Tim-3 Expression by Peripheral Natural Killer Cells and Natural Killer T Cells Increases in Patients with Lung Cancer - Reduction after Surgical Resection

Background: The purpose of this study was to investigate Tim-3 expression on peripheral CD3-CD56+natural killer (NK) cells and CD3+CD56+ natural killer T (NKT) cells in lung cancer patients. Materials andMethods: We analyzed Tim-3+CD3-CD56+ cells, Tim-3+CD3-CD56dim cells, Tim-3+CD3-CD56bright cells, and Tim-3+CD3+CD56+ cells in fresh peripheral blood from 79 lung cancer cases preoperatively and 53 healthy controlsby flow cytometry. Postoperative blood samples were also analyzed from 21 members of the lung cancer patientcohort. Results: It was showed that expression of Tim-3 was significantly increased on CD3-CD56+ cells, CD3-CD56dim cells and CD3+CD56+ cells in lung cancer patients as compared to healthy controls (p=0.03, p=0.03 andp=0.04, respectively). When analyzing Tim-3 expression with cancer progression, results revealed more elevatedTim-3 expression in CD3-CD56+ cells, CD3-CD56dim cells and CD3+CD56+ cells in cases with advanced stages(III/IV) than those with stage I and II (p=0.02, p=0.04 and p=0.01, respectively). In addition, Tim-3 expression wassignificantly reduced on after surgical resection of the primary tumor (p<0.01). Conclusions: Tim-3 expressionin natural killer cells from fresh peripheral blood may provide a useful indicator of disease progression of lungcancer. Furthermore, it was indicated that Tim-3 might be as a therapeutic target.     

流式细胞术在早期乳腺癌外周血微转移检测中的应用

目的 定量检测早期乳腺癌患者外周血的肿瘤细胞含量（ＰＢＣＣ）。方法 采用人细胞角蛋白抗体和ＣＤ４５抗体双色免疫标记外周血标本，应用多参数流式细胞仪方法检测早期乳腺癌患者手术前后外周血中肿瘤细胞含量。结果 术前ＰＢＣＣ与肿瘤大小、腋窝淋巴结状况、病理分期及脉管瘤栓均明显相关性（Ｐ〈０．０１），而术后ＰＢＣＣ与腋窝淋巴结状况、病理分期和脉管瘤栓具有相关性（Ｐ〈０．０５），而与肿瘤大小无关。结论 采用流     

食管鳞癌患者外周血及癌组织中浸润性NK细胞表面受体的表达

目的 分析食管鳞癌患者外周血及组织中NK细胞的比例及其表面受体的表达。方法 流式细胞仪检测食管鳞癌患者外周血及组织中NK细胞的比例及其表面受体的表达。结果 食管鳞癌组织中NK细胞比例明显高于癌旁5 cm以远的非肿瘤正常组织。CD16在食管鳞癌患者外周血中的表达与健康者外周血并无明显差异，但在鳞癌组织中的表达则明显低于正常组织；癌组织中NKp30、NKG2D的表达均明显低于正常组织；NKp44的表达明显高于正常组织。在同一患者体内，与外周血相比，癌组织中的CD16、NKp30、NKG2D的表达更低；而NKp44、NKG2A的表达更高。外周血与癌组织中浸润的NK细胞数量与肿瘤进展呈负相关。结论 食管鳞癌组织中NK细胞表面受体表达失衡可介导食管鳞癌细胞发生免疫逃逸。     

Selective Expansion of Human Natural Killer Cells from Peripheral Blood Mononuclear Cells by the Cell Line, HFWT

An anchorage-dependent Wilnis tumor cell line HFWT was found to stimulate selective and remarkable expansion of human natural killer (NK) cells from human peripheral blood mononuclear cells (PBMC). After PBMC of healthy donors were cultured on irradiated HFWT cells for 10–21 days, the lymphocytes expanded 58- to 401-fold. This NK cell expansion required direct contact of PBMC with live, but not fixed, HFWT cells. The PBMC from an end-stage brain tumor patient also expanded 156-fold, whereas those cultured with irradiated NK-sensitive K562 grew only 30.5-fold. CD16⁺CD56⁺ NK cells accounted for more than 70% of the population expanded on HFWT cells. No essential difference in expression of NK receptors was observed in the expanded NK cells on HFWT and K562 and without feeder cells. The expanded NK cells killed not only fresh HFWT cells but, unexpectedly, also MHC class I-expressing autologous brain tumor cells at an effector/target ratio of 4 for 24 h. These results will contribute to the development of a large-scale preparation method for human NK cells, which will aid studies of NK cell biology and possible treatment of brain tumors.     

CD133 mRNA在胃癌患者外周血单核细胞中的表达及意义

[目的]通过检测胃癌患者外周血单核细胞中CD133的表达,探讨其与临床病理特征的关系。[方法]于术前抽取30例胃癌患者、15例胃溃疡患者、15名健康志愿者外周静脉血,密度梯度离心法分离单核细胞。半定量反转录聚合酶链反应检测CD133 mRNA表达水平。分析胃癌患者外周血CD133 mRNA表达与临床病理特征的关系。[结果]胃癌患者外周血中CD133mRNA表达水平明显高于胃溃疡患者和健康志愿者（0.2804±0.1835 vs 0.0984±0.1321,t=6.724,P〈0.001;0.2804±0.1835 vs 0.0282±0.0597,t=-7.327,P〈0.001）。CD133 mRNA表达与肿瘤最大直径、淋巴结转移、TNM分期有关（P〈0.05）,而与性别、分化程度、淋巴管浸润、血管浸润、浸润深度无关。Spearman相关分析显示,CD133 mRNA表达与淋巴结转移呈正相关（P〈0.01）。[结论]胃癌患者外周血中CD133 mRNA的表达与胃癌的发展、转移及预后密切相关。     

Analysis of the Expression of Surface Receptors on NK Cells and NKG2D on Immunocytes in Peripheral Blood of Patients with Nasopharyngeal Carcinoma

Background: The aberrant expression of surface receptors on immunocytes may represent potential markers of tumorescape for nasopharyngeal carcinoma (NPC). The aim of this study was to investigate the expression of representativereceptors on natural killer (NK) cells and NK group 2, member D (NKG2D) on immunocytes in the peripheral bloodof patients with NPC. Methods: Patients (n = 64) with NPC prior to initiation of treatment were defined as the studygroup. Healthy volunteers (n = 31) served as the control group. The expression of NK cells and NKT cells; the triggeringreceptors NKp30, NKp44, and NKp46 on NK cells; the activating receptor NKG2D on NK cells, CD4+ T cells, andCD8+ T cells; and the inhibitory receptors CD158b and CD159a on NK cells were analyzed by flow cytometry in thetwo groups. Results: Here, our study showed that no differences were observed in terms of the numbers of NK cells orNKT cells, or the expression of CD158b and CD159a on the surface of NK cells between the two groups. Nevertheless,the expression levels of NKp30 and NKp46 on NK cells in the NPC patients were significantly lower than in the healthyindividuals (P < 0.05). No differences existed in the expression of NKG2D on NK cells, but NKG2D on CD8+ T cellsshowed a markedly lower expression in the study group (P < 0.001). Conclusions: Our findings may reflect a possiblemechanism of immune evasion for NPC. The enhancement of immunotherapy concerning NKp30, NKp46, and NKG2Dmay be an innovative treatment strategy for patients with NPC.     

宫颈癌患者外周血树突状细胞的体外扩增和鉴定

 目的 探索体外扩增宫颈癌患者外周血来源的成熟树突状细胞(denelritic cell，DC)的方法，并鉴定DC的形态、结构、表型及生物学活性。方法 淋巴细胞分离液分离宫颈癌患者外周血，得到DC前体细胞(PBMC)，以(3M-CSF、IL-4、TNF-α诱导培养。用光镜和透射电镜观察形态结构，流式细胞仪检测细胞表面特异性分子，MTT法测定DC刺激T细胞增殖的活性。结果 联合应用细胞因子可诱导扩增出源自宫颈癌患者外周血的成熟DC，具有典型形态特征，较高表达HLA-DR、CD1a、CD80，能强烈激发同种异体T细胞增殖反应。结论 宫颈癌患者外周血体外培养可成功获得成熟的DC，为开展宫颈癌临床免疫治疗奠定了基础。     

肺癌患者外周血细胞CD_(25)、CD_4表达和Apo水平的检测

目的 :研究肺癌患者外周血细胞CD2 5、CD4表达规律和Apo水平。方法 :采用流式细胞术对10 1例肺癌患者外周血细胞CD2 5 、CD3 CD4 表达率和Apo水平进行了检测。并分析了肿瘤转移状况和化疗对上述 3项指标的影响。结果 :肺癌患者外周血细胞CD2 5 和CD3 CD4 率均显著低于正常对照组 (P <0 0 1和P <0 0 5 ) ,Apo显著高于正常对照组 (P <0 0 1)。化疗者的 3项指标均显著高于未化疗者 (P <0 0 1,P <0 0 5和P <0 0 5 ) ;在未化疗患者中 ,有转移者和无转移者之间的CD2 5和CD3 CD4表达率差异均无显著性 (P >0 0 5 ) ,而转移者的Apo却显著高于无转移者 (P <0 0 1) ;在化疗患者中 ,转移者的Apo表达率显著高于无转移者 (P <0 0 1) ,CD2 5 表达率低于无转移者 (P <0 0 5 ) ,而CD3 CD4 二者差异却无显著性 (P >0 0 5 )。结论 :肺癌能抑制患者外周血细胞CD2 5 和CD3 CD4 细胞进一步降低 ,但却引起Apo明显升高。化疗可以提高肿瘤患者CD2 5 、CD3 CD4 和Apo的检出率 ,使机体免疫功能能有所恢复     

CIK治疗后肿瘤患者外周血T淋巴细胞亚群和NK细胞变化研究

目的:探讨CIK(CellInductionKiller)治疗后肿瘤患者的免疫功能状态。方法:采用流式细胞术检测20例肿瘤患者外周血T淋巴细胞亚群和NK细胞。结果:肿瘤患者CD3+、CD4+、CD3-/CD16+56+细胞和CD4/CD8比值明显低于对照组并呈显著性差异,而CD8+细胞高于对照组(P<0.01)。CIK生物治疗后CD3+、CD4+、CD3-/CD16+56+细胞和CD4/CD8比值都升高,其中CD4+、CD3-/CD16+56+细胞与治疗前比较呈显著性差异(P<0.05),而CD8+细胞与治疗前比较降低(P<0.05)。结论:肿瘤患者的免疫功能低下,CIK生物治疗能够提高机体的免疫功能。流式细胞术对患者免疫功能的监测,具有快速、灵敏、准确的特点,对于评价疗效、判断预后具有重要价值。     

Granulocyte-Macrophage Colony-stimulating Factor Augments Lymphokine-activated Killer Activity from Pleural Cavity Mononuclear Cells of Lung Cancer Patients without Malignant Effusion

The role of recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) in augmentation of lymphokine-activated killer (LAK) cell induction by interleukin-2 (IL-2) from pleural cavity mononuclear cells (PCMNCs) was examined in sixteen patients with resectable primary lung cancer not associated with malignant effusion. None of the patients had received any anticancer therapy prior to this study. Incubation of PCMNCs of patients without malignant effusion with GM-CSF for 4 days in the presence of IL-2 resulted in a significant increase in LAK activity against natural killer-resistant Daudi cells. This result was obtained by using the 4 h ⁵¹Cr-release assay. PCMNCs and blood mononuclear cells (BMNCs) were harvested simultaneously from pleural cavity lavage fluid and peripheral blood in lung cancer patients. The LAK activity developed from PCMNCs and BMNCs following incubation with IL-2 for 4 days, but the LAK activity from PCMNCs was significantly lower than that from BMNCs ( P < 0.05). Incubation of PCMNCs with GM-CSF augmented the LAK activity from PCMNCs to a level as high as that from BMNCs. These results suggest that the combined use of GM-CSF with IL-2 may result in augmentation of LAK activity developed from PCMNCs of lung cancer patients without malignant effusion.     

外周血人类斯钙素-1基因表达与乳腺癌预后的关系

目的探讨人类斯钙素-1(hSTC-1)基因在乳腺癌患者外周血中的表达与乳腺癌预后的关系。方法采用RT-PCR方法检测乳腺癌患者癌组织及外周血中hSTC-1mRNA的表达。结果30例乳腺癌患者癌组织hSTC-1mRNA阳性率为93.3%,51例乳腺癌患者外周血中hSTC-1mRNA的阳性率为37.3%,血液中的hSTC-1mRNA的表达与多个临床病理因子有显著相关性(P<0.05),包括肿瘤大小、淋巴结转移、TNM分期;而非肿瘤女性患者外周血检测不到hSTC-1mRNA。结论乳腺癌患者外周血中的hSTC-1有望成为判断患者预后的指标。     

乳腺癌患者外周血、淋巴结、癌组织中免疫细胞构成分析

目的分析乳腺癌患者外周血、淋巴结、癌组织免疫细胞构成。方法20例健康志愿者及40例乳腺癌患者外周血、18例乳腺癌患者反应性增生淋巴结及40例乳腺癌肿瘤组织细胞悬液用于流式细胞术检测,分析其单个核细胞群中CD₃⁺、CD₄⁺、CD₈⁺、CD₁₉⁺、CD₅₆⁺细胞百分比及CD₃₄^-Lin^- HLA-DR⁺细胞群中细胞CD_11c⁺ CD₁₂₃^- (pDC₁）、CD_11c^-CD₁₂₃⁺( pDC₂）百分比,计算 CD₄/CD₈比值以及DC₁/DC₂比值。结果乳腺癌患者外周血CD₃⁺、CD₄⁺、CD₈⁺、CD₁₉⁺、CD₅₆⁺细胞百分比、CD₄/CD₈比值与对照组比较,差异均无统计学意义（P>0.05）,pDC₁/pDC₂比值显著低于对照组（P<0.05）;反应性增生淋巴结组织亚群构成为：CD₃⁺（54.5±6.8）%、CD₄⁺（37.8±4.9）%、CD₈⁺（13.1±5.5）%、CD₁₉⁺（5.9±3.4）%、CD₅₆⁺（1.9±0.4）%、CD₄/CD₈为（3.6±1.3）,DC₁/DC₂为（3.6±1.3）%。无法取得正常组织中的值,未作对照分析。瘤组织中CD₃⁺（62.4±17.5）%、CD₄⁺ （32.0±8.2）%、CD₈⁺（26.9±10.5）%、CD₄/CD₈（1.3±0.4）、CD₁₉⁺（2.2±2.0）%、CD₅₆⁺（2.3±1.6）%,DC数不足以分析。结论乳腺癌患者外周血、淋巴结、肿瘤组织中免疫细胞构成,与对照组比较表现为抗肿瘤特征,循环中pDC₁/pDC₂比值减低可能与DC₁在引流淋巴结中分布增加有关。     

氩氦冻融的肺癌细胞对人外周血单个核细胞免疫功能的影响

[目的]观察氩氦冻融的小细胞肺癌细胞NCI-H446对正常人外周血单个核细胞免疫功能的影响。[方法]分离正常人外周血单个核细胞,与氩氦冻融的小细胞肺癌细胞NCI-H446混合培养,设一个空白对照组,培养过程中隔天加入IL-210U,分别在第3、7、14d检测培养上清液中IL-12、IL-10含量及NK细胞、CTL细胞的杀伤活性。[结果]氩氦冻融的肺癌细胞NCI-H446与PBMC混合培养后,第3、7d时,培养上清液中IL-12含量分别为71.8±19.1pg/ml,106.2±13.6pg/ml,对照组IL-12含量为38.5±8.4pg/ml,40.8±8.3pg/ml,试验组较对照组明显升高(P<0.05),在第14d时试验组与对照组IL-12表达相当。IL-10的表达在试验组与对照组之间始终未发现明显差异。试验组NK细胞、CTL细胞的杀伤活性在14d内均明显高于对照组(P<0.05)。[结论]氩氦冻融的肺癌细胞可以上调PBMC对IL-12的表达,而对IL-10的表达无影响,同时可明显增强NK细胞和CTL活性。     

巢式RT-PCR检胃癌患者外周血循环癌细胞的研究

目的　分析胃癌患者外周血中CK 2 0mRNA及CEAmRNA的异常表达及意义。方法　采用RT PCR方法检测 5 2例胃癌患者术前外周血中CK 2 0mRNA及CEAmRNA的表达。结果　胃癌患者外周血中CK 2 0mRNA、CEAmRNA阳性表达率分别为 42 .3 % (2 2 /5 2 )及 5 1.9% (2 7/5 2 ) ,CK 2 0mRNA和CEAmRNA两者中至少 1个基因为阳性的表达率为 63 .5 % (3 3 /5 2 )。胃癌患者外周血中CK 2 0mRNA表达与胃癌的远处转移有关 ,CEAmRNA表达、CK 2 0mRNA和CEAmRNA都表达、CK 2 0mR NA和CEAmRNA两者中至少 1个表达与胃癌的浸润深度、淋巴结转移和远处转移相关 (P <0 .0 5 )。结论　胃癌患者外周血CK 2 0和CEA的RT PCR检测有助于患者预后的判断 ;联合检测CK 2 0mRNA和CEAmRNA可增加外周血循环癌细胞检测的敏感性     

非小细胞肺癌患者外周血淋巴细胞中CD44表达的临床研究

目的 探讨非小细胞肺癌患者外周血淋巴细胞中CD44表达与其临床病理特征的关系。方法 应用流式细胞术对50例肺癌患者外周血淋巴细胞中CD44表达进行荧光免疫检测,通过自身对照分析治疗前后的变化,并与正常对照组（n=20）及肺部良性病变组（n=25）进行对比研究。结果 CD44在3组中均有不同程度的表达。50例肺癌患者外周血淋巴细胞中CD44表达明显高于正常对照组及良性病变组（P＜0.01）。良性病变组和正常对照组CD44的表达无显著性差异（P＞0.05）。手术前外周血淋巴细胞中CD44表达显著高于手术后（P＜0.01）;肺癌淋巴结转移者CD44表达高于不伴淋巴结转移者（P＜0.01）;Ⅲ、Ⅳ期和Ⅰ-Ⅱ期之间CD44的表达有非常显著性差异（P＜0.01）;肺癌组织学分级与CD44表达有明显相关性（P＜0.05或0.01）;肺癌患者外周血中CD44的表达与组织学类型无相关性。结论 应用流式细胞仪检测CD44的表达水平可作为判断肺癌负荷状况、转移及预后的指标。     

恶性肿瘤患者外周血细胞TNF-α和IFN-γ检测的临床意义

目的　探讨恶性肿瘤患者外周血细胞中TNF -α和IFN -γ的表达规律。方法　用流式细胞仪对恶性肿瘤患者外周血细胞的TNF -α和IFN -γ的表达率进行了检测和分析。结果　恶性肿瘤患者 (未化疗者 )外周血细胞的TNF -α和IFN -γ的表达率和正常对照组相比无显著性差异 (P >0 0 5 )。肿瘤化疗者的外周血细胞的TNF -α和IFN -γ表达率显著高于尚未化疗者 (P <0 0 1)。肿瘤转移者血细胞的TNF -α和IFN -γ的表达率均显著低于肿瘤未转移者 (P <0 0 1)。结论　恶性肿瘤未能引起患者外周血细胞中细胞因子含量的明显变化 ;化疗可导致患者血细胞的细胞因子表达水平明显升高 ;肿瘤转移能使患者血细胞的细胞因子表达水平明显降低。这为肿瘤化疗机制提供了新的解释 ,为判断肿瘤恶性度和预后提供了新的指标     

癌抗原激活红细胞调控白细胞CXCR4分子表达与意义

[目的]采用自然和分离实验体系研究大肠癌患者红细胞调控白细胞免疫反应。[方法]癌细胞加入全血细胞悬液(或白细胞悬液)和枸橼酸抗凝新鲜血浆,放37℃作用1h后,观察白细胞CXCR4表达和血清蛋白水平。[结果]癌细胞加入全血细胞和血浆组粒细胞CXCR4表达值(64.14±17.95)明显高于癌细胞加入白细胞和血浆组(41.57±1.58)(P<0.05),全血细胞和血浆组血清蛋白水平(20.63±1.41)明显低于白细胞和血浆组(24.25±1.58)(P<0.05)。大肠癌患者组白细胞CXCR4低于正常人。[结论]红细胞是白细胞CXCR4和分泌免疫球蛋白反应的调节器。大肠癌患者红细胞增强白细胞CXCR4表达能力明显下降。     

乳腺癌患者外周血T淋巴细胞AgNOR活性表达及其临床意义

目的:探讨乳腺癌患者外周血T淋巴细胞AgNOR的活性表达及其与临床病理因素的相关性.方法:利用KL型图像分析系统检测80例乳腺癌患者,40例乳腺纤维腺瘤患者,25例乳腺炎症患者和75例健康人外周血T淋巴细胞AgNOR的活性表达(T淋巴细胞核仁银染面积与细胞核面积比值,即I.S%),并与乳腺癌患者临床病理因素进行相关性分析.双抗体夹心法检测乳腺癌患者CA153和CEA.结果:发现乳腺癌患者外周血T淋巴细胞AgNOR的活性表达(5.88±6.05%)明显低于健康人(7.07±0.81%)、乳腺纤维腺瘤患者(6.58±0.72%)和乳腺炎症患者(9.58±0.68%)(P=0.000);乳腺炎症患者AgNOR的活性表达明显高于健康人、乳腺纤维腺瘤和乳腺癌患者(P=0.000);健康人与乳腺纤维腺瘤患者之间比较无显著性差异(P＞0.05).乳腺癌患者AgNOR阳性率(65.00%)明显优于CA153(17.50%)和CEA(12.50%)的阳性率(P=0.000).AgNOR表达与乳腺癌临床分期有关(P＜0.05),而与年龄、肿瘤大小、淋巴结转移状况无相关性(P＞0.05).结论:AgNOR阳性表达可作为评价乳腺癌发生、发展的客观生物学指标;在乳腺癌诊断、鉴别诊断和病情监测等方面有重要意义.