流式细胞术检测慢性乙型肝炎患者外周血Th17和T_C17细胞

目的建立流式细胞术检测外周血中Th17、TC17细胞的方法,并探讨慢性乙型肝炎(CHB)患者外周血Th17细胞、TC17细胞变化的的意义。方法用三色流式细胞术检测了24例CHB患者和15例健康对照者外周血中Th17细胞和TC17细胞。结果用流式细胞仪结合不同荧光素标记的CD3、CD8和IL-17A单克隆抗体可准确检测外周血中Th17、TC17细胞的数量。对照组中Th17、TC17的检测结果分别为(2.13±0.50)%(1.6%～3.3%)和(0.27±0.13)%(0.2%～0.6%);CHB患者组中分别为(5.58±4.52)%(1.6%～19.0%)和(1.05±0.93)%(0.2%～3.1%)。CHB患者外周血中Th17、TC17细胞显著高于对照组(P=0.007、P=0.004)。结论流式细胞术检测外周血Th17、TC17细胞方法可靠;CHB患者外周血中Th17细胞、TC17细胞与对照组比较显著增高。     

子痫前期患者外周血树突状细胞对Th1/Th17细胞分化的影响

目的 观察正常妊娠妇女和子痫前期患者外周血单个核细胞(PBMC)来源的树突状细胞(DC)对Th1、Th17细胞分化的影响.方法 实验组为子痫前期疾病患者32例;对照组为正常妊娠妇女20例.分离正常妊娠组和子痫前期组PBMC,经贴壁获得单核细胞,加GM-CSF,IL-4和LPS培养诱导为成熟DC,流式细胞术检测表面分子标志CD14,CD80,CD83,CD86的表达,ELISA检测培养上清液中IL-23的含量.磁珠分选出CD4+T淋巴细胞,与正常妊娠孕妇外周血单核细胞来源的树突状细胞(N-DC)共同培养,同时添加细胞因子IL-2,或与子痫前期患者外周血单核细胞来源的树突状细胞(P-DC)、IL-2共同培养;或与N-DC共同培养,同时添加细胞因子IL-1p、IL-6;或与P-DC共同培养,并添加细胞因子IL-1β、IL-6.以上各组培养到第6天用流式细胞术检测CD4+ IFN-γ+ Th1、CD4+ IL-17+ Th17细胞比例.结果 P-DC中CD83、CD80、CD86的表达高于N-DC,差异有统计学意义(P＜0.05).P-DC与不同的细胞因子共同作用,促使CD4+T细胞分化为Th1、Th17细胞的能力高于N-DC,差异有统计学意义(P＜0.01).结论 子痫前期患者外周血DC表型和功能的改变可能与患者免疫失衡有一定关系.     

Th17细胞和Treg细胞比例失衡在AS发展中作用的研究进展

在强制性脊柱炎(ankylosing spondylitis,AS)发病机制的诸多假设中,免疫细胞格局失调被认为是一个重要机制。Th17细胞和Treg细胞是CD4+T细胞的两个亚群,对炎症分别起着促进和抑制的作用。诸多研究提示,Th17细胞和Treg细胞数量及相关细胞因子在AS患者外周血中与健康者有差别,其比例失衡在AS的发展中有重要作用,具体机制仍不清楚。本文介绍了近年来Th17和Treg细胞及其比例失衡在AS发展中作用的研究进展。     

强直性脊柱炎患者Th17细胞与CD4+CD25+FoxP3+调节性T细胞的变化及意义

目的 探讨强直性脊柱炎( ankylosing spondylitis,AS)患者Th17和CD4+ CD25+ FoxP3+调节性T细胞比例及相关细胞因子水平的变化及意义.方法 强直性脊柱炎患者40例,正常同年龄对照37例.采用流式细胞术检测外周血Th17与调节性T细胞的比例,双抗体夹心酶联免疫吸附法( ELISA)检测血清IL-6、IL-23、IL-17和TGF-β水平.结果 AS组患者外周血Th17细胞比例明显高于对照组[(1.02±0.34)％vs(0.68:±0.29)％,P＜0.05],CD+ CD25+ FoxP3+细胞比例明显低于对照组[(3.77±0.81)％ vs (4.69±1.23)％,P＜0.05].AS患者血清中IL-6、IL-23、IL-17水平明显高于对照组[ (6.15±2.71) ng/L vs(3.31±1.65) ng/L; (9.44±3.12) ng/ml vs (5.82±2.61) ng/ml;( 10.53±4.97) ng/L vs (6.78±3、26) ng/L,P均＜0.01];差异有统计学意义.与对照组相比,AS组TGF-β水平有下降的趋势[(4,76±2.15) ng/ml vs(5.16±2.02) ng/ml,P＞0.05],但差异无统计学意义.AS患者血清各细胞因子含量与临床及实验室指标无相关性.结论 强直性脊柱炎患者Th17与CD4+CD35+FoxP3+调节性T细胞比例失衡,血清IL-6、IL-23、IL-17和TGF-β水平变化,这些原因可能参与强直性脊柱炎免疫发病过程.     

类风湿关节炎患者Th17和CD161+Th17细胞水平的变化

目的 观察类风湿关节炎(RA)患者和健康对照组外周血中CD4+ IL-17+T细胞(Th17)和CD4+ CD161+ IL-17+T细胞(CD161+Th17)的水平,探讨其在RA发病过程中的意义.方法 采用流式细胞术测定36例RA患者和11例健康对照组外周血中Th17细胞及CD161+ Th17的细胞百分率.结果 RA患者外周血Th17及CD161+ Th17细胞水平明显高于健康对照组(P＜0.01)且与疾病活动指数(DAS28)、血沉和C-反应蛋白水平呈显著正相关(P ＜0.05);RA患者和健康对照组外周血中CD161+ Th17细胞百分率明显高于Th17细胞百分率(P＜0.01);Th17细胞百分率与CD161+ Th17细胞百分率显著正相关(P＜0.01).结论 Th17及CD161+ Th17细胞在RA患者外周血中均增高且与疾病活动度正相关.     

B细胞活化因子及其受体BR3在强直性脊柱炎患者外周血白细胞中的表达及临床意义初探

为了探讨强直性脊柱炎(AS)患者外周血白细胞中B细胞活化因子(BAFF)及其受体(BR3)的表达水平及其临床意义.采用流式细胞术检测24例AS患者、30名健康体检者外周血白细胞上BAFF、BR3的表达水平,并比较其与红细胞沉降率(ESR)、C反应蛋白(CRP)、免疫球蛋白(IgG、IgA、IgM)的相关性.结果显示.A...     

SLE患者外周血Th17细胞的表达及其临床意义

目的：探讨Th17细胞与系统性红斑狼疮（SLE）的发生及疾病活动性的相关性。方法：采用酶联免疫吸附法（ELISA）检测正常人和SLE患者外周血单个核细胞（PBMCs）刺激培养上清液中IL-17的水平,并用流式细胞术（FCM）分析PBMCs中Th17细胞的频率。结果：与正常对照组相比,稳定期和活动期SLE患者PBMCs刺激后产生IL-17的量明显增高（P〈0.01）,Th17细胞的频率也高于正常对照组（P〈0.01）。与静止期患者相比,活动期SLE患者PBMCs产生IL-17的水平和Th17细胞的频率也明显增高（P〈0.05）。结论：Th17细胞可能通过分泌细胞因子IL-17参与了SLE发病及病情发展,其水平高低部分反映SLE疾病的活动性。     

支气管哮喘患者外周血Th17 与Th9 细胞的变化及临床意义

目的:探讨Th17、Th9 细胞水平在支气管哮喘患者中的表达及其临床意义。方法:选取67 例哮喘患者为研究对象,30 例健康体检者为对照组,根据全球哮喘防治倡议(GINA)将哮喘患者分为间歇与轻度持续组20 例,中度持续组25 例和重度持续组22 例。收集患者临床资料,完成哮喘控制测试(ACT)评分、肺功能和呼出气一氧化氮(FeNO)值测定,流式细胞仪检测外周血PBMC 中CD3+ CD8- IL-17+ 、CD3+ CD8- IL-9+细胞水平。结果:与健康对照组相比,哮喘患者Th17、Th9 细胞水平均明显升高(P<0.05);中重度哮喘患者Th17 及Th9 细胞水平显著高于间歇与轻度哮喘组(P<0.05),而间歇与轻度哮喘组患者与健康对照组比较,差异无统计学意义(P>0.05)。哮喘患者外周血Th17 及Th9 细胞的表达均与FeNO 值正相关(r 值依次为0.501、0.438;P<0.05),与ACT 评分、肺功能FEV1、FEV1/ FVC 及PEF 负相关(r 值依次为-0.441、-0.362;-0.307、-0.377;-0.419、-0.411;-0.428、-0.24;P<0.05),同时Th17 与Th9 细胞在哮喘患者外周血中的表达率正相关(r 值为0.443,P<0.05)。结论:Th17、Th9 细胞在哮喘患者中高表达,具有协同致炎作用,且与症状严重程度相关,提示Th17 及Th9 细胞对哮喘患者病情评估有潜在的参考价值。     

梅毒血清固定患者外周血Th17细胞的检测

目的检测梅毒血清固定患者外周血Th17细胞的细胞比例和相关细胞因子的变化,初步探讨Th17细胞在梅毒血清固定现象中的作用。方法梅毒血清固定患者30例,同时以30例健康体检人作为对照组。采用流式细胞术(Flow cytometry, FCM)检测外周血单个核细胞(peripheral blood mononuclear cel s, PBMC)中Th17细胞在CD4+T细胞中的比例;采用实时定量PCR方法检测Th17细胞特异性转录因子孤独受体γt（orphan nuclear receptor gammat , RORγt）及细胞因子IL-17mRNA的表达。结果梅毒血清固定患者外周血Th17细胞比例及RORγt和IL-17的表达量显著低于健康对照组（P<0.05）。结论 Th17细胞的异常可能是梅毒血清固定现象形成的重要原因之一。     

老年抑郁症伴焦虑患者外周血中Th17细胞与细胞因子的改变

探讨老年抑郁症伴焦虑患者(≥60岁)外周血中Th17细胞发挥的作用。选取28例老年抑郁症伴焦虑患者为实验组,另选取老年健康体检者(≥60岁)28例为对照组。采用流式细胞术检测两组人群外周血中CD3~+、CD4~+ T淋巴细胞亚群及Th17细胞(CD4~+IL-17~+)的比例,ELISA检测患者血清中IL-6、TNF-α、IL-1β、IL-17含量,实时定量PCR检测外周血淋巴细胞中核转录因子IL-17、RORγt表达水平。老年抑郁症伴焦虑患者外周血CD3~+、CD4~+ T淋巴细胞百分率低于对照组(P均0.01),Th17细胞表达比例为(2.05±0.47)%,明显高于对照组(0.69±0.25)%,差异有统计学意义(P0.01);IL-6、TNF-α、IL-1β、IL-17在老年抑郁症伴焦虑患者组的浓度均高于对照组,差异有统计学意义(均为P0.01),老年抑郁症伴焦虑患者外周血淋巴细胞中IL-17、RORγt基因表达水平均高于对照组,差异有统计学意义(均为P0.01)。Thl7细胞与老年抑郁症伴焦虑具有相关性,可能通过分泌以IL-17为主的细胞因子造成患者机体免疫功能紊乱,参与老年抑郁症伴焦虑病情发展。     

CD4~+效应T细胞在自身免疫性溶血性贫血患者外周血的变化研究

目的研究自身免疫性溶血性贫血(AIHA)患者外周血中效应T细胞亚群Th1和Th17数量和比例的变化,探讨其在AIHA发病中的作用。方法选择30例AIHA患者,分离外周血单核细胞(PBMC),采用流式细胞术检测Th1和Th17细胞频率,用ELISA测定培养上清中IFN-γ和IL-17水平,并与健康对照组比较。结果 AIHA患者PBMC中IFN-γ+Th1和IL-17+Th17细胞频率均显著高于健康对照组(P<0.01);而且,AIHA患者外周血IL-17水平明显高于健康对照组(P<0.05),但IFN-γ水平在患者和正常人之间差异不显著。结论Th17与Th1细胞亚群可能参与AIHA患者的免疫学发病机制。     

Increased number of Th22 cells and correlation with Th17 cells in peripheral blood of patients with IgA nephropathy

T-helper (Th) 22 and Th17 cells are involved in the pathogenesis of autoimmune diseases. However, the role of Th22 and correlation with Th17 cells in the pathophysiology of IgA nephropathy (IgAN) remain unknown. In our study, Th22 and Th17 cells in peripheral blood of IgAN patients, non-IgA mesangial proliferative glomerulonephritis (non-IgA MsPGN) patients, and healthy controls were measured by flow cytometry. The concentration of plasma interleukin-22 (IL-22) was examined by enzyme linked immunosorbent assay (ELISA). The results showed that Th22 cells, Th17 cells, and plasma IL-22 were significantly elevated in IgAN patients compared with non-IgA MsPGN patients and healthy controls. Th22 cells showed a positive correlation with the levels of plasma IL-22 in IgAN patients. Moreover, a significantly positive correlation between Th22 cells and Th17 in IgAN patients was observed. Furthermore, IgAN patients with proteinuria showed a higher percentage of Th22 cells than IgAN patients without proteinuria. Our data demonstrated that IgAN had increased frequencies of peripheral Th22, Th17 cells and plasma IL-22, indicating that Th22 along with Th17 cells are involved in the immune responses of IgAN.     

Characterization of peripheral blood TCR repertoire in patients with ankylosing spondylitis by high-throughput sequencing

Ankylosing spondylitis (AS) is a chronic and progressive autoimmune disease affecting the invasion of the spine, sacroiliac joints and peripheral joints. T cells play a vital role in the underlying pathogenesis of AS, which mediated autoimmune and inflammatory responses via specific recognition of autoantigen peptides presented by susceptibility HLA. Antigen-specific T cells triggered by HLA/antigen complexes will undergo a massive expansion that forming an uneven T cell repertoire. To enhance our understanding of T-cell-mediated autoimmune in AS, we applied TCR β chains high-throughput sequencing to AS patients for in-depth TCR repertoire analysis. A significantly lower TCR repertoire diversity was observed in peripheral blood of AS patients relative to controls. And severe patients in our AS cohort have a more restricted TCR repertoire than mild patients, suggesting that the TCR repertoire diversity might be associated with the clinical severity of disease. No V, J and VJ pairs with significant biased usage were identified, which indicated that the usage frequency deviation of certain V/J/V-J genes in AS patients is little. This is a pilot study with potentially interesting observation on reduced diversity of T cells repertoire in peripheral blood of AS patients and further studies are needed.     

类风湿性关节炎患者外周血Th17/Treg细胞比率失衡的研究

目的:观察类风湿性关节炎(RA)患者外周血Th17细胞与Foxp3+CD4+CD25+调节性T(Treg)细胞的平衡状态与疾病状态的关系,初步阐明Th17/Treg细胞比率失衡在RA发病机制中的作用和意义。方法:流式细胞术(FCM)检测RA患者和健康人外周血中Th17细胞和Foxp3+CD4+CD25+Treg细胞的比率。结果:活动期RA患者外周血CD3+CD4+T细胞和Th17细胞的比率均明显高于健康对照组(P均0.05);而Foxp3+CD4+CD25+Treg细胞的比率明显低于健康对照组(P0.05)。随疾病活动性的增加,Th17细胞表达增高(P0.05);而Foxp3+CD4+CD25+Treg细胞表达降低,但无统计学意义(P0.05)。结论:RA患者外周血T细胞紊乱以CD4+T细胞的增加为主,Th17细胞比率的增加和Foxp3+CD4+CD25+Treg细胞比率的降低所致的Th17/Treg细胞比率失衡,可能在RA的发生发展中起重要作用。     

Chlamydial antibody crossreactivity with peripheral blood mononuclear cells of patients with ankylosing spondylitis: the role of HLA B27.

We have previously reported the association of Chlamydia trachomatis with HLA B27+ related diseases. To investigate the possibility that chlamydial antibodies serve to localize the immune response in such diseases, we examined the crossreactivity of chlamydial antibodies (rabbit anti-D and anti-L2 serotypes) with peripheral blood mononuclear cells of patients with ankylosing spondylitis (AS) and anterior uveitis (AU) and with human and bovine ocular tissue and cells in culture. Our results indicate a significantly increased percentage binding of chlamydial antibody (D serotype) to the mononuclear cells of HLA B27+ patients with AS when compared with HLA B27- patients with AS (12.9% +/- 2.2 versus 5.4% +/- 2.2), B27+ controls (5.5% +/- 1.5) and B27- controls (6.1% +/- 1.0). There was no significant difference between controls and HLA B27+ patients with AU (6.6% +/- 1.9) and B27- patients with AU (8.7% +/- 1.1). This crossreactivity could not be blocked by monoclonal HLA B27 antibody. Chlamydial antibodies (D and L2) crossreact with human and bovine conjunctiva but not uvea, tissue culture derived iris fibroblasts or smooth muscle cells. Our results provide additional support for the concept of crossreactivity between antibodies to microbial agents and peripheral blood mononuclear cells of patients with HLA B27+ AS.     

强直性脊柱炎患者外周血T细胞亚群极化状态的研究

目的:分析强直性脊柱炎患者外周血中T细胞亚群的极化状态,进一步阐明强直性脊柱炎的免疫学发病机制.方法:运用流式细胞(FCM)技术,通过CD3/CD4或CD3/CD8设门,检测早、晚期AS患者和健康对照者外周血淋巴细胞的CD3 CD4 CD30 、CD3 CD4 CD30ˉ、CD3 CD8 CD30 、CD3 CD8 CD30ˉT细胞的百分率,分别表达Th1、Th2、Tc1、Tc2细胞,并进行比较分析.结果:在早期AS患者CD4 T细胞和CD4 T/CD8 T细胞的比值均明显低于健康对照组(P＜0.05),Th1细胞百分率及Th1/Th2比值明显降低(P＜0.05),CD8 T细胞的百分率明显高于健康对照组(P＜0.05),Tc2细胞明显升高(P＜0.05),且Tc1/Tc2比值明显降低(P＜0.05);在晚期AS患者Tc2细胞明显升高(P＜0.05),Tc1/Tc2比值明显降低(P＜0.05).结论:早期AS患者外周血CD4 T细胞、CD8 T细胞、CD4 T/CD8 T的变化与健康对照比较具有显著性意义,阐明了AS早期患者体内的免疫功能紊乱,呈现一种免疫功能低下的状态.重要的是Th1细胞、Th1/Th2、Tc1/Tc2的明显减少,提示在早期AS患者体内Th1/Th2、Tc1/Tc2的失调,且以Th2、Tc2占有优势,而晚期AS患者因Tc1/Tc2明显降低导致比例失衡,这些显示Th1/Th2、Tc1/Tc2的比例失衡可能是导致AS发病的重要因素,是AS发病机制之一.