目的探讨EBV mi R-BART17-3p影响原发免疫性血小板减少症(ITP)患儿Treg/Th17平衡的机制。方法收集ITP患儿(ITP组,20例)和健康儿童(对照组,20例)外周血并分离CD_(4)^(+)T细胞。采用实时荧光定量聚合酶链式反应法、Western blot法、酶联...目的探讨EBV mi R-BART17-3p影响原发免疫性血小板减少症(ITP)患儿Treg/Th17平衡的机制。方法收集ITP患儿(ITP组,20例)和健康儿童(对照组,20例)外周血并分离CD_(4)^(+)T细胞。采用实时荧光定量聚合酶链式反应法、Western blot法、酶联免疫吸附法检测EBV mi R-BART17-3p、T细胞免疫球蛋白黏蛋白3(Tim-3)、叉头框蛋白P3(Fox P3)、白细胞介素17A(IL-17A)和转化生长因子-β(TGF-β)的m RNA、蛋白表达水平及含量。采用双荧光素酶报告基因实验考察EBV mi R-BART17-3p对Tim-3表达水平的影响。将15只BALB/C小鼠随机分为空白对照组、模型组、观察组,各5只。腹腔注射抗血小板抗体MWReg30以复制ITP小鼠模型,建模4 d后观察组小鼠予尾静脉注射携带EBV mi R-BART17-3p inhibitor的腺病毒载体。细胞染色并观察形态,检测外周血中TGF-β、IL-17A含量及血小板计数,采用流式细胞仪分别检测CD_(4)^(+)T细胞中Th17和Treg水平,并计算二者百分比。结果与对照组比较,ITP组患儿外周血EBV mi R-BART17-3p表达水平显著升高,Tim-3和TGF-βm RNA表达水平显著降低(P<0.05);Tim-3 m RNA表达水平与EBV mi R-BART17-3p表达水平呈显著负相关(r=-0.732,P<0.001)。Tim-3慢病毒载体p LKO.1-sh-Tim-3(sh-Tim-3)可显著降低Tim-3、Fox P3、TGF-β水平(P<0.05)。mi R-BART17-3p mimic显著升高了CD_(4)^(+)T细胞中mi R-BART17-3p的表达水平,并显著降低了Tim-3、Fox P3、TGF-βm RNA和蛋白表达水平(P<0.05);mi R-BART17-3p mimic可显著降低TGF-β含量,Tim-3+mi R-BART17-3p过表达逆转了mi R-BART17-3p mimic对TGF-β的抑制作用。动物实验结果显示,沉默EBV mi R-BART17-3p可促进Treg分化,减少脾脏和骨髓组织中的巨核细胞计数,并显著增加外周血中血小板计数。结论EBV mi R-BART17-3p可通过Fox P3/Tim-3途径调节ITP患儿Treg/Th17的免疫失衡。展开更多
T cell immunoglobulin and mucin domain 3 （Tim-3） is well known to negatively regulate T cells responses, but its role in burn-induced T cells immune suppression remains unclear. In the present study, in order to ide...T cell immunoglobulin and mucin domain 3 （Tim-3） is well known to negatively regulate T cells responses, but its role in burn-induced T cells immune suppression remains unclear. In the present study, in order to identify the relationship between Tim-3 expression and post-burn T cells immune suppression, C57BL/6 mice were subjected to burn injury or sham injury, and the liver and spleen were harvested at the day 1 after operation. The expression level of Tim-3 on hepatic or splenic T cells and the functional properties of Tim-3＋ T cells were evaluated. It was found burn injury induced dramatically elevated Tim-3 expression on both hepatic and splenic CD4＋ and CD8＋ T cells in contrast with the post-burn depletion of T cells. Furthermore, Tim-3 expression was correlated with the suppressive phenotype of T cells following burn injury, including increased expression of anti-inflammatory cytokine IL-10, decreased expression of pro-inflammatory cytokines IFN-γ and TNF-α, reduced T cell proliferation and elevated co-expression of Tim-3 and PD-1. Moreover, Tim-3＋ T cells subsets were more prone to spontaneous apoptosis than Tim-3- T cells subsets. Our findings reinforce the idea that the up-regulated expression of Tim-3 on T cells after burn injury plays an important role in the development and maintenance of burn-induced T cell immune suppression.展开更多
目的探讨T细胞免疫球蛋白和黏蛋白结构域分子3(T cell immunoglobulin and mucin domain protein 3,Tim-3)在晚期血吸虫病患者外周血CD56+自然杀伤细胞(NK细胞)表面的表达及其与肝纤维化指标的关系。方法采用流式细胞术检测28例临床晚...目的探讨T细胞免疫球蛋白和黏蛋白结构域分子3(T cell immunoglobulin and mucin domain protein 3,Tim-3)在晚期血吸虫病患者外周血CD56+自然杀伤细胞(NK细胞)表面的表达及其与肝纤维化指标的关系。方法采用流式细胞术检测28例临床晚期血吸虫病患者和30例健康者CD56+NK细胞表面Tim-3的表达,应用全自动化学发光免疫分析仪检测肝纤维化血清学三型前胶原N端肽(PIIIP N-P)、层黏连蛋白(LN)、IV型胶原(CIV)和透明质酸(HA)等4项指标,ELISA检测血清中γ-干扰素(IFN-γ)和白细胞介素-4(IL-4)水平,Fibroscan仪检测肝硬度值(LSM)反映肝纤维化程度。统计分析Tim-3表达水平与IL-4和IFN-γ水平、肝脏纤维化程度以及肝纤维化血清指标的相关性。结果流式细胞分析结果显示,晚期血吸虫病患者外周血CD56+NK细胞表面Tim-3表达水平为(62.3±11.4)%,高于健康对照组的(52.1±6.5)%(P<0.01)。晚期血吸虫病患者和健康对照组的PIIIP N-P水平分别为(86.5±29.5)ng/ml和(22.0±7.8)ng/ml,LN分别为(49.3±21.5)ng/ml和(20.4±6.3)ng/ml,CIV分别为(67.5±22.3)ng/ml和(22.0±3.9)ng/ml,HA分别为(645.9±483.1)ng/ml和(54.7±27.7)ng/ml,两组各指标差异均具有统计学意义(P<0.05)。晚期血吸虫病患者和健康对照组IFN-γ水平分别为(93.9±20.1)ng/L和(107.7±24.6)ng/L,IL-4分别为(46.6±11.8)ng/L和(28.9±8.9)ng/L,两组差异均具有统计学意义(P<0.05)。Spearman非参数相关分析结果显示患者CD56+NK细胞表达的Tim-3水平与患者肝纤维化指标LSM、PIIIP N-P、LN、CIV和IL-4水平呈正相关(r=0.528~0.834,P<0.01),与IFN-γ水平呈负相关(r=-0.501,P<0.01),而与HA水平无明显相关性(r=0.352,P>0.05)。结论晚期血吸虫病患者外周血CD56+NK细胞Tim-3表达呈上调,并与患者肝纤维化指标LSM、PIIIP N-P、LN、CIV和IL-4水平呈正相关。展开更多
文摘目的探讨EBV mi R-BART17-3p影响原发免疫性血小板减少症(ITP)患儿Treg/Th17平衡的机制。方法收集ITP患儿(ITP组,20例)和健康儿童(对照组,20例)外周血并分离CD_(4)^(+)T细胞。采用实时荧光定量聚合酶链式反应法、Western blot法、酶联免疫吸附法检测EBV mi R-BART17-3p、T细胞免疫球蛋白黏蛋白3(Tim-3)、叉头框蛋白P3(Fox P3)、白细胞介素17A(IL-17A)和转化生长因子-β(TGF-β)的m RNA、蛋白表达水平及含量。采用双荧光素酶报告基因实验考察EBV mi R-BART17-3p对Tim-3表达水平的影响。将15只BALB/C小鼠随机分为空白对照组、模型组、观察组,各5只。腹腔注射抗血小板抗体MWReg30以复制ITP小鼠模型,建模4 d后观察组小鼠予尾静脉注射携带EBV mi R-BART17-3p inhibitor的腺病毒载体。细胞染色并观察形态,检测外周血中TGF-β、IL-17A含量及血小板计数,采用流式细胞仪分别检测CD_(4)^(+)T细胞中Th17和Treg水平,并计算二者百分比。结果与对照组比较,ITP组患儿外周血EBV mi R-BART17-3p表达水平显著升高,Tim-3和TGF-βm RNA表达水平显著降低(P<0.05);Tim-3 m RNA表达水平与EBV mi R-BART17-3p表达水平呈显著负相关(r=-0.732,P<0.001)。Tim-3慢病毒载体p LKO.1-sh-Tim-3(sh-Tim-3)可显著降低Tim-3、Fox P3、TGF-β水平(P<0.05)。mi R-BART17-3p mimic显著升高了CD_(4)^(+)T细胞中mi R-BART17-3p的表达水平,并显著降低了Tim-3、Fox P3、TGF-βm RNA和蛋白表达水平(P<0.05);mi R-BART17-3p mimic可显著降低TGF-β含量,Tim-3+mi R-BART17-3p过表达逆转了mi R-BART17-3p mimic对TGF-β的抑制作用。动物实验结果显示,沉默EBV mi R-BART17-3p可促进Treg分化,减少脾脏和骨髓组织中的巨核细胞计数,并显著增加外周血中血小板计数。结论EBV mi R-BART17-3p可通过Fox P3/Tim-3途径调节ITP患儿Treg/Th17的免疫失衡。
基金supported by grants from National Natural Science Foundation of China (No. 30700799, 81172803)Doctoral Fund of Ministry of Education of China (No. 20070487119)Natural Science Foundation of Hubei Province (No. 2007AD A201)
文摘T cell immunoglobulin and mucin domain 3 （Tim-3） is well known to negatively regulate T cells responses, but its role in burn-induced T cells immune suppression remains unclear. In the present study, in order to identify the relationship between Tim-3 expression and post-burn T cells immune suppression, C57BL/6 mice were subjected to burn injury or sham injury, and the liver and spleen were harvested at the day 1 after operation. The expression level of Tim-3 on hepatic or splenic T cells and the functional properties of Tim-3＋ T cells were evaluated. It was found burn injury induced dramatically elevated Tim-3 expression on both hepatic and splenic CD4＋ and CD8＋ T cells in contrast with the post-burn depletion of T cells. Furthermore, Tim-3 expression was correlated with the suppressive phenotype of T cells following burn injury, including increased expression of anti-inflammatory cytokine IL-10, decreased expression of pro-inflammatory cytokines IFN-γ and TNF-α, reduced T cell proliferation and elevated co-expression of Tim-3 and PD-1. Moreover, Tim-3＋ T cells subsets were more prone to spontaneous apoptosis than Tim-3- T cells subsets. Our findings reinforce the idea that the up-regulated expression of Tim-3 on T cells after burn injury plays an important role in the development and maintenance of burn-induced T cell immune suppression.
