支气管肺发育不良小鼠肺组织FOXP3^(+)调节性T细胞(Treg)数量减少且向RORγt^(+)FOXP3^(+)Treg转换

目的 探讨调节性T淋巴细胞(Treg)表型转换在支气管肺发育不良(BPD)小鼠肺组织中的作用。方法 C57BL/6新生小鼠随机分成空气组及高氧组,每组10只,高氧组建立新生小鼠高氧暴露的BPD动物模型。在小鼠生后7 d和14 d,每组各处死5只小鼠,取出新鲜肺组织。HE染色观察肺组织病理变化,Western blot法检测肺表面活性蛋白C(SP-C)表达水平;流式细胞术检测肺组织中叉头盒P3(FOXP3)^(+)Treg及维甲酸相关孤核受体γt(RORγt)^(+)FOXP3^(+)Treg占CD4^(+)淋巴细胞百分比,ELISA检测肺组织中白细胞介素17A(IL-17A)、 IL-6的含量。采用Spearman相关分析法分析FOXP3^(+)Treg与SP-C相对表达量的相关性以及RORγt^(+)FOXP3^(+)Treg与IL-17A、 IL-6含量的相关性。结果 与同时间点空气组相比,高氧组肺泡结构简单化,放射状肺泡计数与SP-C相对表达水平均明显下降,高氧组FOXP3^(+)Treg占比减少,RORγt^(+)FOXP3^(+)Treg占比增多,肺组织IL-17A、 IL-6含量明显升高。FOXP3^(+)Treg与SP-C相对表达水平呈正相关,RORγt^(+)FOXP3^(+)Treg与IL-17A、 IL-6含量呈正相关。结论 BPD小鼠肺组织FOXP3^(+)Treg数量减少并向RORγt^(+)FOXP3^(+)Treg转换,可能参与高氧所致肺损伤。

MiR-19a-3p regulates the Forkhead box F2-mediated Wnt/β-catenin signaling pathway and affects the biological functions of colorectal cancer cells

BACKGROUND Colorectal cancer(CRC)is one of the most common malignancies worldwide.AIM To explore the expression of microRNA miR-19a-3p and Forkhead box F2(FOXF2)in patients with CRC and the relevant mechanisms.METHODS Sixty-two CRC patients admitted to the hospital were enrolled into the study group,and sixty healthy people from the same period were assigned to the control group.Elbow venous blood was sampled from the patients and healthy individuals,and blood serum was saved for later analysis.MiR-19a-3p mimics,miR-19a-3p inhibitor,miR-negative control,small interfering-FOXF2,and short hairpin-FOXF2 were transfected into HT29 and HCT116 cells.Then quantitative polymerase chain reaction was performed to quantify the expression of miR-19a-3p and FOXF2 in HT29 and HCT116 cells,and western blot(WB)analysis was conducted to evaluate the levels of FOXF2,glycogen synthase kinase 3 beta(GSK-3β),phosphorylated GSK-3β(p-GSK-3β),β-catenin,p-β-catenin,α-catenin,Ncadherin,E-cadherin,and vimentin.The MTT,Transwell,and wound healing assays were applied to analyze cell proliferation,invasion,and migration,respectively,and the dual luciferase reporter assay was used to determine the correlation of miR-19a-3p with FOXF2.RESULTS The patients showed high serum levels of miR-19a-3p and low levels of FOXF2,and the area under the curves of miR-19a-3p and FOXF2 were larger than 0.8.MiR-19a-3p and FOXF2 were related to sex,tumor size,age,tumor-nodemetastasis staging,lymph node metastasis,and differentiation of CRC patients.Silencing of miR-19a-3p and overexpression of FOXF2 suppressed the epithelialmesenchymal transition,invasion,migration,and proliferation of cells.WB analysis revealed that silencing of miR-19a-3p and FOXF2 overexpression significantly suppressed the expression of p-GSK-3β,β-catenin,N-cadherin,and vimentin;and increased the levels of GSK-3β,p-β-catenin,α-catenin,and Ecadherin.The dual luciferase reporter assay confirmed that there was a targeted correlation of miR-19a-3p with FOXF2.In addition,a rescue experiment revealed that there were no differences in cell proliferation,invasion,and migration in HT29 and HCT116 cells co-transfected with miR-19a-3p-mimics+sh-FOXF2 and miR-19a-3p-inhibitor+si-FOXF2 compared to the miR-negative control group.CONCLUSION Inhibiting miR-19a-3p expression can upregulate the FOXF2-mediated Wnt/β-catenin signaling pathway,thereby affecting the epithelial-mesenchymal transition,proliferation,invasion,and migration of cells.Thus,miR-19a-3p is likely to be a therapeutic target in CRC.

胃癌组织中Foxp3^(+)Tregs和pDCs与胃微生物群失调的关系

目的探讨胃癌组织中Foxp3^(+)Tregs和浆细胞样树突状细胞(p DCs)与胃微生物群失调的关系。方法选择我院2018年2月-2020年2月诊治的胃癌患者97例(收集其胃癌组织标本和癌旁标本)和同期健康者40例,胃癌患者根据类型分为肠型、弥漫性和混合型,分别为31例、28例、38例。采用免疫组化染色、Westernblot法测定胃癌组织中Foxp3^(+)Tregs、p DCs表达,16S r RNA法检测胃微生物群,并分析各指标表达差异及相关性。结果肠型、弥漫性和混合型癌巢组织中Foxp3^(+)Tregs、p DCs表达量逐渐升高(P<0.05)。胃癌患者鞘氨醇单胞菌、幽门螺杆菌、单形拟杆菌水平低于健康者,而痤疮丙酸杆菌、咽峡炎链球菌、产黑普氏菌高于健康者;不同类型胃癌患者氨醇单胞菌、幽门螺杆菌、单形拟杆菌、痤疮丙酸杆菌、咽峡炎链球菌、产黑普菌水平比较差异有统计学意义(P<0.05)。痤疮丙酸杆菌、咽峡炎链球菌、产黑普菌与Foxp3^(+)Tregs、p DCs数量呈正比关系(P<0.05)。结论胃癌癌旁和癌巢组织微生境中细菌丰度降低,不同类型胃癌肿瘤微生境中有不同的优势微生物;组织中Foxp3^(+)Tregs、p DCs与胃微生物群失调相关。

CEACAM6和FOXP3对胰腺癌肿瘤浸润淋巴细胞及预后的影响

目的探讨胰腺癌组织中癌胚抗原相关黏附分子6(CEACAM6)和叉头盒P3(FOXP3)的表达对患者免疫功能及预后的影响。方法采用免疫组织化学法检测40例胰腺癌组织中CEACAM6、FOXP3和CD3、CD8、CD45RO的表达,并分析其与临床病理特征及预后的关系。结果胰腺癌CEACAM6的强阳性率为50%,FOXP3的强阳性率为60%。Ⅲ、Ⅳ期胰腺癌组织中CEACAM6和FOXP3的强阳性率较高,CEACAM6的高表达与CD8和CD45RO细胞的阳性率呈负相关。FOXP3在病理为低分化胰腺癌中强阳性率较高。FOXP3的表达与CD3、CD8、CD45RO阳性T细胞的浸润呈负相关。高表达CEACAM6和FOXP3患者的生存期较短。结论 CEACAM6和FOXP3与胰腺癌的恶性程度有关,其表达对胰腺癌中的肿瘤浸润T细胞具有抑制作用。

FOXP3基因多态性与儿童ITP发病及进展的相关性研究

目的通过对儿童免疫性血小板减少症(immune thrombocytopenia,ITP)患儿Forkhead Box P3(FOXP3)基因单核苷酸多态性检测、了解儿童ITP发病并随访疾病进展情况,探讨FOXP3基因多态性与儿童ITP发生、发展的关系,以期为开展儿童ITP的靶向治疗提供一定理论依据。方法 328例ITP患儿作为实验组,219例健康志愿者作为对照组。实验组根据病程及转归又分为两组(病程在12个月以内组、病程在12个月以上组)。利用Sequenom SNP位点分型检测方法分别检测FOXP3基因rs3761547、rs3761548、rs2232365 3个位点单核苷酸多态性。结果 1)rs2232365位点在性别年龄均匹配实验组AG基因型频率较正常对照组明显增高(P=0.013),其他2个位点单核苷酸多态性在两组之间差异无统计学意义。2)rs3761547位点在12个月以上组GG基因型及G等位基因较12月以内组明显增高(P=0.008,0.001);rs2232365位点A与G基因频率在不同病程间差异有统计学意义(P=0.033)。结论 FOXP3 rs2232365位点携带AG基因型可能为ITP发病的易感因素。携带FOXP3 rs3761547 GG基因型及G等位基因、rs2232365位点G等位基因的ITP患儿更易发展为慢性,病程迁延。rs2232365位点单核苷酸多态性与儿童ITP的发生发展均相关,可能是疾病机制的关键。

Forkhead box P3 and indoleamine 2,3-dioxygenase co-expression in Pakistani triple negative breast cancer patients

BACKGROUND Forkhead box P3(FOXP3)is a specific marker for immunosuppressive regulatory T(T-reg)cells.T-regs and an immunosuppressive enzyme,indoleamine 2,3-dioxygenase(IDO),are associated with advanced disease in cancer.AIM To evaluate the co-expression of FOXP3 and IDO in triple negative breast cancer(TNBC)with respect to hormone-positive breast cancer patients from Pakistan.METHODS Immunohistochemistry was performed to analyze the expression of FOXP3,IDO,estrogen receptor,progesterone receptor,and human epidermal growth factor receptor on tissues of breast cancer patients(n=100):Hormone-positive breast cancer(n=51)and TNBC(n=49).A total of 100 patients were characterized as FOXP3 negative vs positive and further categorized based on low,medium,and high IDO expression score.Univariate and multivariate logistic regression models were used.RESULTS Out of 100 breast tumors,25%expressed FOXP3 positive T-regs.A significant coexpression of FOXP3 and IDO was observed among patients with TNBC(P=0.01)compared to those with hormone-positive breast cancer.Two variables were identified as significant independent risk factors for FOXP3 positive:IDO expression high(adjusted odds ratio(AOR)5.90;95%confidence interval(CI):1.22-28.64;P=0.03)and TNBC(AOR 2.80;95%CI:0.96-7.95;P=0.05).CONCLUSION Our data showed that FOXP3 positive cells might be associated with high expression of IDO in TNBC patients.FOXP3 and IDO co-expression may also suggest its involvement in disease,and evaluation of FOXP3 and IDO expression in TNBC patients may offer a new therapeutic option.

结直肠癌组织STAT3、Foxp3、IL-6表达与临床病理特征的相关性

目的 检测结直肠癌组织中信号转导及转录因子3(STAT3)、叉头盒p3(Foxp3)及白细胞介素6(IL-6)蛋白的表达,分析其与结直肠癌临床病理特征间的关联。方法 选择44例结直肠癌患者的肿瘤组织作为实验组,记录患者的性别、年龄、分化程度、肿瘤类型及淋巴结转移等病理临床参数,同时选择20例患者结直肠癌旁组织作为对照组;应用免疫组织化学法分析实验组和对照组中STAT3、Foxp3及IL-6的表达,分析3种蛋白的表达与临床病理特征的相关性及3种蛋白之前的相互关系。结果 实验组STAT3、Foxp3、IL-6蛋白的表达明显高于对照组,STAT3蛋白的表达与肿瘤分化程度有关,差异有统计学意义(P<0.05);Foxp3蛋白的表达与淋巴结转移有关,差异有统计学意义(P<0.05);IL-6蛋白的表达与肿瘤分化程度有关,差异有统计学意义(P<0.05);结直肠癌肿瘤组织中STAT3、Foxp3和IL-6蛋白的表达无相关性,差异不具有统计学意义(P>0.05)。结论结直肠癌组织中STAT3、Foxp3和IL-6蛋白过量表达提示患者恶性程度高,转移可能性大,可以作为判断结直肠癌预后效果的重要参考指标之一。

FOXP3 and Its Cofactors as Targets of Immunotherapies

Forkhead box P3 (FOXP3) is a “master regulator” of regulatory T cells (Tregs), which are a subset of T cells that can suppress the antigen-specific immune reaction and that play important roles in host tolerance and immune homeostasis. It is well known that FOXP3 forms complexes with several proteins and can be regulated by various post-translational modifications (PTMs) such as acetylation, phosphorylation, ubiquitination, and methylation. As a consequence, the PTMs change the stability of FOXP3 and its capability to regulate gene expression, and eventually affect Treg activity. Although FOXP3 per se is not an ideal drug target, deacetylases, acetyltransferases, kinases, and other enzymes that regulate the PTMs of FOXP3 are potential targets to modulate FOXP3 and Treg activity. However, FOXP3 is not the only substrate for most of these enzymes;thus, unwanted ‘‘on target/off FOXP3” side effects must be considered when inhibitors to these enzymes are used. In this review, we summarize recent progress in the study of FOXP3 cofactors and PTM proteins, and potential clinical applications in autoimmunity and cancer immunity.

继发性肺结核患者治疗初期FOXP3 TSDR DNA去甲基化变化情况分析

目的应用一种 FOXP3 TSDR DNA去甲基化荧光定量PCR技术，并分析该方法在继发性肺结核患者治疗初期变化情况。方法选取2014年6月～2015年5月来深圳市龙华新区慢性病防治中心就诊的继发性肺结核病人47例作为研究组，健康对照40例，分离外周血单个核细胞，筛选 CD4＋CD25＋T细胞，提取基因组 DNA。利用去甲基化 FOXP3 TSDR特异性引物扩增对照组基因组DNA，通过酶切、克隆和回收纯化等过程构建质粒标准品，优化 FOXP3 TSDR去甲基化实时定量PCR技术，建立CD4＋CD25＋T细胞比例与 FOXP3 TSDR PCR 拷贝换算关系，用该方法分析对照组和研究组0周及治疗后2，4和8周时的调节T细胞频率（FOXP3 TSDR去甲基化），应用 spss16.0软件统计分析实验所得数据。结果0周时47例研究组抗酸染色均为阳性，对照组均为阴性。以抗酸染色结果分组：对照组、抗酸（1＋）组、抗酸（2＋）组、抗酸（3＋）组和抗酸（4＋）组其调节 T细胞频率分别为1.63％＆#177;0.70％，1.96％＆#177;0.10％，1.32％＆#177;0.32％，0.86％＆#177;0.21％和0.53％＆#177;0.12％，抗酸（2＋）组与对照组差异无统计学意义，抗酸（3＋）组与对照组差异有统计学意义。研究组0，2，4和8周时调节T细胞频率均值、范围及95％可信区间分别为1.05％（0.32％～2.03％），CI（0.93％，1.18％）；2.04％（0.95％～3.95％），CI（1.85％，2.24％）；3.44％（2.35％～4.95％），CI（3.27％，3.61％）；2.79％，（1.02％～4.27％），CI （2.60％，2.98％）。对照组与研究组0周时人群调节T细胞频率之间差异有统计学意义（t＝4.669，P＜0.05）。单变量方差分析显示治疗时间对研究组外周血调节T细胞频率（FOXP3 TSDR 去甲基化）水平变化有影响（F＝347.2，P＜0.001， df＝3，组内F＝407.4，P＜0.001，df＝3），治疗时间和分组交互效应呈线性关系（F＝678.2，P＜0.001，df＝1）。结论该方法敏感、特异，可以用于继发性肺结核患者的疗效监测。

Triptolide attenuate cardiac hypertrophy and elevate Foxp3 expression in mice

Aim To explore the role of transcription factor Foxp3 and the regulating effect of triptolide （TP） in the progression of myocardial hypertrophy in mice. Methods Fifty male mice were randomly divided into 5 groups, i. e., normal control group, myocardial hypertrophy model group and TP （10, 30, 90μg · kg^-1） treated groups. Myocardial hypertrophy was induced by isoprenaline （ISO） 5 mg kg^-1 once daily for 14 days. Triptolide was giv- en intraperitoneally once daily. Left ventricle tissue was subjected to HE staining and chemiluminescence technique to assess effects on hypertrophy, fibrosis and inflammation, quantitative assessment of hypertrophy regulatory genes were performed by qPCR and WB. Results After 14 days of treatment, myocardial expressions of Foxp3 and CD4 were significantly reduced in the model group compared with controls. The expression level of TGFβ1 in control group was lower, while that in model group increased obviously. TP could significantly lessen myocardial tissue damage, and reduce the heart index and left ventricular index. Compared with model group, TP （30, 90 μg · kg^-1 ） significantly increased myocardial expression ratio of α-MHC to β-MHC, reduced serumal levels of BNP and troponin I, elevated mRNA and protein expressions of Foxp3 and CD4 in myocardial tissue and reduced the protein expression of TGFβ1 by comparison of those in model group. Conclusion TP can effectively ameliorate myocardial damage and inhibit left ventricular remodeling through elevating the expression of CD4 and Foxp3 and decreasing that of TGF-β.

甲炎康泰对自身免疫性甲状腺炎大鼠甲状腺组织RORγt/FOXP3及TGF-β、Smad3的影响

目的:探讨甲炎康泰对自身免疫性甲状腺炎(AIT)大鼠保护作用的机制。方法:制作AIT大鼠模型,随机分为模型组、甲炎康泰组,每组10只。另选取10只为正常组。灌胃干预8周后,ELISA检测大鼠血清中甲状腺过氧化物酶抗体(TPOAb)浓度,甲状腺组织进行HE染色、实时荧光定量PCR检测和免疫组化检测。结果:与模型组比较,甲炎康泰组TPOAb浓度显著降低(P<0.05),淋巴细胞浸润减轻;甲炎康泰组大鼠甲状腺组织中维甲酸相关核孤儿受体γt(RORγt)mRNA及蛋白表达显著降低(P<0.01,P<0.05),叉头翼状螺旋转录因子P3(FOXP3)、转化生长因子-β(TGF-β)、Smad3 mRNA及蛋白表达均显著升高(P<0.01,P<0.05)。结论:甲炎康泰可能通过增强甲状腺组织中TGF-β、FOXP3以及Smad3的表达和抑制RORγt的表达以缓解AIT的发生发展。

CD4^(+)CD25^(+)but not CD4^(+)Foxp3^(+) T cells as a regulatory subset in primary biliary cirrhosis

Increasing evidence indicates a role for regulatory T cells(Tregs)in the immune response and in autoimmune diseases,but the role of Tregs and cytokines in autoimmune hepatic diseases remains largely unclear and controversial,especially in patients with primary biliary cirrhosis(PBC).This study was undertaken to investigate Tregs and different cytokines in the liver and peripheral blood of PBC patients.We found that these patients demonstrated a reduction of CD4^(+)CD25^(+) T cells but elevated CD4^(+)Foxp3^(+) T cells in peripheral blood mononuclear cells(PBMCs)and CD41 T cells.The percentage of CD4^(+)CD25^(+) T cells in PBMCs was negatively correlated with elevated plasma interferon(IFN)-c levels.A liver-specific analysis showed that the frequency of Foxp31 Tregs,transforming growth factor(TGF)-b1 and IFN-c were increased in PBC patients.Our findings suggest that an imbalance between CD4^(+)CD25^(+) Tregs and cytotoxic cytokines plays a crucial role in the pathogenesis of PBC while the role of Foxp3 needs further investigation.

Expansion of GARP-Expressing CD4^(+)CD25^(-)FoxP3^(+)T Cells and SATB1Association with Activation and Coagulation in Immune Compromised HIV-1-Infected Individuals in South Africa

Although antiretroviral treatment lowers the burden of human immunodeficiency virus(HIV)-related disease,it does not always result in immunological recovery.This manifests as persistent chronic inflammation,immune activation or exhaustion that can promote the onset of co-morbidities.As the exact function of regulatory T(Treg)cells in HIV remains unclear,this cross-sectional study investigated three expression markers(Forkhead box protein P3[FOXP3],glycoprotein A repetitions predominant[GARP],special AT-rich sequence binding protein 1[SATB1])and compared their expansion between CD4^(+)CD25^(-)and CD4^(+)CD25^(++)T cells.Age-matched study subjects were recruited(Western Cape,South Africa)and sub-divided:HIV-negative subjects(n=12),HIV-positive na(i|")ve treated(n=22),HIV-positive treated based on CD4 count cells/μL(CD4>500 and CD4<500)(n=34)and HIV-treated based on viral load(VL)copies/mL(VL<1000 and VL>1000)(n=34).Markers of immune activation(CD38)and coagulation(CD142)on T cells(CD8)were assessed by flow cytometry together with FOXP3,GARP and SATB1 expression on CD4^(+)CD25^(-)and CD4^(+)CD25^(++)T cells.Plasma levels of interleukin-10(IL-10;anti-inflammatory marker),IL-6(inflammatory marker)and D-dimer(coagulation marker)were assessed.This study revealed three major findings in immuno-compromised patients with virological failure(CD4<500;VL>1000):(1)the expansion of the unconventional Treg cell subset(CD4^(+)CD25^(-)FOXP3^(+))is linked with disease progression markers;(2)increased GARP expression in the CD4^(+)CD25^(-)and CD4^(+)CD25^(++)subsets;and(3)the identification of a strong link between CD4^(+)CD25^(-)SATB1+cells and markers of immune activation(CD8^(+)CD38^(+))and coagulation(CD8^(+)CD142^(+)and D-dimer).

清温并用法对慢加急性肝衰竭肠源性内毒素血症大鼠结肠组织FoxP3,ROR-γt表达的影响

目的:研究清温并用法(即温阳解毒化瘀方)对慢加急性肝衰竭(ACLF)大鼠结肠组织叉头样转录因子3(FoxP3),维甲酸相关核孤儿受体-γt(ROR-γt)表达的影响,探索其对肝衰竭肠源性内毒素血症(IETM)的可能调节机制。方法:130只SD大鼠随机分为正常组10只,造模组120只。造模组以皮下注射牛血清白蛋白致敏法,腹腔注射D-氨基半乳糖(D-Gal)+脂多糖(LPS)建立ACLF大鼠模型。将成模的大鼠随机分为模型组,清法组(茵陈蒿汤6.68 g·kg^-1),温法组(茵陈术附汤7.09 g·kg^-1),清温并用法组(温阳解毒化瘀方19.53 g·kg^-1)。正常组及模型组予蒸馏水灌胃,其余各组予等容积相应中药灌胃,共1周。检测各项指标在1,12,24 h时间点的数值,流式细胞仪检测外周血调节性T淋巴细胞(Treg)/辅助性T淋巴细胞(Th)17细胞比值变化,实时荧光定量聚合酶链式反应(Real-time PCR)检测结肠组织FoxP3,ROR-γt mRNA表达,原位杂交法、免疫组化法观察大鼠结肠组织FoxP3,ROR-γt mRNA及蛋白表达。结果:与正常组比较,模型组大鼠Treg/Th17细胞在上述时间点显著下降(P<0.01);与模型组比较,清法、温法组Treg/Th17细胞差异无统计学意义,清温并用法Treg/Th17细胞升高(P<0.05);与正常组比较,模型组大鼠FoxP3,ROR-γt mRNA在上述时间点表达均显著升高(P<0.01);与模型组比较,清法、温法组FoxP3,ROR-γt mRNA在上述时间点均有所降低(P<0.05),清温并用法组显著降低(P<0.01);清温并用法组FoxP3,ROR-γt mRNA较清法组、温法组有所下降(P<0.05)。结论:清温并用法治疗肝衰竭IETM的机制与可能与调节FoxP3,ROR-γt表达有关。

宫颈鳞癌患者外周血和肿瘤组织中PD-1/PD-L1及相关免疫细胞的变化特点及其临床意义

目的:探讨PD-1/PD-L1通路及相关免疫细胞在宫颈鳞癌(cervical squamous cell cancer,CSCC)发生、发展中的变化特点及其临床意义。方法:收集2018年12月至2020年9月在福州市第一医院接受手术的CSCC患者和健康体检人员的癌组织/宫颈组织和外周血样本,分为健康对照组、宫颈上皮内癌变(cervical intraepithelial neoplasia,CIN)Ⅱ级组、CINⅢ级组和CSCC组,代表CSCC发生、发展进程各阶段,每组50例。ELISA法检测各组人员的外周血血浆中PD-1、PD-L1、叉头状转录因子P3(FOXP3)的表达水平,FCM法检测各组人员外周血PD-1^(+)CD4^(+)CD25^(+)CD127^(-/low)细胞的数量,应用多色荧光组织染色法检测肿瘤浸润性淋巴细胞(TIL)在CSCC组织中的分布特点。结果:随着模拟的CSCC发生和发展,外周血中PD-1、PD-L1和FOXP3的表达呈上升趋势,术后则呈下降趋势。在CSCC患者抗凝全血中,CD4^(+)、CD4^(+)CD25^(+)CD127^(-/low)以及PD-1^(+)CD4^(+)CD25^(+)CD127^(-/low)细胞占淋巴细胞的比例增加。在CSCC组织中可见大量CD4^(+)、CD8^(+)和FOXP3^(+)细胞浸润,其中CD4^(+)和FOXP3^(+)细胞主要围绕肿瘤细胞聚集区分布、CD8^(+)和PD-L1^(+)细胞则呈广泛弥漫性分布。结论:PD-1、PD-L1、FOXP3和适应性调节性T细胞是促进CSCC发生发展的重要因素,其可作为ESCC免疫治疗的靶点和临床预后的潜在标志物。

Expression of E2A in Mid-secretory Endometrium of Women Suffering from Recurrent Miscarriage

E2A is involved in promoting forkhead box P3（FOXP3） and retinoid-related orphan receptor gamma t（RORγt） gene transcription, which are pivotal transcription factors of T regulatory cells and Th17 cells, respectively. Little is known about the involvement of E2 A in pregnancy process. This study aimed to investigate the expression of E2 A, cytotoxic T-lymphocyte-associated protein 4（CTLA-4）, and Foxp3 in luteal phase endometrium of women suffering recurrent miscarriage（RM）（n=21） and control group（n=11） by immunohistochemistry, with the Vectra？ automated quantitative pathology imaging system for analysis. The percentage of E2 A＋ cells and CTLA-4＋ cells was significantly higher in the endometrium of women with RM than in the controls. There was positive correlation between E2 A and CTLA-4（r=0.523, P=0.002）, E2 A and FOXP3（r=0.380, P=0.032）, and FOXP3 and CTLA-4（r=0.625, P=0.000） in the mid-secretory phase of endometrium for all subjects. It was concluded that the abnormal expression of endometrial E2 A existed in mid-secretory endometrium of women with RM, and there was a positive correlation between E2 A and FOXP3, and E2 A and CTLA-4, suggesting the possible regulation role of E2 A involved in regulating endometrium receptivity.

Regulatory T cells in inflammatory bowel diseases and colorectal cancer

Regulatory T cells(T regs) are key elements in immunological self-tolerance.The number of T regs may alter in both peripheral blood and in colonic mucosa during pathological circumstances.The local cellular,microbiological and cytokine milieu affect immunophenotype and function of T regs.Forkhead box P3+ T regs function shows altered properties in inflammatory bowel diseases(IBDs).This alteration of T regs function can furthermore be observed between Crohn's disease and ulcerative colitis,which may have both clinical and therapeutical consequences.Chronic mucosal inflammation may also influence T regs function,which together with the intestinal bacterial flora seem to have a supporting role in colitis-associated colorectal carcinogenesis.T regs have a crucial role in the immunoevasion of cancer cells in sporadic colorectal cancer.Furthermore,their number and phenotype correlate closely with the clinical outcome of the disease,even if their contribution to carcinogenesis has previously been controversial.Despite knowledge of the clinical relationship between IBD and colitis-associated colon cancer,and the growing number of immunological aspects encompassing sporadic colorectal carcinogenesis,the molecular and cellular links amongst T regs,regulation of the inflammation,and cancer development are still not well understood.In this paper,we aimed to review the current data surrounding the role of T regs in the pathogenesis of IBD,colitis-associated colon cancer and sporadic colorectal cancer.

Protective Effect of Norcantharidin on Collagen-Induced Arthritis Rats

Objective： To observe the effect of norcantharidin（NCTD） on collagen-induced arthritis（CIA） rats. Methods： Sixty Sprague-Dawley（SD） rats were randomly divided into 6 groups（n=10）： normal group, CIA model group（model group）, NCTD low-dose group [1.35 mg/（kg·d）], NCTD middle-dose group [2.7 mg/（kg·d）], NCTD high-dose group [5.4 mg/（kg·d）] and methotrexate（MTX） group [1.8 mg/（kg/w）]. Anesthetized rats were sacrificed by luxation of cervical vertebra after 4 weeks of administration. The arthritis scores were evaluated twice a week. The pathological changes in the ankle joints of rats were observed by hematoxylin-eosin（H＆E） staining. The serum levels of interleukin（IL） 1β, IL-6, tumor necrosis factor（TNF）-α, vascular endothelial growth factor（VEGF）, IL-17 and transform growth factor（TGF） β were detected by enzyme linked immunosorbent assay（ELISA）. The mRNA expression of retinoid-related orphan nuclear receptor γ t（ROR γ t） and forkhead box P3（Foxp3） in peripheral blood lymphocytes were confirmed by real-time polymerase chain reaction. Results： MTX and high-dose NCTD not only decreased the arthritis scores but also alleviated the pathological changes in CIA rats＇ ankle joints compared with the model group（P〈0.05 or P〈0.01）. All doses of NCTD significantly inhibited the serum levels of IL-6, IL-17 and TNF-α in CIA rats（P〈0.05）. Only middle-and high-dose of NCTD prominently decreased serum IL-1β and TGF-β levels of CIA rats（P〈0.05）. However, NCTD has no effect on vascular endothelial growth factor（VEGF） level in CIA rats. The Foxp3 mRNA expression in all NCTD groups were increased significantly than in the model group（P〈0.05）. The mRNA expression of RORγt in NCTD high-dose group was decreased apparently in comparison with the model group（P〈0.05）. Conclusion： NCTD showed therapeutic effect on CIA rats by inhibition of cytokines and regulation of Th17/Treg cells.

Tumor Necrosis Factor a Reduces SNAP29 Dependent Autolysosome Formation to Increase Prion Protein Level and Promote Tumor Cell Migration

Tumor Necrosis Factor α(TNFα) is best known as a mediator of inflammation and immunity, and also plays important roles in tumor biology. However, the role of TNFα in tumor biology is complex and not completely understood. In a human melanoma cell line, M2, and a lung carcinoma cell line, A549, TNFα up-regulates prion protein(PrP) level, and promotes tumor cell migration in a PrP dependent manner. Silencing PRNP abrogates TNFα induced tumor cell migration;this phenotype is reversed when PRNP is re-introduced. Treatment with TNFα activates nuclear factor kappa B(NF-κB)signaling, which then mitigates autophagy by reducing the expression of Forkhead Box P3(FOXP3). Down regulation of FOXP3 reduces the transcription of synaptosome associated protein 29(SNAP29), which is essential in the fusion of autophagosome and lysosome creating autolysosome. FOXP3 being a bona fide transcription factor for SNAP29 is confirmed in a promoter binding assay. Accordingly, silencing SNAP29 in these cell lines also up-regulates PrP, and promotes tumor cell migration without TNFα treatment. But, when SNAP29 or FOXP3 is silenced in these cells, they are no longer respond to TNFα. Thus, a reduction in autophagy is the underlying mechanism by which expression of PrP is up-regulated,and tumor cell migration is enhanced upon TNFα treatment. Disrupting the TNFα-NF-κB-FOXP3-SNAP29 signaling axis may provide a therapeutic approach to mitigate tumor cell migration.

基于Treg细胞功能的稳定性探讨解毒通络生津方对干燥综合征模型鼠免疫调节的作用

目的:观察不同剂量解毒通络生津方对干燥综合征模型NOD/Ltj小鼠血清白细胞介素(IL)-6、肿瘤坏死因子-α(TNF-α)及颌下腺叉头框转录因子P3(FoxP3)表达的影响,探讨解毒通络生津方对NOD/Ltj小鼠免疫调节的作用机制。方法:将30只8周龄NOD/Ltj小鼠随机分为模型组,解毒通络生津方低、中、高剂量组和羟氯喹组共5组,12周龄开始每日分别灌服等量的生理盐水,含生药9、18、36 g·kg^(-1)的解毒通络生津方和60 mg·kg^(-1)的羟氯喹,同时选取6只ICR小鼠作为空白组,给予等量的生理盐水灌胃。实验期间记录各组小鼠第9、12、16周龄的日均饮水量、唾液流量,给药4周后摘取组织计算颌下腺指数、脾脏指数,苏木素-伊红(HE)染色观察颌下腺组织病理形态,Meso Scale Discovery(MSD)法检测小鼠血清IL-6、TNF-α、IL-10水平,免疫组化检测颌下腺FoxP3的表达和分布,蛋白免疫印迹法(Western blot)检测小鼠颌下腺FoxP3蛋白的表达,实时荧光定量聚合酶链式反应(Real-time PCR)检测小鼠颌下腺FoxP3、TNF-α mRNA的表达。结果:与空白组ICR小鼠比较,模型组小鼠饮水量增多、唾液流量减少、颌下腺指数降低、颌下腺组织病理学评分升高,血清IL-6、TNF-α表达升高,IL-10表达降低,颌下腺FoxP3蛋白表达降低,颌下腺FoxP3 mRNA表达降低、TNF-α mRNA表达升高(P<0.05)。与模型组比较,解毒通络生津方低、中、高剂量组饮水量均明显减少,唾液流量均明显增加(P<0.05),且中、高剂量组疗效更显著;解毒通络生津方中剂量组颌下腺指数升高(P<0.05),解毒通络生津方高剂量组脾脏指数降低(P<0.05);解毒通络生津方低、中、高剂量组颌下腺组织病理评分均降低(P<0.05),高剂量组疗效更显著;解毒通络生津方低、中、高剂量组血清IL-6、TNF-α表达均降低,IL-10表达升高,颌下腺TNF-α mRNA表达降低(P<0.05);解毒通络生津方中、高剂量组颌下腺FoxP3蛋白表达升高,颌下腺FoxP3 mRNA表达升高(P<0.05)。结论:解毒通络生津方可能通过促进FoxP3+调节性T细胞(Treg)功能的稳定,抑制炎症细胞因子的分泌,从而缓解干燥综合征全身免疫炎症的进展。