树突状细胞上调MHC Ⅰ类相关抗原A表达对NK细胞活性的影响

目的:观察单核细胞、未成熟和成熟树突状细胞(DCs)表达MHC Ⅰ类相关抗原A(MICA)的情况,以及DCs表达MICA后对NK细胞活性的影响.方法:用流式细胞术分别检测单核细胞、未成熟DC(iDCs)以及LPS、TNF-α、CD40L、IL-15和IFN-α分别刺激的成熟DCs表面MICA的表达,并观察DCs表达MICA后对NK细胞表达CD69、细胞毒活性和分泌IFN-γ的影响.最后用流式细胞术检测重组NKG2D/Fc蛋白和抗IL-12单克隆抗体(mAb)对DCs激活NK细胞的影响.结果:单核细胞不表达MICA,iDCs低表达MICA.LPS、TNF-α和CD40L对DCs表达MICA无影响;但IFN-α和IL-15可促进DC上调MICA表达.DCs表达MICA后可促进NK细胞表达CD69、分泌IFN-γ、杀伤K562细胞.NKG2D/Fe蛋白可抑制NK细胞的杀伤活性和分泌IFN-γ;而抗IL-12mAb仅抑制IFN-γ分泌.结论:MICA在DCs表面的表达受局部微环境影响,且DCs表达MICA后可增强NK细胞的活性.     

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。     

IFN-γ上调的单核细胞表面MHC-I类链相关分子促进NK细胞活化

目的:研究IFN-γ诱导上调表达的单核细胞MHC-I类链相关分子(MICs)分子对NK细胞的活化作用.方法:采用密度梯离离心法分离人外周血单个核细胞(PBMC),以免疫磁珠法从PBMC中特异性分选单核细胞及NK细胞,以细胞因子IFN-γ、TNF-α刺激单核细胞后,再将单核细胞与NK细胞共培养,以流式细胞术(FCM)检测NK细胞表面CD69分子及胞内IFN-γ表达,以51Cr释放试验检测NK细胞对K562细胞的杀伤效应.结果:IFN-γ上调人单核细胞表面MICs表达;IFN-γ刺激的单核细胞能促进异体NK细胞CD69及胞内IFN-γ表达,能增强NK细胞对K562细胞的杀伤效应;单核细胞的这种效应至少部分依赖于IFN-γ上调的MICs分子,因为用抗MIC抗体封闭或用细胞小室阻断细胞接触,可以明显地抑制NK细胞的活化.结论:IFN-γ上调人单核细胞表面MICs分子介导了NK细胞的活化.     

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。     

可溶性MICA在乳腺癌免疫逃逸中的作用

目的: 观察乳腺癌患者血清中可溶性MICA(sMICA)表达情况, 探讨sMICA在乳腺癌免疫逃逸中的作用.方法: ELISA方法检测乳腺肿瘤患者外周血sMICA的分泌.流式细胞术(FCM)检测sMICA及白细胞介素15(IL-15)对NK细胞表面NKG2D表达的影响.MTT法检测NK细胞对乳腺癌细胞的杀伤活性.结果: ELISA结果显示, 血清sMICA含量在健康成年人血清中为阴性, 在乳腺良性肿瘤患者血清中含量为(76.8±22.3)ng/L, 在恶性肿瘤患者血清中含量为(205.36±71.27)ng/L, 乳腺癌患者中血清sMICA含量高低与TNM分期呈正相关.应用含sMICA的血清与NK细胞共培养可明显下调NK细胞的杀瘤活性[(76.2±6.7)% vs (48.4±4.1)%], NK细胞表面NKG2D表达和上清中IFN-λ分泌量也下降.IL-15明显上调NK细胞表面NKG2D表达, 增加培养上清IFN-λ分泌量和增强NK对MCF-7的杀瘤活性.结论: sMICA表达与乳腺癌TNM分期正相关, sMICA通过下调NK细胞NKG2D表达水平而降低NK细胞杀瘤活性, 在肿瘤免疫逃逸中起重要作用.IL-15可以上调NK细胞NKG2D的表达并增强NK细胞杀瘤活性.     

HBV通过上调肝细胞表面Galectin-9的表达抑制NK细胞的功能

为了探讨HBV感染后Tim-3/Galectin-9信号通路对NK细胞功能的影响,我们首先利用RT-PCR和FACS法比较四种不同肝癌细胞系HepG2、HepG2-N1、HBV阳性的HepG2.2.15和HepG2-HBV细胞表面Galectin-9的表达差异,继而观察NK细胞对不同Galectin-9表达水平的靶细胞的杀伤效率和产生IFN-γ能力的变化,并采用Annexin V/PI双染法观察Galectin-9对NK细胞凋亡的影响。结果显示,HBV阳性的HepG2-HBV和HepG2.2.15细胞表面Galectin-9的表达明显高于HepG2和HepG2-N1细胞,NK细胞对高表达Galectin-9的HepG2-HBV和HepG2.2.15细胞的杀伤能力明显低于低表达Galectin-9的HepG2细胞,产生IFN-γ的能力亦明显降低。Galectin-9重组蛋白抑制NK细胞的杀伤效率并促进其发生凋亡;阻断Tim-3/Galectin-9信号可在一定程度上恢复NK细胞的功能。结果表明,HBV感染可通过诱导肝细胞表面Galectin-9的表达抑制NK细胞杀伤和IFN-γ分泌能力,并促使其发生凋亡,这可能是HBV感染易造成机体细胞免疫耐受的重要原因之一。     

重组人IL-18在大肠杆菌中的表达及其抗肿瘤作用

目的：获取rhIL-18原核表达产物并研究其活性。方法：用本室构建的含基因重组表达载体PBV220-IL-18的大肠杆菌DHSot，经42℃热诱导表达和包涵体提取纯化后获取rhIL-18蛋白；采用ELISA试剂盒检测rhIL-18刺激PBMC分泌IFN-γ的活性及MTT法检测其对NK细胞杀伤K562细胞自然杀伤活性；同时用荷瘤小鼠模型检测其体内抗瘤功能。结果：诱导含重组表达载体PBV220-IL-18的大肠杆菌D145ct后，蛋白电泳显示出一条相对分子量（Mτ）为18000的蛋白条带；10μg rhIL-18蛋白体外刺激PBMC后，其分泌IFN-γ的能力与对照组相比提高了8倍，细胞毒性提高3倍；同时rhIL-18蛋白能明显抑制肿瘤生长和延长荷瘤鼠生存期。结论：获得了有免疫调节功能和抗肿瘤活性的rhIL-18蛋白。为今后IL-18重组产物的研制和开展肿瘤的生物治疗提供了实验依据。     

CpG-ODN活化人NK细胞的初步研究

目的：研究D型CpG-ODN对人免疫细胞的活化效应。方法：CpG-ODN体外刺激人外周血单个核细胞(PBMC)，ELBA检测培养液IFN-α及IFN-γ的含量；RT-PCR检测PBMC中TLR9的表达水平；MTF法观察活化的NK对K562的杀伤作用。结果：CpG-ODN有效诱导PBMC分泌IFN-α和IFN-γ，增强活化的NK细胞对K562细胞的杀伤作用，且能显著上调TLR9 mRNA的表达。结论：在TLR9的介导下，CpG-ODN能有效激活NK细胞，参与机体免疫调节。     

不同肿瘤细胞表面MICA的表达及NK细胞杀伤活性的研究

目的：观察不同肿瘤细胞表面MICA的表达及其对NK细胞杀伤活性的影响。方法：以体外培养的K562（人慢性髓原白血病细胞株）、MCF-7（乳腺癌细胞株）、HR-8348（直肠腺癌细胞株）、CNE-2（人鼻咽癌细胞株）、Hela（人宫颈癌细胞株）为靶细胞，流式细胞仪检测细胞表面MICA的表达，以K562细胞作为对照，应用LDH释放法检测不同效靶比情况下体外培养的NK细胞对靶细胞的杀伤活性。结果：K562、MCF-7、HR-8348、CNE-2、Hela细胞表面均表达MICA，体外杀伤试验表明NK细胞对上述肿瘤细胞均有杀伤活性，anti-MICA单抗可部分封闭NK细胞对靶细胞的杀伤活性。结论：NK细胞对K562、MCF-7、HR-8348、CNE-2、Hela细胞具有较高的杀伤活性，可作为一种生物治疗手段。     

低剂量辐射对NK细胞生物学活性的影响

目的：探讨低剂量照射（LDR）对NK细胞体内及体外增殖和杀伤活性的影响,并探讨其相关的信号传导机制,同时将体外LDR后的NK细胞回输小鼠体内,观察LDR后的NK细胞对肿瘤的形成和生长的抑制作用。方法：从小鼠脾脏提出的单个核细胞,应用免疫磁珠的方法分离CD3^＋／CD16^＋／CD56^＋细胞,并应用流式细胞仪鉴定后咀总剂量为20Gy ^60Co γ射线照射后的小鼠脾细胞为饲养细胞,以重组人白介素-2（rhIL-2）为诱导因子。在体外进行NK细胞的扩增培养。培养后的细胞经流式细胞仪进一步鉴定其表型。体外实验将NK细胞分成阴性对照组,和25mGy、75mGy、200mGy和500mGy组,每组又分成照射后4h、24h、48h、72h组;体内实验将小鼠分成阴性对照组和75mGy照射后4h、24h、48h、72h组。体内体外实验均应用流式细胞仪检测各组细胞NK细胞的增殖指数,并应用^3H—TdR掺人入法检测其杀伤活性;体外实验我们同时验证LDR刺激NK细胞生长和活性的信号传导的饥制,将细胞分成阴性对照组、75mGy照射组、照射＋SB203580组、照射＋P79350组,每组细胞检测细胞的增埴啦度、活性变化及相关蛋白磷酸化P38MAPK、IFN-γ、FasL、perforin的变化,并统计它们之间的关系;体内实睑应用K562尾静脉输入和皮下移植制作K562成瘤模型,观察LDR后NK细胞回输到小鼠体内对K562成瘤的抑制作用及已经成瘤的K562小鼠肿瘤的生长的抑制作用。结果：应用上述方法可以成功培养出CD3^＋／CD16^＋／CD56^＋NK细胞,体内、体外低剂量照射后可以增加NK细胞的增殖指数,并增强其对K562细胞的杀伤活性。体外实验75mGy照射后24h最为明显。体内照射48h最明显;LDR后NK细胞表达磷酸化的P38MAPK、IFN-γ、FasL、perforin均增加,且与PI及杀伤活性呈正相关,与未照射的NK细胞比较,差异只有显著性（P〈0．05）,抑制P38MAPK表达后,PI、杀伤活性及相关蛋白IFN-γ,FasL、perfofin均下降;LDR后NX细胞回输到尾静咏注射成瘤模型小鼠中肝、脾CD13^＋、S期细胞及外周血异常细胞所占的百分率都明显下降,与束照射NK细胞同输纽比较,差异具有显著性（P〈0.05）,回输到经皮下抑制成瘤的小鼠模型中肿瘤体积、重量明显下降,小鼠的生存时问和生存率明显延长,与未照射NK细胞回输组比较,差异具有显著性（P〈0．05）。结论：低剂最辅射町必提高体内体外NK细胞的增殖和杀伤活性,机制是LDR可以增强P38MAPK信号传导的途径,LDR后NK细胞回输小鼠体内不但可以抑制K562细胞成瘤作用,还可以抑制已经成瘤的小鼠的肿瘤生长并提高生存时间和生存率。     

膜型/分泌型MICA对NK细胞受体NKG2D的相反调节效应及其对NK细胞受体谱的影响

目的　观察膜型和分泌型MICA对NK细胞受体表达的影响 ,以探讨NK细胞抗肿瘤活化机制及肿瘤细胞表达MICA分子的意义。方法　用MTT法测定人NK细胞系 (NK92 )的细胞毒活性 ;用RT PCR或FACS检测NK细胞受体 (NKG2D ,NKG2A B ,KIR2DL1,KIR2DS1)及NKG2D的识别配体MICA的表达。结果　肿瘤细胞表面的MICA分子可上调NKG2D的表达 ,下调抑制性受体NKG2A B和KIR2DL1的表达 ;而分泌型MICA (sMICA)分子对NKG2D及抑制性受体的表达均有抑制作用。结论　膜型MICA分子可上调NKG2D的表达 ,激发NK细胞对肿瘤细胞的细胞毒效应 ;分泌型MICA分子则通过降低NKG2D的表达下调机体的抗肿瘤免疫效应 ,肿瘤细胞分泌sMICA分子为肿瘤发生免疫逃逸的机制之一。     

Interferon-γ up-regulates major-histocompatibility-complex class I-related chain A expression and enhances major-histocompatibility-complex class I-related chain A-mediated cytolysis of human corneal epithelium by natural killer cells in vitro

Natural killer (NK) cells are important in the ocular surface innate response against viral and bacterial infection. Major-histocompatibility-complex class I-related chain A (MICA) antigens are ligands of natural killer group 2D, an activating or coactivating receptor expressed on NK cells. Recent studies demonstrated that interferon-gamma (IFN-γ) could modulate MICA expression in tumor cells. However, little is known about MICA expression and regulation in human corneal epithelium. Our study assessed whether the proinflammatory cytokine, IFN-γ, affects MICA expression in human corneal epithelium. We identified low levels of surface MICA expression in corneal epithelium using flow cytometry. IFN-γ promoted surface MICA expression in corneal epithelium and increased soluble MICA levels in a dose-dependent manner. IFN-γ also enhanced NK cell-mediated cytotoxicity against the corneal epithelium. Anti-MICA antibodies could further block this process. In summary, we describe a novel IFN-γ function in the regulation of the innate response in ocular surfaces.     

Immobilized MHC class I chain-related protein A synergizes with IL-15 and soluble 4-1BB ligand to expand NK cells with high cytotoxicity ex vivo

Major histocompatibility complex (MHC) class I chain-related protein A (MICA), which is a ligand for human NKG2D, is expressed by a variety of epithelial tumor cells and promotes the activation of natural killer (NK), CD8⁺ and γδ-T cells. Although ectopic expression of MICA on tumor cells elicits anti-tumor responses, soluble MICA downregulates the activities of lymphocytes. In this study, we showed that recombinant, immobilized MICA (iMICA) molecules coated on plastic wells weakly promote peripheral NK cell activation, secretion of interferon (IFN)-γ and degranulation without inducing apoptosis. In addition, iMICA synergized with IL-15 and soluble 4-1BB ligand (s4-1BBL) to expand NK cells 25- to 42-fold in a 13-day culture, whereas NK cells stimulated only with IL-15 and s4-1BBL expanded 10- to 16-fold. In contrast to NK cells expanded by IL-15 and s4-1BBL stimulation, NK cells expanded long term in the presence of iMICA exhibited increased cytotoxicity against leukemia cells. These results suggest that large numbers of NK cells with high cytotoxicity can be generated by stimulation with IL-15 and s4-1BBL in the presence of iMICA and that these cells can be used for adoptive cancer immunotherapy.     

MICA 第5 外显子微卫星多态性与食管癌相关性研究

目的:研究MICA 第5 外显子微卫星多态性与食管癌相关性。方法:采用PCR-STR 微卫星基因分型技术检测103 例食管癌患者和84 例正常对照MICA 基因5 外显子多态性。构建食管癌标本中高频率出现的MICA 等位基因的真核表达载体,转染293T 细胞株,LDH 法检测NK 细胞对不同MICA 等位基因转染的293T 细胞的杀伤作用,效靶比20 1。ELISA法检测转染的293T 细胞上清中sMICA 含量。结果:食管癌患者第5 外显子检测到5 种等位基因,频率分别为:MICA-A4(9.71%),MICA-A5(22.3%),MICA-A5.1(40.8%),MICA-A6(15.5%),MICA-A9(11.7%),其中MICA-A5.1 与对照组对比差异有统计学意义(P<0.05)。MICA 等位基因转染293T 细胞株后,相对于其他第5 外显子A5.1 组对NK 杀伤的敏感性较低[(30.4±6.3)%,P<0.05],上清可溶性MICA 分泌增加(135.7±6.2)pg/ ml。结论:食管癌与MICA 第5 外显子多态性A5.1 显著性相关,其危险性高于其他等位基因。     

内皮细胞表面主要组织相容性复合-Ⅰ类相关链A分子介导的NK细胞对其细胞毒作用的实验研究

目的观察内皮细胞表面主要组织相容性复合体-Ⅰ类相关链A(MICA)分子在NK细胞对其杀伤过程中的作用,并探讨其可能的机制.方法用10 ng/ml MICA重组抗原对实验组人脐静脉内皮细胞(HUVEC)进行诱导培养48 h,对照组加入等量磷酸盐缓冲液(PBS).用流式细胞仪(FCM)检测内皮细胞表面MICA分子的表达情况...     

IL-2-activated haploidentical NK cells restore NKG2D-mediated NK-cell cytotoxicity in neuroblastoma patients by scavenging of plasma MICA

NK group 2D (NKG2D)-expressing NK cells exhibit cytolytic activity against various tumors after recognition of the cellular ligand MHC class I chain-related gene A (MICA). However, release of soluble MICA (sMICA) compromises NKG2D-dependent NK-cell cytotoxicity leading to tumor escape from immunosurveillance. Although some molecular details of the NKG2D-MICA interaction have been elucidated, its impact for donor NK (dNK) cell-based therapy of solid tumors has not been studied. Within an ongoing phase I/II trial, we used allogeneic IL-2 activated dNK cells after haploidentical stem cell transplantation for immunotherapy of patients with high-risk stage IV neuroblastoma. NKG2D levels on activated dNK cells increased strongly when compared with freshly isolated dNK cells and correlated with enhanced NK-cell cytotoxicity. Most importantly, elevated sMICA levels in patients plasma correlated significantly with impaired dNK-cell-mediated cytotoxicity. This effect could be reversed by high-dose infusion of activated dNK cells, which display high levels of surface NKG2D. Our data suggest that the provided excess of NKG2D leads to clearance of sMICA and preserves cytotoxicity of dNK cells via non-occupied NKG2D. In conclusion, our results identify this tumor immune escape mechanism as a target to improve immunotherapy of neuroblastoma and presumably other tumors.     

缺氧微环境下ADAM17与ERp5的高表达对肺癌细胞逃避NK细胞免疫杀伤的机制研究

目的 探讨缺氧条件下ADAM17与ERp5的高表达对肺癌细胞逃避NK细胞免疫杀伤的影响.方法 免疫组织化学法检测肺癌组织和癌旁组织中ADAM17与ERp5的表达;qRT-PCR与Western blot检测缺氧条件下的肺癌细胞中ADAM17、ERp5、MICA与MICB的表达;小干扰RNA内源性敲低ADAM17与ERp...     

A subgroup of lupus patients with nephritis,innate T cell activation and low vitamin D is identified by the enhancement of circulating MHC class I-related chain A

The major histocompatibility complex (MHC) class I-related chain A (MICA) is induced upon stress, and labels malfunctioning cells for their recognition by cytotoxic lymphocytes. Alterations in this recognition and also abnormal natural killer (NK) functions have been found in systemic lupus erythematosus (SLE). MICA can be shed from cells, subsequently acting as a soluble decoy receptor (sMICA). Our purpose was to study circulating sMICA levels in relationship with the activation of innate pathways in PBMC in a cohort of lupus patients. NK cells were characterized by flow cytometry. Gene expression of Toll-like receptors (TLR), interferon (IFN)-I sensitive genes and MICA were separately analyzed in monocytes, T cells and B cells. Serum sMICA was measured with enzyme-linked immunosorbent assay (ELISA). In our cohort, NK cell counts dropped in relationship with disease activity. sMICA showed an inverse trend with NK cell counts, as well as a significant association with activity indices, but not with complement decrease. Levels of sMICA associated to proteinuria and active nephritis. A multivariate regression model revealed anti-nuclear antibody (ANA) titres, the up-regulation of TLR-4 in T cells and lower vitamin D as predictors of sMICA enhancement. Interestingly, vitamin D showed an inverse association with proteinuria and a strong correlation with T cell MICA mRNA levels. According to our data, circulating sMICA identifies a subgroup of lupus patients with low vitamin D, innate activation of T cells and nephritis. We propose that lymphocyte shedding could account for the enhancement of sMICA and reflect an immune evasion mechanism driving disease activation in lupus.     

膜型/可溶型MHC Ⅰ类链相关分子A及其受体NKG2D在骨肉瘤中的表达及其意义

目的探讨膜型／可溶型MHCI类链相关分子A（MICA）及其受体NKG2D在骨肉瘤中的表达及其意义。方法采用免疫组织化学（LSAB法）检测膜型MICA（mMICA）在43例骨肉瘤组织中的表达;用流式细胞仪检测上述病例中的16例患者外周血淋巴细胞NKG2D的表达;用ELISA法检测患者血清中可溶型MICA（sMICA）的水平。结果骨肉瘤细胞广泛表达mMICA（37／43）,外周血淋巴细胞NKG2D的表达普遍下降,两者的表达水平均与骨肉瘤的分化呈正相关（P〈0．05）,与临床分期呈负相关（P〈0．05）。检测16例骨肉瘤患者血清中sMICA含量,其中5例升高,且与骨肉瘤的远处转移及临床分期呈正相关（P〈0．05）。结论（1）骨肉瘤细胞表达mMICA,外周血淋巴细胞NKG2D的表达以及血清中sMICA的含量与骨肉瘤的分化及临床分期相关,因此可望作为骨肉瘤病理诊断和估计预后的生物学指标。（2）MICA-NKG2D介导的免疫监视障碍可能促进了骨肉瘤的进展。