肺动脉高压程度呈正相关,Th17/Treg失衡可能参与了肺动脉高压的发生发展。     

Heligmosomoides bakeri antigen rescues CD4-positive T cells from glucocorticoid-induced apoptosis by Bcl-2 protein expression

Heligmosomoides bakeri infection in mice is associated with a dominant CD4⁺ T‐cell response and with the activity of natural Treg cells with CD4⁺CD25⁺ phenotype. The polarization of Th2 T‐cell phenotype and the increase in the CD4⁺CD25⁺ T cell population are regulated by glucocorticoids that induce apoptosis in CD4⁺CD25^? T cells and inhibit apoptosis in CD4⁺CD25⁺ T cells. However, exposure of mice to H. bakeri antigen induces a high glucocorticoid concentration in serum and a reduction in the number of CD4‐positive; CD4⁺CD25^? and CD4⁺CD25⁺ apoptotic T cells in mesenteric lymph node cells. In this study to evaluate the in vitro effect of the anti‐apoptotic property of H. bakeri antigen on T cells, apoptosis of these cells was induced by glucocorticoids‐dexamethasone (Dex). Excretory–secretory (ES) antigen of the nematode prevented Dex‐induced apoptosis in CD4‐positive T cells with CD4⁺CD25^? and CD4⁺CD25^High phenotype by Bcl‐2 protein expression. Contrary to the effect on CD4‐positive T cells, survival of CD8⁺ T cells was not connected with expression of Bcl‐2 protein. This suggest that H. bakeri antigen modulates CD4‐positive T cell sensitivity to glucocorticoid‐induced apoptosis by induction of Bcl‐2 protein.     

Foxp3及其调控的CD4~+CD25~+Treg细胞在肺腺癌患者中表达的规律及对患者生存的影响

目的探讨Foxp3及其调控的CD4~+CD25~+Treg细胞在肺腺癌患者中表达的规律及对患者生存的影响。方法 Western-blot法检测正常肺组织、肺腺癌、鳞癌组织(按Ⅰ、Ⅱ、Ⅲa分期分层)中Foxp3水平。免疫组化方法观察小细胞肺癌、腺癌、鳞癌中Foxp3蛋白表达情况;流式细胞法检测临床各期肺腺癌患者和健康人群血清中表达Foxp3的CD4~+CD25~+Treg细胞计数,并观察化疗及手术对肺腺癌患者血清中Foxp3水平的影响。使用Kaplan-Meier生存曲线分析血清中CD4~+CD25~+Treg细胞计数与肺腺癌患者生存周期之间的关系。结果肺腺癌各期患者Foxp3表达水平和血清中CD4~+CD25~+Treg细胞计数均明显高于健康人群,与肿瘤分期呈正相关,经手术或化疗干预后患者外周血清中细胞计数下降明显。细胞计数低于平均值或化疗后细胞计数变化值高于平均值的患者平均生存周期明显高于细胞计数值高于平均值或化疗后细胞计数变化值小于平均值的患者。结论 Foxp3调控的CD4~+CD25~+Treg细胞在肺腺癌的发生发展中可能起着重要的作用,对其深入研究将有助于肺腺癌的早期诊断,并可将其作为常规检查及监测患者预后的指标之一。     

不同方法检测肝癌患者外周血中CD4+CD25hi-int Tregs水平的关系及意义

目的:探讨以标记CD127low和Foxp3+两种方法检测肝癌(hepatocellular carcinoma,HCC)患者外周血中CD4+CD25hi-int Tregs水平的相关性,建立两者转换的回归方程.方法:流式细胞术同时用CD127low和Foxp3+两种标志物检测31例HCC患者外周血中CD4+CD25hi-int Tregs,检测32例对照组CD4+ CD25hi-int Foxp3+ Tregs水平.结果:HCC患者与健康对照组外周血中CD4+CD25hi-int Foxp3+ Tregs占CD4+T细胞的比例分别为10.33%±4.47%和7.34%±1.76%,差异有统计学意义(P<0.01).HCC患者外周血中CD4+CD25hi-int CD127low Tregs的比例为10.51%±4.78%.与Foxp3+Tregs比例呈正相关,Pearson相关系数为0.889,相关有统计学意义(P<0.01).两者曲线拟合提示幂模型是最优模型,建立回归方程Y=1.236×(x0.899)(Y为Foxp3+Tregs,x为CD127lowTregs).结论:HCC患者外周血中Tregs水平明显升高,用CD127low和Foxp3+两种方法检测HCC患者外周血Tregs水平呈正相关,两者之间数值转换可利用回归方程.     

自身免疫性甲状腺疾病和CD4~+CD25~+调节性T细胞

自身免疫性甲状腺疾病(AITD)的发生及发展与CD4~+CD25~+调节性T细胞(Treg细胞)的数量和功能密切相关.动物实验证明Treg细胞可抑制AITD的发生.如果清除动物体内的该类细胞,可导致AITD发病或使原有的甲状腺疾病加重,Treg细胞通过抑制效应性T细胞的激活而发挥对AITD的影响作用.无论是胸腺还是外周,不同诱导体系来源的Treg细胞均对AITD有影响作用.     

Control of type 1 diabetes by CD4+Foxp3+ regulatory T cells: lessons from mouse models and implications for human disease

In recent years, there has been a revival of the concept of CD4⁺ regulatory T (T_reg) cells as being a central control point in various immune responses, including autoimmune responses and immunity to transplants, allergens, tumours and infectious microbes. The current literature suggests that T_reg cells are diverse in their phenotype and mechanism(s) of action, and as such, may constitute a myriad of naturally occurring and induced T cell precursors with variable degrees of regulatory potential. In this review, we summarize research from various laboratories, including our own, showing that CD4⁺Foxp3⁺ T_reg cells are critical in the control of type 1 diabetes (T1D) in mouse models and humans. In this review, we also discuss cellular and molecular determinants that impact CD4⁺Foxp3⁺ T_reg cell development and function and consequential resistance to organ‐specific autoimmune disease. Recent advances in the use of CD4⁺Foxp3⁺ T_reg cellular therapy to promote immunological tolerance in the absence of long‐term generalized immunosuppression are also presented. Copyright © 2009 John Wiley & Sons, Ltd.     

The New insights from DPP‐4 inhibitors: their potential immune modulatory function in autoimmune diabetes

Dipeptidyl peptidase‐4 (DPP‐4) inhibitors are a new class of anti‐diabetic agents that are widely used in clinical practice to improve glycemic control and protect β‐cell function in patients with type 2 diabetes. DPP‐4 is also known as lymphocyte cell surface protein CD26 and plays an important role in T‐cell immunity. Autoimmune diabetes, a T‐cell mediated organ‐specific disease, is initiated by the imbalance between pathogenic and regulatory T‐lymphocytes. DPP‐4 inhibitors can suppress pathogenic effects of Th1 and Th17 cells and up‐regulate Th2 cells and regulatory T cells, which play a critical role in ameliorating autoimmune diabetes. This provides a basis for the potential use of DPP‐4 inhibitors in the treatment of autoimmune diabetes. Recent studies suggest that DPP‐4 inhibitors improve β‐cell function and attenuate autoimmunity in type 1 diabetic mouse models. However, there are few clinical studies on the treatment of autoimmune diabetes with DPP‐4 inhibitors. Further studies are warranted to confirm the therapeutic effects of DPP‐4 inhibitors on autoimmune diabetes in humans. Copyright © 2014 John Wiley & Sons, Ltd.     

余甘子提取物对非酒精性脂肪性肝病大鼠外周血Treg细胞及肝组织LXRα/FAS通路表达的影响*

目的 探讨余甘子提取物对非酒精性脂肪性肝病(NAFLD)大鼠外周血Treg细胞及肝组织LXRα/FAS通路表达的影响。方法 将48只SD大鼠随机分对照组、模型组、多烯磷脂酰胆碱及小剂量、中剂量和大剂量余甘子处理组,在造模成功后,给予对照组生理盐水灌胃,给予模型组多烯磷脂酰胆碱处理,另给予不同剂量的余甘子灌胃处理,连续给药6周。采用ELISA法检测血清TNF-α、IL-17和IL-10水平,使用流式细胞术检测血CD4⁺IL-17⁺和CD4⁺CD25⁺Treg细胞百分比,采用WB法检测肝组织LXRα和FAS蛋白表达。结果 与模型组比,大剂量余甘子提取物处理组肝组织病理学损伤显著改善,血清ALT、AST、TC、TG水平显著降低(P<0.05);血清TNF-α和IL-17水平显著降低,分别为(34.7±0.9)pg/ml和(3.2±0.2)pg/mL,而IL-10水平显著升高,为(4.8±0.2)pg/mL(P<0.05);大剂量余甘子处理组CD4⁺IL-17⁺百分比为(1.1±0.6)%,显著低于模型组[(1.9±0.4)%,P<0.05],CD4⁺CD25⁺Treg百分比为(1.5±0.6)%,显著高于模型组[(0.9±0.2)%,P<0.05];小、中、大剂量余甘子提取物处理组肝组织LXRα和FAS蛋白表达显著减弱[分别为(1.8±0.1)和(2.0±0.2),P<0.05]。结论 余甘子提取物可改善NAFLD大鼠肝组织病理学损伤,可能与其对炎性细胞因子、Th17/Treg细胞和LXRα/FAS通路因子的表达产生了某种影响有关。