活动期结核病患者外周血NK细胞免疫反应特征分析

为了探讨自然杀伤(nature killer,NK)细胞在抗结核免疫应答中的特征,我们表达并纯化结核分枝杆菌特异性抗原蛋白ESAT-6,体外刺激结核病患者和正常人外周血单个核细胞(PBMC),采用流式细胞术分析不同亚群NK细胞IFN-γ释放,并与T细胞释放IFN-γ水平进行相关性分析。结果显示,经结核特异性抗原ESAT-6刺激后,结核患者来源的PBMC中有2.80%±0.65%的NK细胞可以释放IFN-γ,而正常人NK细胞释放的比例仅为0.09%±0.01%,两者间有显著差异(P0.001);同时CD56highCD16dim NK亚群细胞分泌IFN-γ的水平(6.50%±0.94%)显著高于CD56dimCD16high NK亚群细胞(1.78%±0.38%);比较同一抗原刺激条件下T细胞释放IFN-γ的能力,显示NK细胞释放IFN-γ的能力高于CD4+T和CD8+T细胞,并与释放IFN-γ的CD4+T细胞的比例呈显著正相关。上述研究结果表明,NK细胞在结核特异性抗原ESAT-6刺激下可通过分泌高水平的IFN-γ协同CD4+T细胞在抗结核感染中发挥重要作用,增强NK细胞的功能,以提高结核病患者的免疫应答水平,将为结核病的治疗提供新的策略。     

卡介苗(BCG)通过诱导IL-12产生和受体表达促进人NK细胞功能

目的:研究卡介苗(BCG)对人自然杀伤细胞(NK)功能的作用及其机制.方法:分离抗结核抗体阴性志愿者外周血PBMC、纯化NK细胞, 分别与BCG、 IL-12、 BCG+IL-12、 BCG+抗IL-12Rβ1 mAb(2B10)培养.利用ELISA方法检测培养上清液IFN-γ、 IL-12p40含量;利用ELISpot方法检测IFN-γ、颗粒酶B产生细胞的频率;利用四甲基偶氮唑盐(MTT)比色法测定杀伤功能.利用流式细胞术检测NK细胞IL-12Rβ1的表达.结果:BCG呈剂量依赖的方式诱导PBMC产生IFN-γ.在BCG刺激条件下, PBMC颗粒酶B分泌细胞数明显高于不加任何刺激剂组(P<0.05).BCG增强PBMC杀伤活性.BCG不能诱导纯化NK细胞产生IFN-γ, 但与IL-12同时刺激则表现出协同作用.纯化NK细胞经BCG刺激后杀伤活性与未刺激相比差异无统计学意义.BCG呈剂量依赖方式诱导PBMC产生IL-12、并促进NK细胞不同亚群表达IL-12Rβ1.2B10抗体抑制BCG对PBMC产生IFN-γ和分泌颗粒酶B的诱导作用.结论:BCG间接地促进NK细胞的生物学活性, 其部分机制是通过诱导单核细胞产生内源性IL-12、并上调NK细胞表达IL-12R.     

子痫前期小鼠动物模型的NK细胞和细胞因子的变化

目的:探讨磷脂酰丝氨酸(PS)/磷脂酰胆碱(PC)诱导的先兆子痫模型小鼠中自然杀伤(NK)细胞在外周血、脾脏及子宫的数量及其表面受体的变化,同时了解外周血中的细胞因子(TGF-β、TNF-α和IFN-γ)的水平.方法:用磷脂酰丝氨酸(PS)/磷脂酰胆碱(PC)诱导小鼠出现先兆子痫(PE)样症状,流式细胞仪检测自然杀伤(NK)细胞在外周血、脾脏及子宫的数量和表面受体,酶联免疫法检测外周血中TGF-β、TNF-α和IFN-γ.结果:我们建立的PE样小鼠模型是成功的;与对照组相比,gd17.5时PS/PC组外周血和脾脏中NK细胞数目增加.gd11.5时子宫中NK细胞数目突然增加,随后逐渐降低,且在gd14.5和gd17.5时PS/PC组比对照组NK细胞数目明显减少.PS/PC组脾脏中表面受体CD122表达显著降低,而CD244、CD94及NKG2D均显著增加;子宫中所有表面受体显著降低.在gd11.5、gd14.5及gd17.5时,与对照组相比,PS/PC组外周血中TGF-β的表达水平显著增加;而TNF-α和IFN-γ表达的水平大多不发生明显变化.结论:PE可能与母胎界面处NK细胞的数量及表面受体改变有关.外周血中TGF-β的增加可能抑制子宫处NKG2D等NK细胞表面受体的表达.关于TNF-α和IFN-γ在PE样小鼠模型中的作用还需进一步研究.     

模拟失重对IL-2诱导的人NK细胞生物活性的影响

目的明确模拟失重对白细胞介素2(IL-2)诱导的人自然杀伤(NK)细胞活性的影响。方法回转细胞模拟失重条件培养人NK细胞,CCK-8法检测NK细胞增殖活性、异硫氰酸荧光素标记的膜联素Ⅴ/碘化丙啶(annexinⅤ-FITC/PI)双标记结合流式细胞术检测细胞凋亡率、ELISA检测γ干扰素(IFN-γ)产生水平,反转录PCR检测NK细胞IL-12β1和IL-12β2受体mRNA水平。结果与正常重力组相比,模拟失重培养24 h和48 h后,IL-2诱导的NK细胞增殖水平分别下降13.6%和31%,凋亡率分别增加8%和19%,IFN-γ分泌水平分别降低25.2%和47.8%,杀伤活性分别下降7%和18%,IL-12刺激IL-2预处理NK细胞产生IFN-γ的水平分别降低21.8%和58.8%,同时,NK细胞IL-12β1和IL-12β2受体mRNA水平显著降低。结论模拟失重抑制IL-2诱导的NK细胞增殖、IFN-γ产生和杀伤活性,并进一步通过下调IL-12受体mRNA表达抑制IL-12诱导的IFN-γ产生。     

慢性HBV 感染者IFN-γ+ NK 细胞与Treg 及IL-10 的关系研究

目的:探讨慢性HBV感染者外周血自然杀伤(NK)细胞分泌的IFN-γ与CD4~+CD25~+Foxp3~+调节性T细胞(Treg)及IL-10等细胞因子含量的关系。方法:收集119例慢性HBV感染肝病患者,其中CHB患者57例,LC患者40例,HCC患者22例,另收集同期健康志愿者(HC) 27例。流式细胞仪检测外周血Treg、NK细胞及IFN-γ~+NK细胞比例,CBA细胞因子(IL-10、IFN-γ、IL-2、IL-4、TNF及IL-5)含量,全自动生化分析仪检测血清ALT、AST水平。结果:CHB、LC、HCC患者外周血Treg百分比和血清中IL-10水平均显著高于HC组, CHB、HCC患者外周血Treg绝对数值显著高于HC组(P0.05),CHB、LC及HCC患者Treg与IL-10呈正相关(P0.01,r=0.564;P0.05,r=0.426;P0.05,r=0.476)。NK细胞绝对数、IFN-γ~+ NK细胞百分比及血清中IFN-γ含量在CHB、LC、HCC组中均显著低于HC组(P0.01)。同时CHB患者组IFN-γ~+NK细胞比例与Treg细胞比例、血清IL-10水平均负相关(P0.05,r=-0.618;P0.05,r=-0.635),血清IFN-γ水平亦与Treg负相关(P0.01,r=-0.342);LC患者中,外周血中NK细胞绝对数值与Treg细胞比例负相关(P0.05,r=-0.317),IFN-γ~+NK细胞比例与血清中IL-10水平负相关(P0.05,r=-0.514);HCC患者组IFN-γ~+NK细胞比例与Treg细胞比例、血清IL-10水平均负相关(P0.01,r=-0.676;P0.01,r=-0.615)。结论:CHB患者及其进展疾病如LC和HCC外周血IFN-γ~+NK细胞比例与Treg比例、血清IL-10水平负相关,提示Treg和IL-10参与抑制IFN-γ~+ NK细胞可能是慢性HBV感染及其进展的重要因素之一,因此逆转Treg及IL-10所致的免疫耐受,将会有力提高NK细胞抗HBV慢性感染及恶性进展。     

NK细胞和NKT细胞在慢性乙型肝炎中的表型和功能特征

目的检测慢性乙型肝炎(CHB)患者外周血自然杀伤(NK)细胞、自然杀伤T(NKT)细胞的比例、NKG2D/NKG2A水平和γ干扰素(IFN-γ)、肿瘤坏死因子α(TNF-α)的水平。方法采集并分离CHB患者外周血单个核细胞(PBMC),流式细胞术检测NK、NKT细胞占PBMC的比例,NKG2D、NKG2A水平,经佛波酯(PMA)、布雷菲德菌素A(BFA)、离子霉素(ionomycin)处理后,流式细包术检测IFN-γ、TNF-α的水平,分析各指标与乙肝病毒载量和血清丙氨酸氨基转移酶的关系,采用独立样本t检验与Pearson相关性分析。结果与正常对照者相比,CHB患者外周血NK细胞和NKT细胞比例明显降低;NKG2A阳性的NK细胞和NKT细胞明显升高;经PMA、BFA、ionomycin刺激后,IFN-γ阳性的NK细胞和NKT细胞明显降低。结论 CHB患者NK、NKT细胞存在不同程度的减少、受体表达失调和细胞因子分泌功能下降等功能紊乱。     

肝星状细胞通过活化γδ T细胞抑制人HepG2肝癌细胞增殖和侵袭北大核心CSCD

目的研究人肝星状细胞(HSC)作用于γδ T细胞进而对肝癌细胞增殖、侵袭能力产生的影响。方法利用密度梯度离心法及流式细胞术分离获取健康志愿者与肝癌患者的外周血γδ T细胞,使用TranswellTM小室共培养LX-2细胞和γδ T细胞。ELISA检测γδ T细胞上清液中γ干扰素(IFN-γ)含量,CCK-8法检测HepG2的增殖能力,Transwell^(TM)侵袭实验检测HepG2细胞侵袭能力。结果与LX-2细胞共培养后,γδ T细胞分泌IFN-γ量增加,健康志愿者组IFN-γ分泌量增加比肝癌患者组更明显。共培养后γδ T细胞对HepG2细胞增殖、侵袭的抑制能力增强,且健康志愿者组抑制效果增强较肝癌患者组更明显。结论 HSC可以活化γδ T细胞、促进其分泌IFN-γ,进而增强γδ T细胞抑制肝癌细胞增殖和侵袭的能力。     

大蒜素对大鼠NK细胞肿瘤杀伤活性的影响

目的: 研究大蒜素对体外培养的大鼠NK细胞肿瘤杀伤活性的影响,并初步分析其可能机制。方法: 免疫磁珠分选法分离大鼠脾脏NK细胞,流式细胞仪检测不同浓度的大蒜素对NK细胞的增殖和凋亡的影响,ELISA检测大鼠脾脏NK细胞IFN-γ的分泌水平,乳酸脱氢酶(LDH)法检测大鼠脾脏NK细胞对小鼠淋巴瘤Yac-1细胞的杀伤活性。结果: 大蒜素对体外培养的NK细胞有明显的促进增殖和抑制NK细胞自然凋亡的作用,并能提高NK细胞分泌IFN-γ的水平和增强对Yac-1细胞的杀伤毒性,且在一定的浓度范围内呈剂量依赖性,其中30 mg/L是大蒜素的相对适宜浓度。结论: 大蒜素可能通过上调NK细胞分泌IFN-γ的水平,增强其体外肿瘤杀伤能力。     

阻断KIR2DL4(CD158d)增强NK细胞的杀伤功能

目的利用抗体阻断人原代培养的自然杀伤(NK)细胞表面的杀伤细胞免疫球蛋白样受体2DL4(KIR2DL4),抑制其与配体人白细胞抗原G(HLA-G)的结合,观察对NK细胞杀伤功能的影响。方法用免疫磁珠法分离人NK细胞并培养,流式细胞术检测所得NK细胞的纯度;流式细胞术检测NK细胞表面KIR2DL4的表达水平和人乳腺癌SK-BR-3细胞表面HLA-G的表达水平;将NK细胞与SK-BR-3细胞共培养,并加入KIR2DL4的阻断抗体,ELISA检测NK细胞分泌γ干扰素(IFN-γ)的水平,利用流式细胞术检测NK细胞表面CD107a的表达水平,以检测其脱颗粒情况。结果分离得到的人原代NK细胞纯度可达90%以上;共培养实验发现,KIR2DL4的阻断抗体可促进NK细胞分泌IFN-γ,且NK细胞表面CD107a的表达水平也明显提高,提示其脱颗粒能力增强。结论阻断KIR2DL4信号可明显促进NK细胞的杀伤功能,KIR2DL4在NK细胞杀伤靶细胞的过程中发挥抑制性作用。     

Foxp3转染小鼠CD4+CD25-T细胞抑制NK细胞活性

目的： 通过逆转录病毒载体转染Foxp3基因到小鼠CD4+CD25-T细胞,以研究体外诱导获得的调节性T细胞对NK细胞免疫活性的调节作用及其机制。方法： 携带Foxp3基因的逆转录病毒转染初始CD4+CD25-T细胞,以获得持续性高表达Foxp3的CD4+ T细胞模型。CD4+Foxp3+ T细胞与NK细胞共培养后,用［51Cr］标记的YAC-1细胞检测NK细胞的杀伤毒性。在TGF-β阻断实验中,通过Transwell共培养实验以及向细胞共培养体系加入抗TGF-β抗体,并检测NK细胞杀伤毒性。结果：逆转录病毒转染初始CD4+CD25-T细胞,成功建立了表达Foxp3的CD4+T细胞模型,转染后1周Foxp3阳性表达的T细胞比例为38.0%。CD4+Foxp3+ T细胞在与NK细胞共培养的24 h和48 h后,对NK细胞的细胞毒性杀伤效应的抑制率分别为42.9%和22.7%。在CD4+Foxp3+ T细胞与NK细胞的共培养体系中加入抗TGF-β抗体后,其抑制率分别由原来的42.9%(24 h)、22.7%(48 h)变为3.2%(24 h)、2.1%(48 h)。在Transwell共培养实验中与NK细胞直接接触的Foxp3+CD4+T细胞可以诱导NK细胞免疫抑制,而没有直接接触的Foxp3+CD4+T细胞则不能抑制NK细胞的杀伤作用。结论：强制性表达Foxp3的CD4+CD25-T细胞可以在体外发挥免疫抑制作用,可以抑制NK细胞的细胞毒性杀伤作用。转染 Foxp3的CD4+CD25-T细胞对NK细胞发挥作用依赖于细胞之间的直接接触,与转染后T细胞表面表达TGF-β有关。     

Clear cell basal cell carcinoma

We describe a case of clear cell basal cell carcinoma of the superficial type, presenting as a crusted eruption on the abdomen. Histological examination showed a solid proliferation of clear cells attached to the under-surface of an atrophied epidermis. In addition, distinct pagetoid infiltration was seen within the overlying epidermis. A focal connection between the clear cell portion and a deeper lying nodular basal cell carcinoma was demonstrated, elucidating the true nature of the lesion. Immunohistochemical studies and electronmicroscopy confirmed the epithelial derivation of the tumour. The clear cell appearance was due to multiple cytoplasmic electronlucent vacuoles which were not surrounded by membranes.     

Absolute dependence of T cell receptorhi cell generation and relative dependence of T cell receptorint cell generation on the thymus

Recent evidence indicates that conventional T cells are generated by the mainstream of T cell differentiation in the thymus and acquire a high density of T cell receptor expression (i.e. TCR^hi). In contrast, primordial T cells (or NK1.1⁺ T cells) are generated by the extrathymic pathways or an alternative intrathymic pathway and express an intermediate density of TCR (i.e. TCR^int). To obtain further evidence, it was examined how thymus grafting influenced the distribution of T cell populations in athymic nude mice. When BALB/c nu/nu mice were engrafted with thymocyte-depleted BALB/c+/+ fetal thymi, two changes emerged after grafting: nude mice generated TCR^hi cells de novo in the periphery as well as in the grafted thymi, and the absolute number of interleukin-2 receptor β chain⁺ TCR^int cells increased prominently in number in the periphery. Among thymic hormones tested, the administration of thymosin α induced a slight expansion of CD3^int cells in nude mice. To examine a possible interaction of TCR^int cells with TCR^hi cells in the periphery, B6 nu/nu mice (Ly5.2⁺) were injected with TCR^hi cells purified from the spleen of B6 Ly5.1 congenic mice. In this case, TCR^int (Ly5.2⁺) cells expanded well in all tested organs of nude mice. These results suggest that the generation of TCR^hi cells is absolutely dependent on the thymus and that TCR^int cells expand under the influence of the thymus (humoral) and due to interaction with thymus-derived conventional T cells.     

树突状细胞对调节性T细胞的调控作用

树突状细胞(DCs)是目前已知的体内功能最强大的专职性抗原提呈细胞,具有启动免疫应答和诱导免疫耐受的双重特性.近年来树突状细胞对调节性T细胞的调控作用是免疫学领域的一个研究热点.Foxp3+ Tregs是一群同时具有免疫低反应性和免疫抑制性功能两大特征的T淋巴细胞,它在维持机体内环境稳定、预防自身免疫性疾病、抑制移植排斥反应等病理生理过程中发挥着重要作用.越来越多的研究结果证实DCs和Tregs二者在维持外周免疫耐受中存在着紧密联系,DCs可以诱导抗原特异性Tregs的生成并增加后者的抑制活性,其中参与该调节机制的分子主要包括相关细胞因子、Toll样受体、共刺激分子及维甲酸等.对DCs在接触共生和致病微生物时诱导和调控Tregs细胞有了一些新发现.     

具有透明细胞乳头状肾细胞癌形态特征的透明细胞性肾细胞癌的临床病理特征

目的探讨具有透明细胞乳头状肾细胞癌形态特征的透明细胞性肾细胞癌(clear cell papillary renal cell carcinoma-like clear cell renal carcinoma, CCPRCC-like CCRCC)的临床和病理特征, 旨在提高对该类肿瘤的认识。方法回顾性分析浙江大学医学院附属第一医院诊断的3例CCPRCC-like CCRCC的临床资料、组织学形态及免疫表型, 随访患者预后并复习相关文献。结果男性2例, 女性1例, 平均年龄43岁。肿块最大径9 cm。镜下观察3例病例均包含透明细胞乳头状肾细胞癌样(CCPRCC-like)及透明细胞性肾细胞癌样(CCRCC-like)两种区域, 前者在整个瘤体所占比例为20%～90%。CCPRCC-like区域含有囊、乳头、腺管状结构。细胞核远离基底膜, 细胞核WHO/国际泌尿病理协会(ISUP)分级1级。在CCPRCC-like区域, 细胞角蛋白(CK)7表达范围10%～80%, 中等及以上强度表达。CD10表达均为100%, 主要为顶端胞膜表达。在CCRCC-like区域, 1例CK7阴性, 其余...     

梭形细胞间变性大细胞淋巴瘤

1.病例简介:患者男,40岁.2个月前自觉右腹股沟区疼痛,行走时加剧,于2009年6月在外院行B超检查发现腹股沟区多枚淋巴结肿大,直径约4.0～5.0 cm,胰周、腹主动脉及髂血管旁多发低回声实性结节,最大直径为4.5 cm;行CT检查,提示腹膜后、肝门部多发肿大淋巴结.血常规检查:白细胞、中性粒细胞升高,淋巴细胞减少.2009年7月23日手术切除右腹股沟淋巴结.原单位病理诊断:考虑为指状突树突状细胞肿瘤.因多家医院诊断结果存在分歧,遂来我院会诊,详细询问病史,患者既往无其他脏器肿瘤病史.     

Galectins: versatile modulators of cell adhesion, cell proliferation, and cell death

 Lectins, or carbohydrate binding proteins, recognize specific oligosaccharide structures on glycoproteins and glycolipids. Several families of animal lectins have been identified; for some of these lectins, functions such as leukocyte adhesion and microbial opsonization have been described. The galectins are a family of lectins found in species ranging from sponges and nematodes to humans. Members of the galectin family have been proposed to mediate cell adhesion, to regulate cell growth, and to trigger or inhibit apoptosis. The expression pattern of different galectins changes during development, and this pattern is also altered at sites of inflammation and in breast, colon, prostate, and thyroid carcinomas. In addition, the level of expression of some galectins by tumor cells has been shown to be correlated with metastatic potential. The mechanisms by which galectins exert these diverse effects remain largely unknown. Some glycoprotein counterreceptors recognized by certain galectins have been identified; this is an important first step in understanding the cell-type specific effects of different galectins. This review discusses the way in which the modulation of galectin activity may affect strategies for treatment of a variety of human diseases, including autoimmunity and cancer. Received: 4 April 1997 / Accepted: 24 September 1997     

Between-observer repeatability of sputum differential cell counts. Influence of cell viability and squamous cell contamination

BACKGROUND: Induced sputum differential cell counts have been advocated as a method of noninvasively assessing airway inflammation in asthma and other airway diseases. Relatively little is known about the between-observer repeatability of sputum differential cell counts and the factors that influence it. OBJECTIVE: To assess the between-observer variability of induced sputum cell counts. METHODS: Sputum was induced and processed using standard techniques. Forty-two slides from 38 patients (31 with asthma, seven normal subjects) were randomly selected. Slides were classified as good (<20% squamous cells and >50% viability; n = 24); low viability (<50% viability; n = 10) and high squamous cell contamination (>20% squamous cells; n = 8). Two blinded observers counted between 200 and 400 nonsquamous cells and agreement was assessed by the intraclass correlation coefficient (ICC) and the standard deviation of between-observer differences (SD). RESULTS: The overall ICC were 0.9, 0.89, 0.9 for eosinophils, neutrophils and macrophages and 0.29 and 0.69 for lymphocytes and epithelial cells. Repeatability was greater in slides classified as good compared with slides with low cell viability and particularly excess squamous cell contamination. CONCLUSIONS: We have shown that the overall between-observer repeatability of the differential eosinophil, neutrophil and macrophage cell counts is good. Low cell viability and particularly excess squamous cell contamination reduce between-observer repeatability suggesting that techniques that ensure high cell viability and reduce squamous contamination would be an advantage.