卵巢肿瘤患者外周血NK细胞受体NKG2A、NKG2D及NK活性检测的临床意义

通过研究卵巢癌及良性卵巢肿瘤患者外周血NK细胞表面受体的表达情况及NK活性的变化,分析探讨宿主NK细胞受体与肿瘤免疫逃逸的关系及其临床价值。分离受检者外周血单个核细胞,应用MTT法检测NK细胞的细胞毒活性,流式细胞术检测NK细胞受体NKG2D和NKG2A的表达,并结合临床病理因素作比较分析。结果显示,与良性卵巢肿瘤组和正常组相比,卵巢癌患者外周血NK细胞的细胞毒活性降低,NK细胞表面NKG2D的表达水平降低,而NKG2A的表达水平明显升高,其变化与卵巢癌的病情进展有关。此结果表明,卵巢癌患者机体NK细胞杀伤活性下降,NKG2D与NKG2A二者之间的平衡表达可能对NK细胞的功能状态起着重要的调节作用。     

卵巢肿瘤患者自然杀伤细胞活化性受体NKG2D与其配体MICA表达的初步研究

目的:研究卵巢癌、良性卵巢肿瘤患者外周血NK细胞表面活化性受体NKG2D的表达及局部组织中相应配体MICA的表达情况,并结合临床病理因素分析探讨宿主NK细胞受体NKG2D在抗卵巢癌中的作用及其与肿瘤免疫逃逸的关系。方法:对4 2例卵巢癌、2 3例良性卵巢肿瘤及2 0例正常妇女,采用流式细胞术检测外周血NK细胞NKG2D的表达状况,RT PCR技术检测在上述部分相应组织标本中MICA的表达。结果:恶性、良性卵巢肿瘤患者及正常人外周血NK细胞NKG2D的表达水平分别为( 94 2 3±6 0 2 ) %、( 98 70±0 98) %、( 98 6 1±1 5 9) % ,恶性组与另两组之间比较,差异有统计学意义(P <0 0 5 ) ;相应配体MICA在卵巢癌组织中的表达率较良性卵巢肿瘤中明显增高,差异有统计学意义(P <0 0 1) ;在卵巢癌病人是否绝经、不同组织类型、分化程度、手术分期及是否淋巴转移等各组临床病理情况下,其表达率未见明显差异(P >0 0 5 )。结论:卵巢恶性肿瘤患者外周血NK细胞活性降低,其活化性受体NKG2D表达的下降是NK细胞活性下降的原因之一。NKG2D的配体MICA的基因表达可能与卵巢癌的恶性转化有一定的相关性,卵巢癌的免疫逃逸可能与NKG2D表达下调及其配体MI CA的表达升高有关     

NKG2D真核表达载体的构建及其对YT细胞生物学功能的影响

目的：建立NK细胞受体NKG2D真核表达载体，通过转染NK细胞系YT，初步探讨NKG2D分子对YT细胞系杀伤功能的增强作用。方法：用RT-PCR方法从NK-92细胞中调取NKG2D基因片段，克隆到pGEM-TEasy载体并对克隆的DNA片段进行序列分析。用限制内切酶EcoRⅠ和BamHⅠ消化pGEM-T Easy／NKG2D重组质粒，分离NKG2D片段，并插入真核表达质粒pEGFP-N1的相应限制酶位点，酶谱分析鉴定重组表达载体pEGFP-N1／NKG2D。然后经脂质体介导转染CHO细胞和YT细胞。应用荧光显微镜观测、Western blot方法和免疫组化染色对转染细胞内pEGFP-NilNKG2D的表达进行鉴定，MTF方法观察YT细胞对肿瘤细胞的杀伤功能。结果：RT-PCR扩增获得650bp基因片段，经DNA序列分析证明所获得的DNA序列与文献报道的NKG2D序列一致。转染的CHO细胞在荧光显微镜下发出强绿色荧光，Western blot分析显示重组蛋白能特异地与抗人NKG2D单克隆抗体结合；免疫组化检测显示，转染的CHO中有棕色颗粒，证明所构建的NKG2D真核表达载体可以在细胞中表达；转染NKG2D真核表达载体的YT细胞对乳腺癌细胞具有更强的杀伤效果。结论：所获得的表达NKG2D分子的真核表达载体，通过转染YT细胞，初步鉴定所表达NKG2D分子具有生物学功能，可以提高NK细胞对肿瘤细胞的杀伤活性。     

IL-2促进PBMC来源的NK、NKT、CD8+T细胞高表达NKG2D

目的 观察大剂量IL-2活化的人外周血单个核细胞（PBMC）中，NKG2D在NK细胞、T细胞和NKT细胞表面的表达规律。方法 使用三重免疫荧光标记的流式细胞术检测NKG2D的表达情况。使用sMICA蛋白与人PBMC共同培养，之后使用流式细胞术分析NKG2D在NK细胞中的表达情况。使用半定量RT-PCR方法检测大剂量IL-2活化的人PBMC中NKG2D及其锚定蛋白DAP10 mRNA的表达变化。结果 使用大剂量IL-2活化人PBMC细胞后，NKG2D在NK细胞、CD^＋T细胞和NKT细胞表面的表达均增加，但是在CD4^＋T细胞表面始终不表达。同时IL-2可以拮抗sMICA对NKG2D的下调作用。半定量RT-PCR结果显示，使用大剂量IL-2活化人PBMC之后，NKG2D及其锚定蛋白DAP10的mRNA水平并不发生明显变化。结论 大剂量IL-2培养人PBMC之后，NKG2D在NK细胞、CD8^＋T细胞和NKT细胞表面的表达均增加，可能是PBMC活化并获得广谱抗肿瘤效应的机制之一．     

结核杆菌耐热抗原对人外周血淋巴细胞NKG2A/NKG2D的影响

目的：以结核杆菌耐热抗原为刺激剂，观察人外岗血淋巴细胞NKG2A受体和NKG2D受体表达量的变化情况。方法：用结核杆菌耐热抗原刺激人外周血淋巴细胞，用流式细胞技术检测NKG2A受体和NKG2D受体的变化。使用RT-PCR和ELISA检测PBMCs中IFN-γ的表达情况。结果：在IL-2和抗原联合刺激下，NKG2A受体的表达量在第12天大幅度上升，NKG2D受体的表达量始终变化不大。NKG2A受体的表达在T细胞、NK细胞、αβT细胞和γδT细胞表面都有不同程度的增加。结论：NKG2A/NKG2D比例的上升有可能会对免疫系统造成一些影响，其中IFN-γ起着不可忽视的作用。     

氧化应激可选择性诱导细胞的NKG2D配体的表达

目的 探讨氧化应激与细胞NKG2D配体表达的关系,分析氧化应激对NK细胞功能的影响。方法 加H2O2诱导培养的肿瘤细胞处于氧化应激状态。用RT-PCR、Real-time PCR和流式细胞仪等方法分析细胞多种NKG2D配体的表达。用CCK-8法检测NK92细胞对肿瘤细胞的杀伤活性。结果 氧化应激可诱导肿瘤细胞多种NKG2D配体的表达,不同的肿瘤细胞诱导表达的NKG2D配体不同;NKG2D配体表达上调可有效提高NK细胞的细胞毒活性,此效应可被抗NKG2D抗体所阻断。结论 NKG2D配体可能在机体的免疫应答中发挥正向的调节作用。     

NKG2D及其配体研究进展

NKG2D是较为独特的NK细胞活化性受体，其配体具有多样性，因而其识别机制较独特；其表达范围不仅局限于NK细胞，还在T细胞、巨噬细胞、树突状细胞中有表达，功能上，不仅有直接刺激作用，还能作为协同刺激分子传递第二信号。NKG2D及其配体的研究对抗肿瘤免疫、抗感染免疫、自身免疫病的认识具有重要意义。本文对NKG2D及其配体的研究进展作一综述。     

HIV原发感染者自然杀伤样T细胞NKG2A/NKG2D变化的研究

目的深入了解人类免疫缺陷病毒(human immunodeficiency virus,HIV)原发感染者(primary HIV infection,PHI)NKT样细胞表面NKG2A/NKG2D受体表达的变化。方法选取25例未经高效抗逆转录病毒治疗的HIV原发感染者和27例HIV抗体阴性健康对照,用流式细胞仪检测研究对象外周血NKT样细胞表面NKG2D和NKG2A的表达。结果 HIV原发感染者NKT样细胞绝对数和百分率显著低于健康对照(P<0.01)。HIV原发感染者NKT样细胞表面NKG2A、NKG2D受体表达与健康对照并无显著差异。HIV原发感染者病毒调定点低组NKG2A+NKT样细胞、NKG2A+NKG2D-NKT样细胞以及NKG2A+NKG2D+NKT样细胞百分率均显著低于病毒调定点高组(P<0.05);NKT细胞绝对数和百分率、NKG2D+NKT样细胞、NKG2D+NKG2A-NKT样细胞百分率在两组间相似,没有显著性差异。NKG2A+NKT细胞的百分比与病毒载量正相关(R=0.430,P=0.032)。结论 NKT样细胞数量以及其表面NKG2A受体的表达可作为HIV疾病进程的预测指标之一。     

妊娠子宫微环境中uNK细胞NKG2A和NKG2D及其配体表达的研究

目的：研究妊娠子宫微环境中子宫自然杀伤细胞（uNK细胞）NKG2A和NKG2D及其相应配体的表达，探讨NKG2A与NKG2D的不平衡表达在母胎免疫耐受形成中的作用。方法：选择30例孕6-9周的正常妊娠妇女,分离其新鲜蜕膜组织，除去绒毛,分离蜕膜和外周血单个核细胞，采用流式细胞仪测定NK细胞的数量及NKG2A与NKG2D的表达；采用RT-PCR技术检测滋养层组织NKG2A与NKG2D配体人类白细胞抗原-E(HLA-E)、主要组织相容性复合体-Ⅰ类分子相关蛋白A(MICA)mRNA的表达结果：妊娠子宫蜕膜淋巴细胞中NK细胞约占70%,流式细胞分析的结果显示，子宫自然杀伤细胞NKG2A的表达显著高于外周血NK细胞，分别为97.86%±1.75%与33.35%±10.92%（〖AKx-D〗±s），两者差异显著（P<0.05），在滋养层细胞中检测到其配体HLA-E的表达；而与外周血相比，uNK细胞表面NKG2D的表达与之较为相近，分别为93.21%±4.52%与97.80%±1.72%，但两者仍有显著差异（P<0.05）。在滋养层组织未检测到其相应配体MICA mRNA的表达结论：蜕膜中的淋巴细胞主要为NK细胞，其免疫学表型与外周血NK细胞有较大的区别，妊娠期子宫自然杀伤细胞表面高表达抑制性受体NKG2A，同时滋养层组织表达相应的配体人类白细胞抗原-E，这可能是维持母胎界面免疫耐受的重要因素。     

NKG2D及其配体研究进展

NKG2D是较为独特的NK细胞活化性受体 ,其配体具有多样性 ,因而其识别机制较独特 ;其表达范围不仅局限于NK细胞 ,还在T细胞、巨噬细胞、树突状细胞中有表达 ,功能上 ,不仅有直接刺激作用 ,还能作为协同刺激分子传递第二信号。NKG2D及其配体的研究对抗肿瘤免疫、抗感染免疫、自身免疫病的认识具有重要意义。本文对NKG2D及其配体的研究进展作一综述。     

活化性受体NKG2D及其配体的研究进展

NK细胞是肌体免疫系统至关重要的组成部分,其表达多种活化性和抑制性细胞表面受体。NKG2D是较为独特的活化性受体,属C型凝集素家族跨膜蛋白,分布较广,NK细胞、T细胞和其他免疫细胞都可以产生,其配体具有多样性,MHCⅠ类相关分子（MIC）是人类NKG2D识别的配体之一,应激性表达在一些肿瘤细胞或病原体感染细胞的表面。NKG2D既能直接活化NK细胞,又能以协同刺激的方式促进CD8^＋αβT细胞的活化,在抗肿瘤免疫和病毒感染等方面发挥重要作用。     

The activating receptor NKG2D of natural killer cells promotes resistance against enterovirus‐mediated inflammatory cardiomyopathy

In enterovirus‐induced cardiomyopathy, information regarding the detailed impact of natural killer (NK) cells on the outcome of the disease is limited. We therefore hypothesized that NK cells and certain NK cell receptors determine the different outcome of coxsackievirus B3 (CVB3) myocarditis. Here, we demonstrate in murine models that resistance to chronic CVB3 myocarditis in immunocompetent C57BL/6 mice is characterized by significantly more mature CD11b^high NK cells, the presence of NKG2D on NK cells, and enhanced NKG2D‐dependent cytotoxicity compared to CVB3‐susceptible A.BY/SnJ mice. The highly protective role of NKG2D in myocarditis was further proven by in vivo neutralization of NKG2D as well as in NKG2D‐deficient mice but was shown to be independent of CD8⁺ T‐cell‐dependent immunity. Moreover, the adoptive transfer of immunocompetent C57BL/6 NK cells pre‐ (day ?1) as well as post‐infectionem (day +2) displayed the potential to prevent permissive A.BY/SnJ mice from a progressive outcome of CVB3 myocarditis reflected by significantly improved cardiopathology and heart function. Altogether, our results provide firm evidence for a protective role of NKG2D‐activated NK cells in CVB3 myocarditis leading to an effective virus clearance, thus offering novel therapeutic options in the treatment of virus‐induced myocarditis. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd     

The CD94/NKG2A inhibitory receptor educates uterine NK cells to optimize pregnancy outcomes in humans and mice

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Women with Pre-Eclampsia Have an Altered NKG2A and NKG2C Receptor Expression on Peripheral Blood Natural Killer Cells

Problem  Preeclampsia, a pregnancy disorder, is associated with exaggerated inflammation and increased serum monokines. Uterine natural killer (NK) cells are implicated in preeclampsia pathology, but little is known regarding peripheral NK cells in the disease.
Method of Study  We examined blood NK cells at delivery in women with preeclampsia, in healthy pregnant women and in healthy non-pregnant blood donors as a reference.
Results  Although the percentages of both NKG2A- and NKG2C-positive NK cells were normal in preeclamptic women, the levels of NKG2A and NKG2C on NK cells were significantly up-regulated in these women. In vitro stimulation of PBMCs from healthy pregnant women and blood donors with monokines resulted in increased percentage of NKG2A⁺ NK cells and increased NKG2A levels, while levels of NKG2C were decreased.
Conclusions  Our results suggest that the peripheral NK-cell pool is skewed in preeclampsia and possibly under the influence of monokines like interleukin (IL)-15 and IL-12.     

Balance between activating NKG2D,DNAM‐1, NKp44 and NKp46 and inhibitory CD94/NKG2A receptors determine natural killer degranulation towards rheumatoid arthritis synovial fibroblasts

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation and synovial hyperplasia leading to progressive joint destruction. Fibroblast‐like synoviocytes (FLS) are central components of the aggressive, tumour‐like synovial structure termed pannus, which invades the joint space and cartilage. A distinct natural killer (NK) cell subset expressing the inhibitory CD94/NKG2A receptor is present in RA synovial fluid. Little is known about possible cellular interactions between RA‐FLS and NK cells. We used cultured RA‐FLS and the human NK cell line Nishi, of which the latter expresses an NK receptor repertoire similar to that of NK cells in RA synovial fluid, as an in vitro model system of RA‐FLS/NK cell cross‐talk. We show that RA‐FLS express numerous ligands for both activating and inhibitory NK cell receptors, and stimulate degranulation of Nishi cells. We found that NKG2D, DNAM‐1, NKp46 and NKp44 are the key activating receptors involved in Nishi cell degranulation towards RA‐FLS. Moreover, blockade of the interaction between CD94/NKG2A and its ligand HLA‐E expressed on RA‐FLS further enhanced Nishi cell degranulation in co‐culture with RA‐FLS. Using cultured RA‐FLS and the human NK cell line Nishi as an in vitro model system of RA‐FLS/NK cell cross‐talk, our results suggest that cell‐mediated cytotoxicity of RA‐FLS may be one mechanism by which NK cells influence local joint inflammation in RA.     

Variable NKG2 expression in the peripheral blood lymphocytes of rhesus monkeys

To provide a basis for beginning to explore the CD94/NKG2 family of molecules in rhesus monkeys, we sought to characterize the expression of these inhibitory and activating cell signalling molecules in peripheral blood mononuclear cells (PBMCs) from healthy rhesus monkeys. We developed and employed a semiquantitative polymerase chain reaction (PCR)-based assay to evaluate mRNA expression levels of nine NKG2 molecules in PBMCs from the monkeys. In addition to quantitating NKG2A, NKG2B, NKG2C2, NKG2C and NKG2D expression, mRNA expression of transmembrane-deleted forms of these molecules was also evaluated. Significant variability in NKG2 mRNA expression in the PBMCs was detected, with 15 unique NKG2 expression level profiles detected in a study of 15 monkeys. We also found that the ratio of the expressed levels of mRNA of the four NKG2 splice variants, NKG2A, NKG2B, NKG2ADeltatm, and NKG2BDeltatm, was variable between the monkeys as well as in an individual monkey over a period of 1.5 years. These findings indicate the dynamic nature of NKG2 mRNA expression in the rhesus monkey.