四聚体技术检测原发性胆汁性肝硬化患者血循环中抗原特异性T细胞

目的定量检测原发性胆汁性肝硬化(PBC)患者体内抗原特异性T淋巴细胞含量,探讨其在PBC发病机制中的作用。方法采用四聚体技术检测15例HLA-A0201阳性(A2 )PBC患者外周血单个核细胞(PBMC)经抗原肽诱导生长的细胞毒性T淋巴细胞(CTL)中PDC-E2159~167aa与PDC-E2165~174aa特异性CD8 T细胞频率,以A0201阴性(A2-)PBC患者与A2 的其他慢性肝病和健康自愿者为对照组。结果在A2 PBC患者PDC-E2159~167aa与PDC-E2165~174aa诱导的CTL中可检测到其相应的四聚体/CD8 细胞,平均频率分别为0.42%±0.24%(0.17%~1.08%)、0.27%±0.17%(0.05%~0.56%),各对照组的四聚体阳性细胞频率均低于0.1%,差异非常显著(P<0.001);A2 PBC组中PDC-E2159~167aa特异性的CTL频率与PDC-E2165~174aa特异性的CTL无显著性差异(P>0.05)。处于临床Ⅰ、Ⅱ期的A2 PBC患者中CD8 特异性CTL频率均较Ⅲ期的要高(P<0.05)。PDC-E2159~167aa特异性CTL与PDC-E2165~174aa特异性CTL频率在抗-PDC阳性PBC组和抗-PDC阴性组之间均无显著性差异(P>0.05)。结论HLA-A0201限制性的PDC-E2159~167aa和PDC-E2165~174aa特异性CD8 CTL在PBC疾病进展中起重要作用,抗线粒体抗体阴性或抗-PDC阴性PBC患者与阳性患者可能有着相似的T细胞介导的免疫发病机制。     

流式细胞术检测肿瘤患者外周血T淋巴细胞亚群研究

目的 探讨肿瘤患者T淋巴细胞亚群的变化及临床意义。方法 采用流式细胞术检测27例肿瘤患者和25例正常对照组的T淋巴细胞亚群。结果 肿瘤患者CD3^＋T细胞、CD4^＋T细胞和CD4^＋／CD8^＋比值明显低于对照组并有统计学意义，而CD8^＋T细胞显著高于对照组（P〈0．01）。结论 肿瘤患者的免疫功能下降。流式细胞术对肿瘤患者的免疫功能的检测具有快速、敏感、准确的特点，对于评价疗效、判断预后具有重要价值。     

肺癌患者外周血T淋巴细胞亚群变化特点及临床意义

目的:探讨肺癌患者外周血T淋巴细胞变化及其临床意义。方法:采用流式细胞术检测法检测56例肺癌患者(其中20例早中期肺癌、36例晚期肺癌);18例健康人群外周血单个核细胞(Peripheral blood mononuclear cells,PBMC)中总T细胞(CD3+)、Th细胞(CD3+CD4+)、Tc细胞(CD3+CD8+)、NKT细胞(CD3+CD16+CD56+)、NK细胞(CD3-CD16+CD56+)、调节性T细胞(Treg,CD4+CD25+Foxp3+)占CD4+T细胞比例和CD3+γδT细胞比例。结果:56例肺癌患者总T细胞(CD3+)比例明显低于健康组(61.41%±7.88%vs71.63%±5.59%,P0.001),肺癌患者Tc细胞比例明显低于健康组(23.58%±7.18%vs28.44%±5.20%,P0.05),肺癌患者Treg比例明显高于健康组(6.20%±1.63%vs3.65%±2.00%,P0.001);肿瘤组外周血CD3+γδT细胞比例显著低于健康组(3.35%±1.41%vs5.53%±1.87%,P0.01)。但肺癌患者晚期组与肺癌早中期组比较外周血CD3+γδT细胞比例显著增高(3.70%±1.89%vs2.64%±1.41%,P0.05),肺癌患者晚期组与早中期组比较外周血Treg细胞比较显著增高(6.78%±2.64%vs5.06%±1.22%,P0.05)。肺癌患者外周血中NK细胞低于健康组(15.02%±7.61%vs18.74%±6.39%,P0.05),而Th细胞和NKT细胞都有所降低,但差异尚不具统计学意义(P0.05)。结论:肺癌患者总T细胞、Th细胞、Tc细胞和NK细胞都有所降低,Treg细胞有所上升,提示肺癌患者处于免疫抑制状态。Treg细胞和CD3+γδT细胞与肺癌的临床病程具有一定的关联性。     

Circadian changes of T lymphocyte subsets in human peripheral blood

The circadian variations in circulating T cell subsets defined by monoclonal antibodies in eight healthy male volunteers were evaluated in whole blood using a flow cytometry. In all subjects, the number of lymphocytes showed a clear rhythmicity with high values at night and low values during the day. This circadian variation in circulating lymphocytes appeared to reflect largely a change in the number of T cells rather than B cells. The percentage of OKT3+ and OKT11+ cells showed a similar fluctuation with a peak at night and a depression during the day. It was found that the percentage of OKT4+ cells varied in parallel with that of T cells, particularly of OKT3+ cells, but the OKT8+ subset was not appreciably altered over a 24 h period. Thus, a circadian variation of T cells could be largely accounted for by a circadian change of OKT4+ cells. Plasma cortisol levels showed an expected circadian variation. It was also shown that there might be an intimate relationship between these circadian changes of T cell subsets and plasma cortisol levels.     

基因修饰树突状细胞诱导前列腺癌患者外周血T细胞亚群重排的研究

目的探讨以腺相关病毒（AAV）为载体,前列腺特异性抗原（PSA）基因转染树突状细胞（DC）诱导前列腺癌患者外周血T细胞亚群变化特点及临床意义。方法抽取30例前列腺癌患者外周血,采用密度梯度离心法分离外周血单个核细胞,以rAAV/PSA感染DC前体细胞,采用系列细胞因子诱导DC前体细胞成熟。第6天收集成熟DC并与T细胞按比例混合培养,诱导细胞毒性T淋巴细胞（CTL）。分别于DC与T细胞混合培养前后应用流式细胞术分析外周血T细胞亚群及调节性T细胞（CD4^＋CD25^＋FoxP3^＋Treg）的表达水平。结果 PSA基因转染DC刺激T淋巴细胞爆发增殖,与培养前比较,混合培养6d后CD8^＋、CD8^＋CD69^＋、CD8^＋CD28^＋T细胞的比例和CD8^＋/CD4^＋比值均明显增高,差异有统计学意义（P〈0.01）;而CD8＋CD28-T细胞和Treg细胞的比例均显著降低,差异有统计学意义（P〈0.01）。CD4^＋T细胞比例较前略有升高,但差异无统计学意义（P〉0.05）。结论 PSA基因转染DC能够有效地激活CD8＋抗原特异性CTL,下调免疫抑制性T细胞,提高患者的细胞免疫功能,为前列腺癌的免疫治疗提供新的有效策略。     

类风湿性关节炎患者2H4+T淋巴细胞亚群的流式细胞仪分析

了解类风湿性关节炎患者免疫功能的异常变化，探讨其发病机制。方法，使用间接免疫荧光和直接免疫荧光方法对１１例ＲＡ患者和１２例健康人的外周血Ｔ淋巴细胞进行了研究。结果：ＲＡ患者与对照组比较ＣＤ３＾＋Ｔ细胞和ＣＤ＾＋４Ｔ细胞地明显变化，ＣＤ８＾＋Ｔ细胞明显减少，     

Alterations of T-cell subsets in primary biliary cirrhosis.

To determine whether abnormalities of immunoregulatory T cells are associated with primary biliary cirrhosis, we characterized peripheral blood mononuclear cells in 16 patients with primary biliary cirrhosis and compared them with 30 normal controls. For this analysis we used monoclonal antibodies to the surface antigens on helper/inducer (T4+) and suppressor (T8+) T cell subsets and to a common T cell antigen (T3+). In contrast to normal persons, patients with primary biliary cirrhosis had reduced percentages of T3+ cells. More importantly, there was a relative decrease in helper/inducer (T4+) cells in 9/16 patients and a decrease in suppressor (T8+) cells in 5/16 patients. Furthermore, clinical studies indicated that patients with a decreased suppressor cell population (increased T4+ : T8+ ratio) had more advanced disease, as reflected by serum bilirubin levels (P less than 0.05) and histological changes in the liver (P less than 0.001), than those patients with a reduced helper T cell population (decreased T4+ : T8+ ratio). These data suggest that abnormalities of immune responsiveness in primary biliary cirrhosis may have a more complex origin than a uniform alteration in one immunoregulatory T-cell subset and that these immunoregulatory cell changes vary according to the severity of the disease.     

Gene expression profiles of peripheral blood mononuclear cells in primary biliary cirrhosis

Previous studies on gene expression profiles in primary biliary cirrhosis (PBC) have exclusively focused on liver tissue or intrahepatic cells. Since the pathological process is systemic, other complementary studies in blood cells seemed to be reasonable. In this research, we try to explore differentially expressed genes in peripheral blood mononuclear cells (PBMCs) of PBC patients. Nine PBC patients and 9 healthy controls were recruited as Cohort 1 for a microarray study of screening. Total RNA of PBMCs from each individual was isolated and screened by oligonucleotide microarray (22 K). Then, differentially expressed genes were categorized into signaling pathways. Expression levels of three important genes, tyrosine kinase binding protein (TYROBP), C–C motif chemokine 5 (CCL5) and cathepsin L (CTSL) were confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) in a second Cohort 2 (30 PBC patients and 20 healthy controls). Results show that sixty-five genes differentially expressed in PBC were identified, 20 of which were up-regulated and 45 of which were down-regulated. Twenty-seven signaling pathways were identified. TYROBP and CCL5 were proved to be down-regulated in PBC, and CTSL was proved to be up-regulated (p < 0.05) in PBC, which were all consistent with the screening study. In conclusions, the analysis of gene expression in PBMCs of PBC and the comparison of gene profiles between PBMCs and the liver may provide new clues to the pathogenesis of the disease.     

Isolation of T lymphocyte subsets from peripheral blood using monoclonal antilymphocyte antibodies

Monoclonal antisera against helper (serum OKT4) and suppressor (serum OKT8) T cells were employed in a complement-dependent lymphocytotoxicity reaction to isolate helper and suppressor T cells from peripheral blood mononuclear cells. When prepared by this method, helper and suppressor T cells had a purity of 67.8 percent and 78.8 percent, respectively. Approximately 56 percent of viable suppressor and 33 percent of viable helper T cells were lost during the procedure.     

T cell subsets in patients with acute and chronic HBV infection,primary biliary cirrhosis and alcohol induced liver disease

The proportions of inducer and cytotoxic/suppressor T cells and their concentrations in peripheral blood have been determined in patients with acute and chronic type B hepatitis, hepatitis B virus (HBV) carriers with normal hepatic histology, patients with alcohol-induced liver disease (ALD), primary biliary cirrhosis (PBC) and chronic extrahepatic cholestasis. During acute type B hepatitis the inducer/suppressor ratio was decreased due to an increase in suppressor cell concentrations. When this ratio returned to normal the HBs antigen was cleared and HBs antibody was detectable. Similar abnormalities were found in patients with HBs + ve chronic hepatitis. In HBs antigen-positive patients with normal histology, normal T cell subsets were found.In some patients with primary biliary cirrhosis the ratio of inducer to suppressor cells was low due to a reduction in the concentration of inducer cells and in others high due to a reduction in suppressor cells. Administration of cyclosporin A to the latter group produced an increase in the concentration of suppressor cells and there was an improvement in liver biochemistry.In alcohol-induced hepatitis and cirrhosis the ratio of inducer/suppressor cells was normal.Whether these imbalances of the regulatory cells of the immune system in patients with chronic HBV-induced hepatitis and PBC are of primary or secondary importance is uncertain. The relationship of the depressed ratio to persistence of the hepatitis B virus is worthy of further study.     

2型糖尿病合并肺结核患者外周血T淋巴细胞亚群分析及临床护理对策

目的 探讨2型糖尿病合并肺结核患者外周血T淋巴细胞亚群的特点,为临床护理提供证据支持.方法 利用流式细胞术对41例2型糖尿病合并肺结核患者（DM＋ LTB组）及60例单纯肺结核患者（LTB组）的外周血淋巴细胞亚群进行检测,并以健康成年人为对照组,对检测结果进行统计学分析.结果 DM＋ LTB组和LTB组的外周血淋巴细胞亚群绝对值均显著低于正常对照组（P＜0.05）,DM＋ LTB组CD3＋CD4＋/CD3＋CD8＋比值显著高于LTB组（P＜0.05）.结论 2型糖尿病合并肺结核患者存在比较严重的细胞免疫功能紊乱.护理人员应从加强健康教育、心理及饮食等方面加强护理,提高患者的机体免疫力.     

NKT细胞、NK细胞和T细胞亚群在活动性肺结核患者外周血的表达及意义

目的:探讨活动性肺结核病人外周血CD3+T、CD3+CD4+Th、CD3+CD8+Tc、CD3-CD16+56+NK、CD3+CD16+56+NKT、Th/Tc的表达变化及其临床意义。方法:采用流式细胞术分析45例肺结核病人、16例肺部感染病人及30例健康人外周血CD3+Th、CD3+CD4+Th、CD3+CD8+Tc、CD3-CD16+56+NK、CD3+CD16+56+NKT、Th/Tc的表达和变化情况。结果:初、复治肺结核患者的外周血NKT比例明显高于健康人(P<0.05);Th低于健康人(P<0.05);Tc高于健康人(P<0.05);Th/Tc低于健康人(P<0.05)。初、复治肺结核患者之间T、Th、Tc、NK、NKT、Th/Tc未见明显差异。肺部感染、活动进展Ⅰ期、活动进展Ⅱ期病人NKT阳性细胞数高于健康人和吸收好转期病人且结果有统计学意义(P<0.05);活动进展Ⅱ期组高于肺部感染组且结果有统计学意义(P<0.05)。活动进展Ⅰ期、活动进展Ⅱ期病人Tc阳性细胞数高于健康人组且结果有统计学意义(P<0.05)。Th/Tc吸收好转期、肺部感染、活动进展期、活动进展Ⅱ期均低于健康人组且结果有统计学意义(P<0.05)。轻症、中症、重症患者,重症者Th升高、Tc下降、Th/Tc升高。结论:NKT细胞与肺结核的发展和转归有关。Tc细胞的升高是免疫系统对于结核杆菌的反应,与肺结核病变的发展、范围和程度紧密相关。重点监测外周血NKT细胞和Tc细胞对于掌握活动性肺结核发展和预后有特殊的意义。     

Peripheral blood T lymphocyte subsets in children with congenital asplenia

The aim of the current study was to examine whether a congenital lack of the spleen changes distribution, state of activation and function of peripheral lymphocyte T subsets. Seven children with congenital asplenia (CA) aged 1.5–17 years and seven age-matched controls were tested. By triple-color flow cytometry we examined: (1) the expression of CD3⁺, CD4⁺, CD8⁺, CD19⁺, and CD56⁺ on lymphocytes; (2) the distribution of CD45RA⁺ and CD45RO⁺ in CD4⁺ and CD8⁺; (3) the expression of CD27⁺ in the CD4⁺ and CD8⁺ T-cell-bearing CD45RA⁺, CD45RO⁺, or CD45RB⁺. Lymphocyte proliferative responses and cytokines production (IFN-gamma, IL-6, TNF-alfa, and IL-10) in anti-CD3-induced peripheral blood mononuclear cells were tested. The results indicate (1) a normal distribution of the basic lymphocyte subsets, (2) low CD3⁺/CD8⁺ percentage but expressing CD8^+high and non-significantly elevated CD4⁺/CD8⁺ ratio, (3) CD45RA^+high and CD27^+high in the CD4⁺ and CD8⁺ T cell, and (4) CD45RB^+high in the CD4⁺ and CD45RO^+high in the CD8⁺. The distribution of CD27⁺ in the CD45RA⁺ and CD45RO⁺ CD4⁺ T cells remained unchanged. However, the percentage of CD8⁺/CD45RO⁺/CD27⁺ T cells tended to be elevated. Altogether, these data indicate that CA is connected with (1) the presence CD4⁺ T cells expressing the “naive” phenotype (CD45RA^+high RB^+high and CD27^+high), (2) high numbers of activated CD8⁺ T cells shifted toward the memory phenotype (CD45RO^+high) but still showing high CD27⁺ expression, which may indicate failure in T CD8⁺ cytotoxic effectors differentiation, and (3) a tendency to the rather pro-inflammatory status of cells, low IL-10 expression, and suboptimal lymphocytes responses to mitogenic stimulation.     

淋巴结外恶性淋巴瘤外周血T淋巴细胞亚群与免疫指标测定分析

目的：检测原发性淋巴结外恶性淋巴瘤（Primary extranodal lymphoma，PENL）患者外周血T淋巴细胞亚群比例和免疫球蛋白（Ig）水平的变化。方法：分别应用流式细胞术（FCM）和免疫速率比浊法测定T淋巴细胞亚群比例和免疫球蛋白（Ig）以及补体C3、C4含量。结果：PENL组CD3^＋、CD4^＋和CD8^＋明显降低（P〈0．01），原发性结性恶性淋巴瘤（结性组）CD4^＋／CD8^＋比值和NK细胞比较其他两组有显著意义（P〈0．01）；提示PENL组、结性组中CD3^＋、CD4^＋和NK细胞与IgA、IgG、C3比较有显著的相关性（P〈0．01）。结论：结外恶性淋巴瘤患者免疫功能的改变，在其发病机制中的作用不容忽视，可以作为病情进展的免疫学指标。     

埃索美拉唑四联方案治疗胃溃疡的临床疗效与患者的外周血T细胞亚群水平的关联性分析

为探讨外周血T细胞亚群水平对埃索美拉唑四联方案治疗胃溃疡临床疗效的影响,选取2014年6月至2016年11月间广元市第一人民医院消化内科诊治的86例胃溃疡患者为研究对象。采用流式细胞仪测定CD4~+T细胞和CD8~+T细胞亚群的变化,以CD4~+T细胞的中位数为分界值,将患者分别分为高CD4~+T细胞组和低CD4~+T细胞组,每组43例;以CD8~+T细胞的中位数为分界值,将患者分别分为高CD8~+T细胞组和低CD8~+T细胞组,每组43例。比较埃索美拉唑四联方案治疗不同水平CD4~+T细胞和CD8~+T细胞组患者的临床疗效以及CD4~+T细胞、CD8~+T细胞水平与临床指标的相关性。结果显示,CD4~+T细胞水平与患者临床症状指标之间均成显著负相关(P0.05),而CD8~+T细胞水平与患者临床症状指标之间均成显著正相关(P0.05);低CD4~+T细胞组患者的总体有效率为79.07%,显著低于高CD4~+T细胞组的95.35%(χ2=5.108,P=0.024);而低CD8~+T细胞组患者治疗总体有效率为97.67%,明显高于高CD8~+T细胞组的81.39%,差异具有统计学意义(χ2=6.081,P=0.014);此外,高CD4~+T细胞组患者疼痛、烧灼感、恶心呕吐、嗳气和腹胀的评分值也明显低于低CD4~+T细胞组(P0.05);但CD8~+T细胞组疼痛、烧灼感、恶心呕吐、嗳气和腹胀的评分值均明显高于低CD8~+T细胞组,差异均有统计学意义(P0.05)。上述结果表明,CD4~+T细胞和CD8~+T细胞水平可影响埃索美拉唑四联方案治疗胃溃疡患者的临床疗效。     

AFP-L3阳性肝细胞癌患者肿瘤分期和外周血淋巴细胞亚群的研究

目的: 分析小扁豆凝素结合型甲胎蛋白(AFP-L3)阳性的肝细胞癌患者肿瘤分期及外周血淋巴细胞的改变,探讨AFP-L3水平与肿瘤分期及免疫状态的关系。方法:采集60例肝细胞癌患者外周血,检测AFP及AFP-L3含量。根据外周血AFP-L3比例将60例原发性肝细胞癌患者分为2组:AFP-L3(+)组和AFP-L3(-)组。流式细胞术检测外周血中各淋巴细胞亚群(NK、CD4⁺、CD8⁺、CD4/CD8、Treg)的比例。结果:AFP-L3(+)组患者UICC分期Ⅲ+Ⅳ期为29例,AFP-L3(-)组为7例;AFP-L3(+)组患者BCLC分期C+D期为20例,AFP-L3(-)组为3例。AFP-L3(+)组患者发生血管侵犯为17例,远高于AFP-L3(-)组(1例)。AFP-L3(+)组患者外周血中NK细胞比例及CD4/CD8比值下降,CD8⁺T和Treg细胞显著比例上升。结论:外周血AFP-L3阳性的肝癌患者容易发生血管侵犯,且分期较差,可能与淋巴细胞亚群发生变化、免疫系统受到抑制有关。     

T cell immunity and primary biliary cirrhosis

T lymphocytes play a pivotal role in the autoimmune response in primary biliary cirrhosis (PBC). Recent studies have shown that there is overlapping in the PDC-E2-specific T and B cell epitopes. In addition, helper T and cytotoxic T cell epitopes all contain a shared peptide sequence. In addition, recognition of exogenous antigens including bacterial antigens by autoantigen-specific T cell and the mechanism of molecular mimicry provide a clue to clarifying the pathogenesis of PBC. Furthermore, the findings that autoantigen-immune complexes cross present and also that the presentation of autoantigen is of a higher relative efficiency, define a unique role of autoantibodies in the pathogenesis of the autoimmune disease. The mechanism of immune-mediated bile duct damage in PBC, including the possible role of T cell-mediated cytotoxicity and molecular mimicry is discussed.     

Clonal analysis of liver-derived T cells of patients with primary biliary cirrhosis.

Lymphocyte infiltration in liver tissue is one important histological finding in primary biliary cirrhosis (PBC). So far, functional analyses of lymphocytes in PBC have focused on circulating lymphocytes, whereas lymphocytes at the involved site, the liver, have not been examined functionally. We have established interleukin 2 (IL-2)-dependent T lymphocyte lines (TLL) and clones (TLC) from liver biopsies of 14 patients with PBC. Phenotypic analysis using the monoclonal antibodies MT910 (CD2), MT811 (CD8), and MT151 (CD4) revealed that in nine of 14 TLL cytotoxic-suppressor T cells predominated (CD8+:52-84%; CD4+:14-48%), whereas in five of 14 TLL a preponderance of the CD4+ subpopulation was found (CD4+:56-73%; CD8+:28-45%). From 10 patients 137 TLC were generated which phenotypically correlated to the TLLs. We have tested the cytotoxic potential of seven TLL and 43 TLC in LDCC (lectin-dependent cell-mediated cytotoxicity), NK (natural killing) and ADCC (antibody-dependent cell-mediated cytotoxicity) assays. All TLL and all but one CD8+ TLC tested showed high activity in the LDCC assay, reflecting the cytolytic activity of cytotoxic T cells (CTL). CD4+ clones with LDCC activity were rarely found. NK activity and K cell activity could only be found in two clones. For the first time TLC and TLL from liver tissue of PBC patients could be generated. The high cytotoxic activity displayed by T cells derived from the liver indicates an important role for this immunological mechanism in the tissue damaging process.