小鼠γδ T细胞亚群在不同组织器官的分布特性及在感染后的变化

目的:关注不同γδT细胞亚群在各组织器官的分布特点及在鼠伤寒沙门氏菌肠道感染中的变化,为探讨γδT细胞组织学分布的生理学意义和在感染性疾病中的作用提供依据和新的思路。方法:采用流式细胞术与PCR技术检测胸腺、脾脏、淋巴结、肝脏、皮肤以及小肠上皮内淋巴细胞中γδT细胞及其不同亚群的比例;通过PMA与离子霉素体外刺激,流式细胞术检测不同组织γδT细胞分泌细胞因子IFN-γ与IL-17a的差异;通过灌胃建立鼠伤寒沙门氏菌肠道感染模型,流式细胞术检测肝脏与小肠上皮内淋巴细胞中不同γδT细胞亚群比例的变化。结果:γδT细胞富含于小肠上皮内、皮肤与肝脏中,而在胸腺、脾脏与淋巴结中比例较低。不同亚群在各组织器官中的分布存在较大差异,皮肤主要存在Vγ5+γδT细胞亚群,小肠中主要存在Vγ1+、Vγ4+与Vγ7+γδT细胞亚群。肝脏γδT细胞主要分泌IL-17a,而小肠上皮内γδT细胞主要分泌IFN-γ。在鼠伤寒沙门氏菌肠道感染时,小肠上皮内γδT细胞的比例明显升高,其中Vγ1+γδT细胞比例升高更为显著;肝脏总γδT细胞比例无显著变化,但Vγ1+γδT细胞比例降低,Vγ4+γδT细胞比例明显升高。结论:γδT细胞富含于小肠、皮肤与肝脏中,且其不同亚群具有组织分布特异性。不同组织的γδT细胞产生细胞因子的能力存在较大差异。鼠伤寒沙门氏菌感染时,小肠与肝脏γδT细胞亚群的分布变化也存在较大差异。     

γδT 细胞亚群在慢性HCV 感染中的特征

目的:探讨γδT 细胞及其亚群γδ和γδT 细胞在慢性HCV 感染中的作用。方法:采用流式细胞术检测人外周血γδT 细胞及其亚群γδ和γδT 细胞比例变化。结果:慢性HCV 感染病人外周血中酌啄T 细胞及其亚群γδ和γδT细胞比例与健康对照者相比无显著差异。相关性分析显示HCV 感染患者γδT 细胞数目与肝脏损伤成正相关而与病毒滴度不相关。慢性HCV 感染病人γδT 细胞处于活化状态,CD107a 表达高于对照组。结论: γδT 细胞亚群参与慢性HCV 感染所致肝损伤。     

急性HIV 感染者γδT 细胞及其细胞亚群的表型分析

目的:分析急性HIV 感染者外周血γδT 细胞的表型,诠释γδT 细胞在急性HIV 感染中的作用。方法:流式细胞术检测γδT 细胞表面CD38、程序性细胞死亡蛋白-1(PD-1)、CD160、CD95 和肿瘤坏死因子相关凋亡诱导配体-死亡受体5(TRAIL-DR5)的表达。结果:与健康对照组相比,急性HIV 感染者Vδ1 T 细胞的比例显著增加,Vδ2 T 细胞的比例显著降低,但总γδT 细胞的比例无显著变化。CD38+ γδT 细胞、CD38+ Vδ1 T 细胞和CD38+ Vδ2 T 细胞的比例较健康对照显著增加。此外,PD-1+ γδT 细胞和DR5+ γδT 细胞的比例较健康对照组显著增加,而Vδ1、Vδ2 T 细胞亚群PD-1 和DR5 的表达与健康对照相比差异无显著性。但在急性HIV 感染者中,CD38+ Vδ1 T 细胞、CD160+ Vδ1 T 细胞、PD-1+ Vδ1 T 细胞、CD95+ Vδ1 T 细胞和DR5+ Vδ1 T 细胞的比例均较Vδ2 T 细胞显著增加。结论:急性HIV 感染者γδT 细胞,尤其是Vδ1 T 细胞表面活化、抑制和凋亡分子的表达显著增加,提示Vδ1 T 细胞在急性HIV 感染及疾病进展中发挥更重要的作用。     

IκBα转基因小鼠肝脏免疫细胞亚群的初步分析

目的:分析IκBα转基因小鼠肝脏组织形态、免疫细胞分群、凋亡状态以及表型分子等。方法:HE 组织染色分析野生型小鼠和IκBα转基因小鼠肝脏组织形态变化;利用流式细胞术分析野生型小鼠和I资B琢转基因小鼠肝脏组织中NK、NKT、T 细胞比例和表型分子,进一步采用Annexin V 标记法检测小鼠肝脏免疫细胞的凋亡状态;定量PCR 法检测趋化因子和细胞因子的表达含量。结果:与野生型小鼠相比, IκBα-转基因鼠肝脏组织损伤严重; IκBα转基因小鼠肝脏NK、NKT、T 细胞比例均显著下降,NK 细胞凋亡的比例增加,并且NKp46 在IκBα转基因小鼠肝脏NK 细胞中表达降低。在此过程中,还观察到IκBα转基因小鼠肝脏细胞因子谱发生改变,IFN-γ、CCL2 等表达显著下降,而IL-2、IL-15 等并没有显著变化。结论: IκBα转基因小鼠肝脏组织形态、免疫细胞亚群比例、表型及细胞因子谱均发生改变。     

通过抑制STAT3磷酸化促进γδT细胞CCR5的表达

目的： 研究糖原合酶激酶-3β抑制剂4,6-二取代吡咯并嘧啶(TWS119)促进γδT细胞趋化因子受体CCR5表达的分子机制。方法：用TWS119诱导从健康人外周血中分离培养获得γδT细胞48 h后,用流式细胞仪检测γδT细胞CCR5的表达;Western blot检测GAPDH和pSTAT3的表达。结果：培养10 d后的γδT细胞纯度达到85.79%±5.01%。TWS119浓度在0~8.0 μmol/L范围内能显著促进趋化因子受体CCR5的表达,且剂量依赖性抑制STAT3的磷酸化。同时,用STAT3磷酸化抑制剂Stattic (0.5 μmol/L)预处理γδT细胞也可以促进CCR5的表达,并能协同增强TWS119促进趋化因子受体CCR5的表达。结论： TWS119通过抑制STAT3磷酸化促进γδT细胞CCR5的表达。     

IL-17+γδT细胞在相关疾病中的免疫作用

IL-17是一类重要的促炎症因子,近年来大量研究发现,除了Th17细胞外,γδT细胞也是IL-17的重要来源。IL-17+γδT细胞可以参与多种疾病的诱发和发展,在感染性疾病、自身免疫性疾病和肿瘤等的发生发展中发挥重要作用。     

Vδ2 γδ T细胞亚群在慢性HCV感染中的特征

目的:探讨γδT细胞及其亚群Vδ1和Vδ2 T细胞在慢性HCV感染中的作用。方法:采用流式细胞术检测人外周血γδT细胞及其亚群Vδ1和Vδ2 T细胞比例变化。结果:慢性HCV感染病人外周血中γδT细胞及其亚群Vδ1和Vδ2 T细胞比例与健康对照者相比无显著差异。相关性分析显示HCV感染患者Vδ2 T细胞数目与肝脏损伤成正相关而与病毒滴度不相关。慢性HCV感染病人Vδ2 T细胞处于活化状态,CD107a表达高于对照组。结论:Vδ2 T细胞亚群参与慢性HCV感染所致肝损伤。     

树突状细胞在银屑病中的作用研究进展

银屑病是一种由于免疫系统异常活化导致的慢性皮肤炎症,其发病机制仍未完全阐明。树突状细胞、巨噬细胞、Th17 细胞、γδT 细胞、3 型固有淋巴细胞等多种类型的免疫细胞在银屑病中发挥重要作用,而树突状细胞作为炎症和免疫应答的始动环节,在其中起到关键的调控作用。不同亚群的树突状细胞具有不同的功能,本文旨在总结树突状细胞及其不同的亚群在银屑病发生发展过程中发挥的作用及其机制的研究进展。     

慢性粒细胞白血病患者外周血高表达CD266~+Foxp3~+γδ~+T细胞亚群

目的分析慢性粒细胞白血病慢性期(CML-CP)患者外周血γδT细胞及其亚群的表达情况,及其与临床一线酪氨酸激酶抑制剂(TKI)药物治疗疗效的相关性。探讨成纤维细胞生长因子诱导因子14(Fn14,又称CD266)在γδT细胞功能亚群中的表达情况及其与临床疗效的相关性。方法采用流式细胞术检测CML-CP患者以及正常对照组外周血γδT细胞及其功能亚群的表达情况,并探讨γδT细胞各亚群表达比例与临床一线TKI药物治疗疗效的相关性。结果 CML-CP患者外周血γδT细胞功能亚群CD266~+γδ~+T细胞、Foxp3~+γδ+T细胞、CD266+Foxp3~+γδ~+T细胞、CD266~+Vδ1~+T细胞、Foxp3~+Vδ1~+T细胞、CD266~+Foxp3~+Vδ1~+T细胞、CD266~+Vδ2~+T细胞、Foxp3+Vδ2+T细胞和CD266~+Foxp3~+Vδ2~+T细胞的表达比例均显著高于正常对照组。经过一线TKI药物治疗后达到完全血液学反应的基础上,达到警告治疗反应或治疗失败的CML-CP患者外周血CD266~+Foxp3~+Vδ2~+T细胞亚群比例显著增高。Logistic回归分析亦显示CD266~+Foxp3~+Vδ2~+T细胞亚群的表达比例为CML-CP患者经过一线TKI药物治疗后发生难治的危险因素。结论 CD266~+Foxp3~+γδT细胞亚群与CML-CP患者的临床一线TKI药物治疗疗效密切相关。Fn14(CD266)信号通路可能成为复发难治CML患者的新的治疗靶点。     

分析胃癌组织淋巴细胞亚群及其表达细胞因子比例的变化特征

目的探讨胃癌组织淋巴细胞亚群及其表达细胞因子比例的变化特征。方法胃癌患者110例,取手术切除的胃癌样本,采用流式细胞术比较胃癌组织与正常组织之间NK细胞、B细胞、CD4+T细胞、CD8+T细胞的比例,分析γδT细胞亚群表达TNF-α、IL-1β和IL-10的γδT细胞数量及其在胃癌不同分期组织的比例变化。结果在胃癌组织中,B细胞和NK细胞的比例与正常胃部组织相比无明显变化;CD4+T细胞在胃癌组织中的比例显著大于在正常组织(P0.05);CD8+T细胞在胃癌组织中的比例低于正常组织(P0.05)。表达TNF-α和IL-1β的γδT细胞在胃癌部位的比例低于正常组织中的比例(P0.05);而表达IL-10的γδT细胞在胃癌组织中的比例高于正常组织(P0.05)。对胃癌不同分期组织进行检测,结果发现,在Ⅲ/Ⅳ期胃癌组织中,表达TNF-α和IL-1β的γδT细胞的比例均低于Ⅰ/Ⅱ期胃癌组织(P0.05);而表达IL-10的γδT细胞的比例则高于Ⅰ/Ⅱ期胃癌组织(P0.05)。结论在胃癌组织中CD4+T细胞数量增加,CD8+T细胞下降;表达TNF-α和IL-1β的γδT细胞随着胃癌的进展而不断减少;表达IL-10的γδT细胞随着胃癌的进展而不断增加。通过对胃癌组织淋巴细胞亚群的分析检测,说明淋巴细胞亚群与胃癌组织的发生发展密切相关。     

川楝素与γδ T细胞协同抗结直肠癌的作用及机制研究

目的:探讨γδT细胞对结直肠癌细胞的杀伤活性,并研究川楝素对γδT细胞的协同效应。方法:体外培养人γδT细胞并用流式细胞术进行鉴定。乳酸脱氢酶(LDH)释放实验检测γδT细胞和川楝素对人结直肠癌SW480细胞的杀伤活性。Western blot实验检测川楝素对SW480细胞中Bcl-2、Bcl-x L和MCL-1表达水平的影响。ELISA实验检测川楝素是否影响γδT细胞肿瘤坏死因子相关凋亡诱导配体(TRAIL)和Fas配体(Fas L)的分泌。流式细胞术检测川楝素和γδT细胞处理后SW480细胞的线粒体膜电位和凋亡情况。Western blot实验检测川楝素和γδT细胞处理后SW480细胞caspase-9和caspase-3的活化。结果:体外培养的γδT细胞高表达CD3和γδT细胞受体(TCR)。川楝素处理能显著增强γδT细胞对SW480的杀伤活性。川楝素不影响γδT细胞TRAIL和Fas L的表达。川楝素不影响SW480细胞中Bcl-2和Bcl-x L的蛋白水平但显著抑制MCL-1的表达。转染MCL-1质粒能显著抑制川楝素对γδT细胞的协同效应。川楝素通过抑制MCL-1的表达促进γδT细胞对SW480细胞线粒体膜电位的损伤和凋亡的诱导。川楝素通过抑制SW480细胞MCL-1的表达促进γδT细胞对SW480细胞caspase-9和caspase-3的活化。结论:川楝素通过抑制MCL-1的表达发挥对γδT细胞的协同抗结直肠癌作用。     

Epithelial defence by gamma delta T cells

Gamma delta T cells constitute a separate lineage of T lymphocytes which differ from conventional alpha beta T cells with regard to T cell receptor (TCR) repertoire and tissue localization. In murine skin, gamma delta T cells expressing a canonical V gamma5 TCR are abundant and contribute as so-called dendritic epidermal T cells to local immune surveillance. In humans, major subsets of gammadelta T cells are recognized on the basis of their TCR V delta usage. While V delta2 cells dominate in the peripheral blood, V delta1 cells are preferentially localized in mucosal tissue including the intestinal epithelia. In this article we summarize basic features of intraepithelial gamma delta T cells and discuss their possible role in epithelial defence.     

口服伤寒杆菌后小鼠肠道黏膜上皮内淋巴细胞的变化

目的研究经口服伤寒杆菌后,小鼠肠道黏膜上皮内淋巴细胞中T细胞亚群的变化,探讨肠道黏膜免疫应答机制。方法将8～10周龄的BALB/C小鼠100只随机分为对照组和实验组。实验组灌胃伤寒杆菌悬液分两次进行免疫,在第二次灌胃后第3、5、7、9、11d分别处死小鼠。通过免疫组化染色对小鼠肠道上皮内淋巴细胞进行表型分析,并按表型进行计数。对照组给予同体积PBS。结果实验组小鼠上皮内淋巴细胞中CD4+、CD8+、TCRαβ+、TCRγδ+亚群在3、5、7、9、11天呈增高趋势,其中从第5天开始CD8+、TCRγδ+T细胞亚群数量(个/100肠上皮细胞)与对照组相比,差异有统计学意义(P<0.01),CD4+、TCRαβ+T细胞数量与对照组相比。差异无统计学意义(P>0.05)。结论肠道黏膜上皮内淋巴细胞中CD8+、TCRγδ+T细胞增殖明显,提示其在抗感染免疫应答中起主导作用。     

Oestrogen Treatment Depletes Extrathymic T Cells from Intestinal Lymphoid Tissues

Different lineages of thymic and extrathymic T cells are found in the epithelial layer and in the lamina propria of the small and large intestine of euthymic and athymic mice. A single subcutaneous injection of oestradiolvalorat (Progynon^®-Depot-10, Schering, Berlin, Germany) into athymic mice led to a dose-dependent depletion of extrathymic T cells from the intraepithelial and lamina propria compartments of the small and large intestine. TCRαβ and TCRγδ, CD4⁺ and CD8α⁺ T cell subsets were affected. The depletion of intraepithelial, extrathymic T cells by oestradiol treatment was striking. Oestrogen, therefore, has an effect not only on genital mucous membranes, but also on the large, diffuse lymphoid tissues of the gut, in that it selectively depletes the intestinal, extrathymic T-cell subsets.     

Age-dependent variation in the proportion and number of intestinal lymphocyte subsets, especially natural killer T cells, double-positive CD4+ CD8+ cells and B220+ T cells, in mice

The age-dependent variation in the proportion and number of lymphocyte subsets was examined at various extrathymic sites, including the liver, small intestine, colon and appendix in mice. In comparison with young mice (4 weeks of age), the number of total lymphocytes yielded by all tested organs was greater in adult (9 weeks) and old (40 weeks) mice. The major lymphocyte subset that expanded with age was interleukin-2 receptor (IL-2R) beta+ CD3int cells (50% of them expressed NK1.1) in the liver, whereas it was CD3+ IL-2Rbeta- NK1.1- cells at all intraepithelial sites in the intestine. Although NK1.1+ CD3+ cells were present at intraepithelial sites in the intestine, the proportion of this subset was rather low. The ratio of CD4 to CD8 tended to decrease among natural killer T (NKT) cells and T cells at all intraepithelial sites in the intestine with age. A unique population of double-positive CD4+ CD8+ cells in the small intestine increased in old mice. B220+ T cells were found mainly in the appendix and colon, and the proportion of these T cells decreased in old mice. Conventional NKT cells were very few in Jalpha281-/- and CD1d-/- mice in the liver, while NKT cells which existed in the appendix remained unchanged even in these mice. This was because unconventional CD8+ NKT cells were present in the intestine. The present results suggest that despite the fact that both the liver and intraepithelial sites in the intestine carry many extrathymic T cells, the distribution of lymphocyte subsets and their age-associated variation are site-specific.     

T lymphocytes in host defense against bacterial translocation from the gastrointestinal tract.

Flow cytofluorometric analyses of lymphocytes harvested from the mesenteric lymph node (MLN), mucosal epithelium, and lamina propria of C57BL/6 mice demonstrate that expression of alpha/beta or gamma/delta T-cell receptors (TCR) and CD4 or CD8 molecules by T lymphocytes in the intestinal immune system varies depending upon their anatomic location. The MLN contained equivalent numbers of CD4+ and CD8+ T cells, the vast majority of which were alpha/beta TCR positive (alpha/beta TCR+). The lamina propria T cells were predominantly CD4+ and alpha/beta TCR+, while the intestinal intraepithelial lymphocytes consisted of equivalent numbers of alpha/beta and gamma/delta T cells, the majority of which were CD8+. There were no significant changes in these T-cell phenotypic profiles when the mice were antibiotic decontaminated or monoassociated with Escherichia coli. Mice were depleted of CD4+ T cells and/or CD8+ T cells in vivo by intraperitoneal injections of monoclonal antibody GK 1.5 (rat anti-mouse CD4) and/or monoclonal antibody 2.43 (rat anti-mouse CD8). T-cell depletion was confirmed in the MLN, lamina propria, and the intestinal epithelium by flow cytometry. E. coli C25 translocation from the gastrointestinal (GI) tract to the MLN was significantly increased in mice depleted of CD4+ T cells, CD8+ T cells, or both. T-cell-deficient athymic beige/nude mice also exhibited greater levels of E. coli C25 translocation to the MLN than beige/het euthymic littermates. Salmonella typhimurium translocation also was increased following CD4+ and CD8+ T-cell depletion in mice monoassociated with S. typhimurium. Depletion of CD4+ and/or CD8+ T cells also increased the translocation to the MLN of certain indigenous GI flora bacteria. These results confirm that T-cell-mediated immunity is involved in the host defense against bacterial translocation from the GI tract.