调节性CD4~+T细胞及非调节性CD4~+T细胞与HIV/AIDS患者疾病进展关系的研究

目的分析比较中国HIV感染者CD4+CD25+Foxp3+调节性T细胞(CD4+regulatory T cells,Treg)及非Treg细胞与疾病进展的关系,探讨Treg细胞在HIV感染过程中的作用。方法选取76例HIV/AIDS患者,根据其CD4+T细胞计数水平不同分为3组,A组CD4<200个/μL,B组CD4200～400个/μL,C组CD4>400个/μL。采用流式细胞仪胞内染色技术检测CD4+CD25+Foxp3+调节性T细胞的水平,并分析其与CD4计数及病毒载量的相关性。结果随着疾病的进展,Treg细胞百分比逐渐升高,各组间差异有统计学意义;Treg细胞及非Treg细胞的绝对计数均明显下降,且以非Treg细胞下降为主;Treg绝对计数与病毒载量呈负相关(P<0.05)。结论中国HIV感染者随着疾病进展,辅助性T细胞等非Treg细胞的数量下降,对机体免疫保护能力降低,而Treg细胞数量及功能的下降使其对机体的过度免疫活化的抑制作用减弱,病毒复制,加剧病情进展。     

HIV/AIDS患者治疗前后CD4~+、CD8~+细胞水平检测结果分析

目的了解HIV/AIDS患者高效逆转录抗病毒(HAART)治疗前后CD4+、CD8+细胞计数水平变化情况。方法利用流式细胞分析技术对随机选取在本院住院治疗的177例HIV/AIDS患者进行治疗前后CD4+、CD8+检测,观察其治疗前后CD4+、CD8+细胞计数水平的变化。结果经过6个月的HAART治疗后,在选取的177例HIV/AIDS患者中,CD4+、CD8+细胞计数均较抗病毒治疗前有上升的趋势,治疗前后细胞计数比较差异具有非常显著统计学意义(P<均0.01)。结论 HIV/AIDS患者在持续HAART治疗后,CD4+、CD8+细胞计数将会提升到一定的水平,其可更好地提高HIV/AIDS患者的免疫力,在延长患者生命中具有重要的现实意义。     

人外周血CD4~+CD25~+Foxp3~+调节性T细胞分选及扩增方法的建立

目的建立人外周血CD4~+CD25~+Foxp3~+调节性T细胞(Treg)的免疫磁珠分选法(MACS)及体外扩增方法 ,并对扩增前后Treg细胞纯度及表型进行鉴定。方法流式细胞术检测人外周血Treg细胞占CD4~+T细胞的比例;免疫磁珠法分选CD4~+CD25~+Treg细胞,用CD3/CD28单抗包被的Dynalbeads联合IL2共同刺激CD4~+CD25~+Treg细胞进行体外扩增培养,在扩增培养的第0、7、14、21天分别进行细胞计数及检测细胞的活性,取扩增培养第0天及第14天细胞行流式细胞术检测扩增前后细胞纯度、主要表面标记及Foxp3的表达情况。结果正常人外周血CD4~+CD25~+Foxp3~+Treg水平占CD4~+T细胞(7.94±1.86)%,MACS能够成功分选出CD4~+CD25~+Foxp3~+Treg细胞,分选平均纯度达(95.86±0.65)%,细胞活性95%;以CD3/CD28单抗包被的Dynalbeads联合IL2共同刺激CD4~+CD25~+Treg细胞进行外扩增培养后,细胞有明显扩增。经2周扩增后,细胞扩增倍数达到原细胞数量的(21.33±1.53)倍,扩增后细胞Foxp3的表达由(95.86±0.65)%下降至(93.71±1.30)%,但差异无统计学意义(P0.05)。结论免疫磁珠分选法能够分选出高纯度的CD4~+CD25~+Foxp3~+调节性T细胞,体外成功扩增CD4~+CD25~+Foxp3~+调节性T细胞,扩增前后Treg细胞的纯度及表型无明显变化。     

HIV感染者调节性T细胞消耗可导致HIV特异CD8~+细胞凋亡

目的了解在HIV感染患者CD4+CD25+FoxP3+调节性T细胞(Treg)的降低是否能够引起HIV特异CD8+细胞过度凋亡。方法本研究对75例没有治疗的HIV患者进行研究,检测其外周血中Treg细胞计数及CD8+细胞的活化及凋亡情况并分析其相关性;同时纯化Treg细胞及CD8+细胞,在体外进行共培养,用五聚体检测HIV特异CD8+细胞的活化情况。结果在HIV感染患者,Treg细胞与CD8+细胞的活化及凋亡呈负相关,Treg细胞能够抑制HIV特异的CD8+细胞的凋亡。结论 HIV感染患者Treg细胞的降低可导致CD8+细胞、尤其HIV特异CD8+细胞的凋亡增加。     

CD4~+ CD25~+ Foxp3~+调节性T细胞在成人过敏性哮喘急性发作期和缓解期外周血中的表达

目的:探讨成人过敏性哮喘急性发作期和缓解期外周血中CD4+CD25+Foxp3+调节性T细胞的表达情况及与病情的相关性。方法:收集健康人、过敏性哮喘急性发作期和缓解期患者外周血,流式细胞术测定CD4+CD25+Foxp3+Treg的比例。结果:CD4+CD25+Foxp3+Treg细胞在三组间的差异有统计学意义。哮喘缓解期组、哮喘急性期组较健康对照组该细胞比例均明显下降,哮喘急性期组较哮喘缓解期组更低。CD4+CD25+Foxp3+Treg细胞数量与哮喘病情严重程度呈负相关。结论:CD4+CD25+Foxp3+调节性T细胞可能在过敏性哮喘的发病过程中起重要作用,它的降低导致的免疫缺陷可能与哮喘的发生相关。     

CD4+CD25+Foxp3+:与SLE疾病相关的Treg标志

目的:研究与系统性红斑狼疮(systemic lupus erythematosus,SLE)病理活动相关的调节性T细胞(regulatory T cell,Treg)标志?方法:分离正常人和SLE患者外周血单个核细胞(PBMCs),用三色流式细胞术(flowcytometer,FCM)检测CD4和CD8 T细胞亚群的CD25?Foxp3表达,分析T细胞Foxp3+/CD4+T?CD25+Foxp3+/CD4+?Foxp3+/CD4+CD25+?Foxp3+/CD8+?CD25+Foxp3+/CD8+?及Foxp3+/CD8+CD25+的比值变化,及其与SLE疾病活动指数(SLE disease activity index,SLEDAI)?抗-dsDNA阳性?补体C3/C4水平降低?血清IgG水平增高?肾脏损害?关节病变?白细胞减少?疾病初/复发的关系?结果:①与正常人相比,SLE患者T细胞CD4+CD25+/CD4+比值无明显改变,但中/重度SLE患者外周血T细胞Foxp3+/CD4+?CD25+Foxp3+/CD4+?及Foxp3+/CD4+CD25+比值均显著下降,并与SLEDAI呈负相关;其中CD25+Foxp3+/CD4+比值还与SLE的抗-dsDNA阳性?补体C3/C4水平降低?血清IgG水平增高相关,在疾病初发?肾脏损害?白细胞减少患者该比值下降更为显著;②SLE患者CD8+ T细胞中CD25+?Foxp3+亚群比例未见明显减少,仅中/重度活动性SLE患者Foxp3+/CD8+CD25+比例减少,但与SLEDAI无相关性?结论:以CD25+Foxp3+作为Treg标志,可以明显反映出SLE患者CD4+ Treg存在数量缺陷,并与疾病活动性?免疫功能紊乱和病理损害密切相关,故将CD4+CD25+Foxp3+作为标志进行Treg检测对辨明SLE患者免疫系统功能状态具有一定的临床参考价值?     

吸入激素对哮喘患者外周血CD4~+CD25~+调节性T细胞水平及Foxp3 mRNA表达的影响

目的观察支气管哮喘患者外周血CD4+CD25+调节性T细胞(CD4+CD25+Treg)、Foxp3 mRNA的水平及吸入糖皮质激素对其的影响。方法流式细胞仪检测非急性发作期哮喘患者吸入激素前、后和健康志愿者(正常对照组)外周血单个核细胞(PBMCs)中CD4+CD25+Treg的比例,RT-PCR检测Foxp3 mRNA表达变化,肺功能仪检测第1秒用力呼气容积占预计值百分比(FEV1%pred)和呼气峰流量(PEF)。结果哮喘组治疗前CD4+CD25+Treg水平及Foxp3 mRNA的表达显著低于正常对照组(P<0.05),治疗后CD4+CD25+Treg水平及Foxp3 mRNA的表达较治疗前显著升高(P<0.05);哮喘组治疗前FEV1%pred、PEF显著低于正常对照组(P<0.05),治疗后FEV1%pred、PEF较治疗前显著增加(P<0.05)。结论哮喘患者外周血中具有免疫抑制活性的CD4+CD25+Treg数量减少,功能下降,参与了哮喘的发病过程;吸入激素可以上调哮喘患者外周血CD4+CD25+Treg及Foxp3 mRNA的水平,改善哮喘患者的肺功能。     

硒对特应性皮炎小鼠CD4~+CD25~+Foxp3~+调节性T细胞的影响

目的分析膳食硒对特应性皮炎小鼠CD4+CD25+Foxp3+调节性T细胞的影响。方法 40只BALB/c小鼠随机分为致敏组和对照组,致敏组(G1、G2、G3)分别以缺硒(0.01μg/m L)、中量硒(0.25μg/m L)和高量硒(3.00μg/m L)饲料喂养,对照组(G4)以中量硒饲料喂养。8周后致敏组采用DNCB诱导特应性皮炎症状,G4组作为对照。致敏期间监测皮炎症状。致敏3周后,检测血浆总Ig E、全血细胞计数、脾脏CD4+CD25+Foxp3+调节性T细胞水平和组织硒含量,分析组间各指标差异和变量间关系。结果 G1、G2、G3组小鼠第6天皮炎症状评分、第8天抓挠计数、第21天血浆总Ig E和全血炎症细胞数均高于G4组,差异有统计学意义(P0.05)。G1、G2、G3、G4组CD4+CD25+Foxp3+调节性T细胞占CD4+CD25+T细胞百分比分别为(79.6±7.6)%、(59.0±27.5)%、(37.6±5.4)%、(85.26±9.2)%,差异有统计学意义(P=0.000);其中G3组降低最为明显,与G1、G2和G4组比较差异具有统计学意义(t=0.174,40.828,1.466,P0.01);相关回归分析发现,CD4+CD25+Foxp3+调节性T细胞水平与皮肤组织硒含量呈直线负相关(r=-0.623,P=0.001)。结论膳食硒通过其免疫学效应影响特应性皮炎小鼠CD4+CD25+Foxp3+调节性T细胞的增殖分化水平。     

溃疡性结肠炎患者CD4~+CD25~+调节性T细胞的表达及意义

目的溃疡性结肠炎(ulcerative colitis,UC)是一种病因及发病机制迄今尚未完全明确的累及肠道的慢性非特异性炎症,近年来,研究发现免疫异常与其发病密切相关。通过分析UC患者外周血和肠黏膜中CD4+CD25+调节性T细胞(regulatory T cells,Treg)及其特异性标志物Foxp3的表达特点,探讨CD4+CD25+Treg和Foxp3在UC发病机制中的作用。方法 20例UC活动期患者,15例健康体检者作为正常对照组,分别抽取外周静脉血和经电子肠镜活检肠黏膜组织,以流式细胞术检测外周血单个核淋巴细胞(peripheral blood mononuclear cell,PBMC)和肠固有层单个核细胞(lamina propria mononu-clear cell,LPMC)中CD4+CD25+T细胞和CD4+T细胞比例,应用免疫组化方法检测肠黏膜中Foxp3的表达,并检测血清中IL-10的水平。结果 UC患者外周血中CD4+CD25+T细胞占CD4+T细胞比例明显低于正常对照组(P<0.05),肠黏膜CD4+CD25+T细胞占CD4+T细胞比例和Foxp3+T细胞表达明显高于正常对照组(P<0.05),两组PBMC和LPMC中CD4+T细胞比例无明显差异(P>0.05),两组血清IL-10水平无显著差异(P>0.05)。结论 CD4+CD25+Treg在UC的发病机制中起重要作用,外周血Treg数量减少可能是疾病发生的重要因素。     

肺癌恶性胸水中CD4+CD25+Foxp3+调节性T细胞检测及其临床意义

目的 检测晚期肺癌患者外周血及恶性胸水中CD4+ CD25+ Foxp3+调节性T细胞(CD4+CD25+ Foxp3 +Treg,Foxp3 +Treg)及相关细胞因子的表达情况并探讨其临床意义.方法 通过密度梯度离心法获得26例晚期肺癌患者外周血、胸水中淋巴细胞及上清和10例健康志愿者(对照组)外周血淋巴细胞及上清...     

Alu-LTR PCR方法检测HAART治疗系列各细胞亚群的整合型HIV-1 DNA

目的应用Alu-LTR PCR方法对高效抗反转录病毒治疗(highly active antiretroviral therapy,HAART)系列的CD4+T,CD8+T及B细胞进行检测,明确不同细胞亚群及HAART治疗的不同阶段是否存在整合的HIV-1 DNA。方法收集20例HIV-1感染患者HAART治疗过程中0、4、12周及10例健康对照的抗凝血标本,纯化CD4+T,CD8+T及B细胞并提取DNA,应用Alu-LTR PCR方法进行检测。结果 20例HIV-1感染者HAART治疗系列中CD4+T细胞及CD8+T细胞成功扩出目的片段,CD8+T细胞所扩条带亮度均低于CD4+T细胞,HAART治疗系列0、4、12周标本所扩条带亮度没有明显差异。B细胞及10例健康者均为阴性。结论 CD4+T及CD8+T细胞存在整合型HIV-1 DNA,HIV储藏库主要存在于CD4+T细胞内。B细胞内不存在整合型HIV-1 DNA。随着HAART治疗的进程,整合型HIV-1 DNA仍然存在,进一步证明HAART不能根除HIV储藏库。     

HLA-DR expression on regulatory T cells is closely associated with the global immune activation in HIV-1 infected subjects naïve to antiretroviral therapy

Background  The frequencies of regulatory T cells (Tregs) increased over the HIV infection but its counts actually decreased. We proposed that the decrease of Treg counts may cause the reduction of inhibitory effect and thereby account for the over-activation of Tregs during HIV infection. However, it remains unknown whether Tregs are also over-activated and thereafter the activation induced death may lead to the decrease of Tregs.

Methods  Tregs were defined as CD4⁺CD25⁺CD127^lo/- T cells. Eighty-one HIV-1 infected patients were enrolled in our study, and twenty-two HIV-1 seronegative donors were recruited as the control. The levels of HLA-DR on Tregs were determined by FACSAria flow cytometer.

Results  Compared to HIV-1 seronegative donors, the levels of HLA-DR on CD4⁺CD25⁺CD127^lo/- Tregs were significantly increased in HIV-1 infected patients, and its increase was positively associated with viral loads (r=0.3163, P=0.004) and negatively with CD4 T-cell counts (r=−0.4153, P <0.0001). In addition, significant associations between HLA-DR expression on CD4⁺CD25⁺CD127^lo/- Tregs and the percentages of HLA-DR, CD38, Ki67 expressing CD4⁺ and CD8⁺ T cells were also identified.

Conclusion  HLA-DR on Tregs is a good marker for viral replication and disease progression. The over-activation of Tregs might result in the decrease of Tregs.

     

丙型肝炎患者基线T细胞分化状态与快速病毒学应答的相关性分析

目的探讨慢性丙型肝炎患者基线T细胞分化状态与抗病毒治疗快速病毒学应答(RVR)的关系。方法采用流式细胞仪检测CD27和CD28在CD8+T细胞上的表达,根据其表达差异,判断CD8+T细胞的不同分化状态:早期(CD28+/CD27+)、中期(CD28-/CD27+)和晚期(CD28-/CD27-);比较抗病毒治疗获得RVR组和非RVR组的基线CD27、CD28的表达水平及T细胞分化状态的差异。结果获得RVR组的CD27、CD28在CD8+T细胞的表达均高于未获得RVR组,差异有统计学意义(P=0.004,P=0.01)。T细胞的分化状态在RVR组以早、中期为主,而在非RVR组以晚期T细胞分化状态为主,2组之间差异有统计学意义(P<0.01)。结论基线T细胞的分化状态与干扰素联合利巴韦林抗病毒治疗慢性丙型肝炎RVR有一定相关性,可能与机体T细胞免疫在干扰素的免疫调节下参与病毒清除有关。     

慢性丙型肝炎患者DNT细胞及T细胞亚群的研究

目的探讨外周血中CD3+CD4-CD8-(double negative T,DNT)细胞及T淋巴细胞亚群在慢性丙型肝炎(chronichepatitis C,CHC)患者中的表达及意义。方法利用流式细胞仪对92例CHC患者、93例正常健康人外周血中的DNT细胞及T细胞亚群进行检测,并对比分析两者的差异。结果 CHC患者DNT细胞百分比(8.57%±6.16%)高于正常对照组(6.78%±4.50%)(P<0.05),其绝对数在两者间比较,差异无统计学意义(P>0.05)。CHC患者CD4+%,CD4+/CD8+比值低于正常对照组(P值均<0.001),CD8+%高于正常对照组(P<0.001)。结论 CHC患者DNT细胞百分比显著升高及T淋巴细胞亚群比例改变,可能是丙型肝炎病毒感染慢性化的重要原因之一。     

Early changes of CD4+CD25+Foxp3+ regulatory T cells and Th1/Th2, Tc1/Tc2 profiles in the peripheral blood of rats with controlled hemorrhagic shock and no fluid resuscitation

Background  Hemorrhagic shock induces immune dysfunction. Regulatory T cells (Tregs), T-helper (Th) cells, and cytotoxic T-lymphocytes (CTLs) can execute many crucial actions in immune and inflammatory responses. This study was conducted to investigate the early pathophysiological changes of CD4⁺CD25⁺Foxp3⁺ Treg and Th1/Th2, Tc1/Tc2 profiles in the peripheral blood of rats with controlled hemorrhagic shock and no fluid resuscitation.

Methods  A rat model of controlled hemorrhagic shock with no fluid resuscitation was established. Peripheral blood samples were taken before and four hours after hemorrhagic shock with no fluid resuscitation. Three color flow cytometry was used to detect Tregs, Th1, Th2, Tc1 and Tc2 cells in the samples.

Results  In the peripheral blood of rats, the percentage of Tregs four hours after hemorrhagic shock was significantly lower than before hemorrhagic shock (P=0.001). The ratios of Th1/Th2 and Tc1/Tc2 were changed from (23.08±8.98)% to (23.91±15.36)%, and from (40.40±21.56)% to (65.48±23.88)%, respectively.

Conclusions  At an early stage, the advent of hemorrhagic shock is related to an early decrease of Tregs, and a mild shift in the Th1/Th2, Tc1/Tc2 balance toward Th1 and Tc1 dominance. These changes are part of a hyper-inflammatory state of the host, and will deteriorate the maintenance of immune balance. Further influences and detailed mechanisms need to be investigated.     

对539例HIV感染者皮肤黏膜表现的临床分析

目的通过研究H IV感染者/AIDS患者皮肤黏膜表现的种类数及严重程度及其与CD4+T淋巴细胞计数、CD4+/CD8+比值的关系,评估皮肤黏膜病变作为预测和评估患者免疫状态的临床指标的可行性。方法对北京佑安医院2001年5月~2006年4月收集的539例住院及门诊H IV感染者/AIDS患者的临床资料作回顾性研究,采用流式细胞仪技术检测350例出现不同皮肤黏膜表现及189例无皮肤黏膜表现患者的CD4+T细胞计数、CD4+/CD8+比值,观察H IV感染人群中各种皮肤病的发生率,并研究出现皮肤病的种类数、严重程度与艾滋病患者免疫功能的相关性。结果H IV阳性患者的皮肤病发生率为65%,其中口腔黏膜念珠菌感染最常见。H IV阳性有皮肤黏膜表现组CD4+T细胞计数的平均数与CD4+/CD8+比值(分别为53.04个/μL与0.202 4)明显低于无皮肤黏膜表现组(分别为281.97个/μL与0.312 9),2组间比较差异具有明显的统计学意义(P<0.05),且CD4+T细胞计数与所患皮肤病的种类数及严重程度呈负相关。结论H IV感染者/AIDS患者的常见皮肤病临床表现,即患者皮肤疾病的种类数、发生率及严重程度与CD4+T细胞计数呈负相关,某些皮肤黏膜表现可以作为预测和评估资源有限国家患者免疫状态的临床指标。     

二黄胶囊对实验性自身免疫性脑脊髓炎小鼠细胞免疫的影响

目的观察二黄胶囊对实验性自身免疫性脑脊髓炎小鼠(EAE)细胞免疫的影响。方法皮下注射髓鞘少突胶质细胞糖蛋白35-55(MOG35-55)与完全弗氏佐剂(CFA)混合的抗原,辅以腹腔注射百日咳毒素(PTX),建立EAE模型。造模后每天记录各组小鼠体质量及神经功能评分;用流式细胞仪检测小鼠外周血T淋巴细胞亚群及自然杀伤(NK)细胞;采用HE染色进行脑组织病理学观察。结果模型组小鼠造模后第9天陆续发病,第22天达到高峰,发病急性期(造模后第25天)T淋巴细胞亚群CD3+、CD4+明显比正常组增高(P<0.01),CD4/CD8比值也有所升高(P<0.05);发病缓解期(造模后第40天)T淋巴细胞亚群CD8+和NK细胞明显下降(P<0.05);病理检查发现EAE小鼠脑组织神经元细胞核固缩,小静脉周围炎性细胞浸润形成袖套样改变。二黄胶囊治疗后,EAE小鼠神经功能评分比模型组明显降低(P<0.05),脑组织炎性浸润程度减轻,发病急性期外周血T淋巴细胞亚群CD4+/CD8+比值显著降低(P<0.01);恢复期T淋巴细胞亚群(CD3+、CD8+)及NK细胞明显增加(P<0.05)。结论二黄胶囊可在EAE小鼠发病的不同时期通过调节T淋巴细胞亚群(CD3+,CD4+,CD8+,CD4+/CD8+)及NK细胞而起到防治EAE作用。     

Study of T Cell subsets and IL-7 protein expression in HIV-1-infected patients after 7 years HAART

Objective

To study the changes in T cell subsets and IL-7 in HIV-1-infected patients after seven years of highly active antiretroviral therapy (HAART).

Methods

Seventy-five individuals were included in this study (25 with effective HAART, 18 with ineffective HAART, 17 untreated HIV+ patients, and 15 volunteers in the HIV negative control group). The counts of CD4⁺, CD8⁺, CD8/CD38⁺, and CD8/HLADR⁺ T cells as well as the IL-7 protein expression was measured at 5 time points during a period of seven years in patients starting HAART (baseline) and in the HIV negative control group. The expression of CD127 on CD3⁺ T cells was measured by flow cytometry at a single time point (after 7 years) in patients with HAART and was compared with untreated HIV+ patients and the HIV negative control group.

Results

At baseline CD4⁺ T cell counts of HIV-1-infected patients were lower than that in the control group (p < 0.01), whereas the CD8⁺, CD8/HLADR⁺ and CD8/CD38⁺ T cell counts were higher than those in the control group (p < 0.01). After seven years of effective HAART, the CD4⁺ T cell counts had increased and the CD8⁺ T cell count had decreased, although not to the normal levels (p < 0.05). Both the CD8/HLADR⁺ and CD8/CD38⁺ T cell counts had gradually approached those of the control group (p > 0.05). In the ineffective HAART group, the CD8/CD38⁺ T cell count had not decreased significantly, and CD8/HLADR⁺ T cell count gradually decreased. Before treatment, IL-7 serum levels of patients were significantly higher than that in the control group (p < 0.01). After seven years of effective HAART, IL-7 levels had gradually decreased, but were still higher than in the control group (p < 0.01). The CD127 expression on CD3⁺ CD8⁺ T cells in effective HAART patients was higher than in untreated HIV+ patients (p < 0.05), but was lower than that in the control group (p < 0.05). CD127 expression on CD3⁺ CD4+ T cells was not significantly different among the control group, untreated HIV+ patients and effective HAART group.

Conclusion

After seven years of effective HAART, the quantity and capacity of T cell subsets and IL-7 in HIV-1-infected patients had been partially restored, and the abnormal immune activation has significantly diminished.