脐血中自然杀伤T细胞的免疫学特性及抗肿瘤功能

自然杀伤（NK）T细胞具有NK受体和T细胞受体且显示NK细胞和T细胞两方面性质的淋巴细胞。脐血中的NK T细胞具有较低的免疫原性和较高的分化潜能，具有通过细胞识别途径介导的抗肿瘤细胞的细胞毒性，调节1gE产生和清除自身反应性T细胞等功能，因而在肿瘤的免疫治疗领域可能具有广泛的应用前景。     

肾母细胞瘤患儿外周血调节性T细胞和自然杀伤T细胞的表达研究

目的 研究肾母细胞瘤患儿外周血CD4+CD25+CD127low 调节性T细胞（Treg）和CD3+CD16+CD56+自然杀伤T细胞（NKT）的表达变化,初步探讨发生改变的原因及其临床意义。方法 选取2015年11月至2016年7月就诊的21例肾母细胞瘤患儿作为病例组,21例于本院体检的同年龄段健康儿童为健康对照组,采用流式细胞术检测两组儿童外周血中 CD4+CD25+CD127low T细胞占CD4+T细胞百分比和CD3+CD16+CD56+T细胞占CD3+T细胞百分比,分别代表Treg水平和NKT水平。结果 病例组肾母细胞瘤患儿外周血Treg水平低于健康对照组（P < 0.05）;病例组肾母细胞瘤患儿外周血NKT水平高于健康对照组（P < 0.05）。结论 Treg和NKT水平变化与肾母细胞瘤的发生与发展有关,Treg和NKT水平可能是反映肾母细胞瘤患儿免疫功能状态的较好指标。     

支气管哮喘患儿外周血自然杀伤T淋巴细胞的变化及临床意义

目的 探讨支气管哮喘(哮喘)患儿外周血自然杀伤T淋巴细胞(NKT细胞)数量及体外扩增活化后产生细胞因子水平的变化.方法 选取2009年2月至2012年3月在上海交通大学医学院附属仁济医院儿科门诊确诊的哮喘患儿52例(哮喘组),其中轻度间歇26例,轻度持续14例,中度持续12例.另选20例体检健康儿童作为健康对照组.收集所有研究对象外周静脉血标本,采用密度梯度离心法分离获得外周血单个核细胞,应用免疫荧光标记和流式细胞术检测哮喘组和健康对照组儿童外周血NKT细胞和CD4+ NKT细胞的比例,观察NKT细胞和CD4+ NKT细胞与过敏指标总IgE、嗜酸性细胞阳离子蛋白(ECP)的相关性.经α-半乳糖神经鞘胺醇扩增培养活化后用ELISA法检测外周血细胞因子IFN-γ、IL-4、IL-10水平.比较哮喘组和健康对照组之间的差异.结果 哮喘组患儿的NKT细胞和CD4+ NKT细胞比例均较健康对照组显著下降(P均＜0.01);在不同病情哮喘患儿中,NKT细胞和CD4+ NKT细胞的比例无明显变化(P＞0.05).NKT细胞、CD4+NKT细胞的比例与总IgE、ECP均无明显相关性(P均＞0.05).扩增培养活化后,哮喘组患儿NKT细胞分泌的IL-4水平较健康对照组明显升高(P＜0.01),IL-10水平显著下降(P＜0.01),而IFN-γ水平与健康对照组比较无统计学差异(P＞0.05).结论 哮喘发病可能与NKT细胞和CD4+ NKT细胞数量的低下及功能严重失调相关.     

儿童NK/T细胞淋巴瘤的免疫表型及其与EB病毒的关系

目的探讨儿童NK/T细胞淋巴瘤的免疫表型特征及其与EB病毒（EBV）感染的关系。方法NK/T细胞淋巴瘤5例标本,采用免疫组织化学链霉素抗生物素-过氧化酶连接法（SP法）检测CD45RO、CD3ε、CD56、CD20、T细胞内抗原（TIA-1）、丝氨酸蛋白酶-粒酶B（Granzyme B）确定肿瘤细胞免疫表型及EBV潜伏膜蛋白（LMP-1）,原位杂交技术检测EBV编码的RNA（EBER1/2）。结果NK/T细胞淋巴瘤5例CD45RO、CD3ε、TIA-1和Granzyme B全部阳性,CD56阳性2例,CD20全部阴性。EBER1/2阳性4例,LMP-1阳性3例。结论EBV感染与儿童NK/T细胞淋巴瘤关系密切,EBV感染在儿童NK/T细胞淋巴瘤的发生发展中可能起重要作用。     

支气管哮喘患儿外周血自然杀伤T淋巴细胞的变化及临床意义北大核心CSCD

目的探讨支气管哮喘（哮喘）患儿外周血自然杀伤T淋巴细胞（NKT细胞）数量及体外扩增活化后产生细胞因子水平的变化。方法选取2009年2月至2012年3月在上海交通大学医学院附属仁济医院儿科门诊确诊的哮喘患儿52例（哮喘组）,其中轻度间歇26例,轻度持续14例,中度持续12例。另选20例体检健康儿童作为健康对照组。收集所有研究对象外周静脉血标本,采用密度梯度离心法分离获得外周血单个核细胞,应用免疫荧光标记和流式细胞术检测哮喘组和健康对照组儿童外周血NKT细胞和CD4＋NKT细胞的比例,观察NKT细胞和CIM＋NKT细胞与过敏指标总IgE、嗜酸性细胞阳离子蛋白（ECP）的相关性。经α-半乳糖神经鞘胺醇扩增培养活化后用ELISA法检测外周血细胞因子IFN-γ、IL4、IL-10水平。比较哮喘组和健康对照组之间的差异。结果哮喘组患儿的NKT细胞和CD4＋NKT细胞比例均较健康对照组显著下降（P均〈0．01）;在不同病情哮喘患儿中,NKT细胞和CD4＋NKT细胞的比例无明显变化（P〉0．05）。NKT细胞、CD4＋NKT细胞的比例与总IgE、ECP均无明显相关性（P均〉0．05）。扩增培养活化后,哮喘组患儿NKT细胞分泌的IL-4水平较健康对照组明显升高（P〈0．01）,IL-10水平显著下降（P〈0．01）,而IFN-γ水平与健康对照组比较无统计学差异（P〉0．05）。结论哮喘发病可能与NKT细胞和CD4＋NKT细胞数量的低下及功能严重失调相关。     

白细胞介素15和粒-单核集落刺激因子激活的自然杀伤T细胞对神经母细胞瘤细胞的杀伤作用

目的：研究白细胞介素15(IL-15)和粒-单核集落刺激因子(GM-CSF)对健康儿童自然杀伤T细胞(NKT)增殖和对NKT细胞介导的神经母细胞瘤(NB)细胞的细胞毒作用的影响。方法：采集分离健康儿童外周血T细胞,分别加入IL-15,GM-CSF培养,MTT法检测T细胞增殖程度的变化,流式细胞仪检测NKT/T比例并分选出NKT细胞,MTT法检测NKT细胞对NB细胞毒作用。结果：IL-15刺激组、GM-CSF刺激组和IL-15+GM-CSF刺激组T细胞增殖程度分别是对照组的 1.25 倍、1.2 倍和 1.4 倍(均P<0.01);NKT/T比例分别是对照组的 2.56 倍、3.27 倍和 4.39 倍(P<0.01或0.05);GM-CSF 组和IL-15+GM-CSF组NKT细胞毒作用高于对照组(均P<0.01),IL-15组NKT细胞毒作用与对照组比较差异无显著性(P>0.05)。与GM-CSF组比较,IL-15+GM-CSF组T细胞增殖程度增高但NKT/T比例和NKT细胞毒作用差异无显著性。与IL-15组比较,IL-15+GM-CSF组NKT/T比例和NKT细胞毒作用增高但T细胞增殖程度差异无显著性。IL-15组与GM-CSF组T细胞增殖程度、NKT/T比例和细胞毒作用差异无显著性。结论：NKT细胞对NB具有细胞毒作用,IL-15和GM-CSF联合应用可以促进健康儿童外周血NKT细胞增殖,增强NKT细胞对NB细胞毒作用。     

儿童鼻型NK/T细胞淋巴瘤1例并文献复习

目的 报道罕见儿童鼻型NK/T细胞淋巴瘤1例。方法 总结分析儿童鼻型NK/T细胞淋巴瘤1例的临床及组织病理学特征。详细采集患儿体格检查、鼻窦CT和MRI、骨髓像、免疫学及组织病理学检查等资料,并进行文献复习。结果 患儿,男,15岁,因“发热、皮疹及鼻堵1个月”入院。 入院查体可见全身散在大小不等皮疹,略高出皮肤,直径1～5 cm,呈鲜红色及紫红色,以双下肢多见,左下肢胫骨内侧可见一直径约7 cm皮肤结节。鼻翼肿胀,鼻腔内有增生物,有异味及黄色分泌物。鼻窦CT及MRI均提示鼻腔占位性病变。患儿入院后腹痛进行性加重,腹部X线检查提示肠穿孔,行直肠穿孔修补、阑尾切除及回肠提吊造瘘术。左下肢皮疹病理学检查示：真皮小血管及皮肤附件周围可见异形淋巴细胞浸润;左下肢胫骨内侧皮肤结节穿刺涂片见大量肿瘤细胞;切除肠管病理学检查示：部分盲肠壁黏膜及肌间可见灶状异形淋巴细胞浸润,其形态与左下肢皮疹病理学检查中所见相似。免疫组化示：CD45RO(++),CD3(++)~（+++), CD56(++)~（+++);GranzymeB、CD20、CD79a和CD68均(-)。结合临床表现、病理学检查镜下形态及免疫组化结果,确诊为鼻型NK/T细胞淋巴瘤。患儿家属放弃治疗,患儿于出院2个月后死亡。结论 鼻型NK/T细胞淋巴瘤主要累及鼻腔及面中线部位,并可表现为多系统受累。患儿如有呈进行性面部中线破坏性病变应高度警惕本病的可能。     

儿童NK/T细胞淋巴瘤与survivin基因的表达

NK／T细胞淋巴瘤(NK／T cell lymphoma)是一种少见的结外原发性非霍奇金淋巴瘤,是与EB病毒(Epstein-Barrvirus,EBV)相关的具有高度侵袭性的恶性肿瘤。survivin是凋亡抑制蛋白(inhibitor of apoptosis proteins,IAP)家族的新成员,主要通过抑制caspase-3、caspase-7的活性而发挥抗凋亡作用。我们采用原位杂交技术检测EBV编码的RNA(EBERl／2),免疫组织化学技术检测survivin基因和EBV潜伏膜蛋白(LMP-1)表达,     

甲状腺激素T3诱导K562细胞铁蛋白表达与细胞凋亡和生长的关系

目的：探讨甲状腺激素T3对K562细胞铁蛋白表达及其对细胞凋亡和增殖的影响。方法：收集各组细胞胞浆蛋白，用放射免疫法检测铁蛋白含量，并和流细胞术分析各组细胞凋亡百分率及细胞周期变化。结果：中等浓度及高浓度的T3均能增加K562细胞铁蛋白表达。与空白对照相比有显著性差异（P＜0．05），且随T3浓度增加，铁蛋白的表达亦增高。同一浓度T3与K562细胞共培养，随时间延长，铁蛋白表达增加，与空白对照相比具有显著性差异（P＜0．01）。24h培养组细胞凋亡率较空白对照略有下降，差异有统计学意义，72h培养组T3＝200nM时凋亡百分率明显下降。T3各处理组中S＋G2期细胞比例较空白对照组明显减少（P＜0．01），且72h各组G2＋S细胞百分率较24h明显下降。结论：甲状腺激素T3能诱导K562细胞铁蛋白表达增加，且能干预细胞凋亡和细胞增殖周期。     

树突细胞联合细胞因子诱导的杀伤细胞生物学特性和体外杀瘤机制

目的 探讨树突细胞联合细胞因子诱导的杀伤细胞(DC-CIK)生物学特性和体外杀瘤机制.方法　从白血病患儿外周血分离单个核细胞,经过γ干扰素(IFN-γ)、抗CD3单克隆抗体(CD3McAb)、IL-2 诱导并与树突细胞(DC)共培养后,获得DC-CIK.采用MTT法检测DC-CIK对多种白血病细胞株的杀伤作用.在予以10 mg·L-1、20 mg·L-1小鼠抗人淋巴细胞功能相关抗原-1(LFA-1)单克隆抗体处理后,流式细胞仪检测CD4+CD25+Treg细胞比例,RT-PCR与Western blot方法检测Foxp3基因表达水平.结果 诱导后的DC-CIK细胞形态规则,其对肿瘤细胞B95、Jhhan、M07e 均表现出杀伤活性.其中对B95细胞的杀伤作用较强,在效靶比为51、101 时DC-CIK对B95细胞的杀伤作用均>60%,而对Jhhan、M07e的作用不明显.在予以10 mg·L-1、20 mg·L-1LFA-1单克隆抗体处理后,与阴性对照组比较,2个处理组均使CD4+CD25+ Treg细胞表达增加(t=6.783、7.282,Pa＜0.05).RT-PCR与Western blot结果表明与阴性对照组比较,2个处理组均使Foxp3基因mRNA表达水平增加(t=11.671、10.909,Pa＜0.05),Foxp3基因蛋白表达水平增加(t=16.734、9.562,Pa＜0.05).结论　DC-CIK具有强大的体外杀瘤作用,Foxp3基因参与LFA-1介导的DC-CIK杀瘤途径,其杀瘤机制表现为CD4+CD25+Treg细胞途径受抑制.     

Natural Killer Cell Function in Atopic Dermatitis

ABSTRACT. The natural killer cell activity was studied in 41 children with mild, moderate and severe atopic dermatitis (AD) and in 37 controls. The natural killer cell function of lymphocytes was reduced in atopic children (mean ± SD 21.92±6.18% vs. 43.87±5.80%; p <0.0001). This decrease was not related to the IgE serum level. A negative correlation was found between natural killer cell activity and the clinical severity of AD ( r =0.73; p <0.001). Natural killer cell function was re-evaluated after 9 months in 27 children during a quiescent phase of AD; it was still low, but to a lesser degree (27.66±5.42%, in the quiescent phase, vs. 43.87±5.80, controls; p <0,0001). The reduced natural killer cell activity seems related to the disease activity.     

缺氧缺血性脑病新生儿自然杀伤细胞与T细胞亚群的变化及其相关因素

目的了解缺氧缺血性脑病(HIE)新生儿自然杀伤(NK)细胞及T细胞亚群的变化,观察其与各种因素的相关性。方法应用流式细胞仪测定50例HIE和20例正常新生儿血NK细胞、CD3 、CD4 、CD8 细胞及CD4 /CD8 比值。结果HIE患儿NK细胞和T细胞亚群的表达均明显低于正常新生儿(P均<0.05);中、重度HIE患儿NK细胞和CD3 、CD4 细胞及CD4 /CD8 比值明显均低于轻度HIE(P均<0.05),而不同分度HIE的CD8 细胞则无统计学差异(P均>0.05);NK细胞、CD3 、CD4 及CD4 /CD8 与HIE病情分度均呈负相关(P均<0.05)。NK细胞表达与胎龄呈正相关;CD3 细胞与羊水污染呈负相关(P<0.05);CD4 细胞与羊水污染呈负相关,与出生体质量和Apgar评分呈正相关(P<0.05);CD4 /CD8 比值与分娩方式存在线性相关(P<0.05)。结论缺氧缺血可导致HIE患儿NK细胞活性抑制及T淋巴细胞亚群紊乱,某些因素对细胞免疫功能有一定影响。     

Natural Killer Cell Activity of Peripheral Blood and Bone Marrow Mononuclear Cells from Patients with Childhood Acute Lymphoblastic Leukemia

ABSTRACT. Patients with untreated acute lymphoblastic leukemia had highly significantly reduced natural killer cell activity in peripheral blood ( p <0.01) and bone marrow ( p <0.001) mononuclear cells compared with that in peripheral blood mononuclear cells in normal healthy controls. Patients in remission had normal killer cell activity ( p >0.5) both in peripheral blood and bone marrow mononuclear cells. Thus, the natural killer cell activity correlated well with the disease activity in the patients.     

Natural Killer Activity in Two Cases of Ataxia Telangiectasia

ABSTRACT. We report two cases of ataxia telangiectasia in which a small number of T cells and a slightly diminished response to phytohaemagglutinin or concanavalin A were observed. In one case, serum IgA was undetectable. In spite of T and/or B cell involvement, natural killer activity and the percentage of natural killer cells determined by monoclonal antibodies Leu 7 were normal in both cases.     

Successful Treatment of Genetically Profiled Pediatric Extranodal NK/T‐Cell Lymphoma Targeting Oncogenic STAT3 Mutation

Extranodal natural killer (NK)/T‐cell lymphoma (ENKTCL) is a distinct type of non‐Hodgkin lymphoma predominantly observed in Asian and Latin American adult males. A 12‐year‐old Hispanic female diagnosed with ENKTCL was enrolled in our genomic profiling research protocol. We identified specific somatic alterations consistent with diagnosis of ENKTCL as well as oncogenic mutations in MAP2K1 and STAT3. To our knowledge, this is the first report of an immunophenotypically confirmed and genetically profiled case of ENKTCL in a female pediatric patient in the United States, including its unique treatment and favorable outcome.     

Proliferating Cell Nuclear Antigen in Human Placenta and Trophoblastic Disease

Expression of proliferating cell nuclear antigen (PCNA), an auxiliary factor for the DNA polymerase δ, is closely related to cell proliferation. Using the monoclonal anti-PCNA antibody 19F4, we examined the distribution of PCNA in formalin-fixed, paraffin-embedded sections of mature and first-trimester placentas, 8 hydatidiform moles, and 7 choriocarcinomas. In normal placentas there was strong expression of PCNA in cytotrophoblastic nuclei, while the syncytium and the amnionic epithelium were PCNA unreactive. Staining of stromal cells was variable. These results are in complete agreement with the autoradiographic localization of [³H]thymidine incorporation and demonstrate that immunoreactivity for PCNA accurately defines areas of proliferative activity in routinely processed placental tissues. In choriocarcinomas and hydatidiform moles PCNA was predominantly expressed in the cytotrophoblast and intermediate trophoblast. Again, the syncytium was largely unreactive for PCNA. These findings indicate that even after neoplastic transformation the syncytium has comparatively little proliferative activity whereas the mononuclear trophoblastic cells divide actively.     

Analysis of Killer Cell Activities in Epstein-Barr Virus Infections

Using spontaneously established autologous lymphoblastoid B cell lines (LCL), killer cell activities were studied in children with severe infectious mononucleosis (IM), chronic IM, and acute IM, and compared with those in EBV-seropositive normal controls. Natural killer (NK) cell activity of fresh peripheral blood mononuclear cells (PBMC) was normal in acute IM patients, but it was low in four of six patients with severe or chronic IM. Recombinant inter-leukin 2 (rIL-2)-activated PBMC from normal controls showed lymphokine-activated killer (LAK) cell activity against the respective autologous LCL. The levels of LCL lysis by LAK cells were significantly higher in acute IM patients, lower in chronic IM patients, and much lower in severe IM patients. In contrast to the fact that PBMC stimulated in vitro with autologous LCL (IVS cells) from normal controls and acute IM patients showed potent killing of autologous LCL, IVS cells from severe or chronic IM patients showed lower levels of LCL lysis, which were markedly augmented in three patients by rIL-2 addition to the cultures. These killer cell dysfunctions appear to be responsible for the severe or chronic course of EBV infection.     

Cell-Mediated Immunity in Children with Hodgkin's and Non-Hodgkin's Lymphomas

Natural killer (NK) cell activity and lymphocyte proliferation to mitogens might have diagnostic implications that will help to distinguish between benign and malignant lymphadenopathy in children. In addition, we suggest that NK activity and especially the lowering of NK activity after chemotherapy might be of prognostic value in NHL children.     

Follicular Center Cell Lymphoma of Childhood in Japan

Following a histologic review of 108 patients with childhood lymphoma from 20 hospitals in the Osaka, Hyogo, Nara, and Kyoto prefectures of Japan during the period 1964-1989, 16 cases (15%) were classified as follicular center cell (FCC) lymphomas. None had a follicular pattern of proliferation. The age range was from 2 to 15 years (median 10) with no peak incidence. The male/female ratio was 1.3:1. Presentation was nodal in 10 patients and extranodal in 6. Chronic infection and other predisposing factors for the development of FCC lymphomas in childhood are discussed, with a review of the literature.