NKG2D配体在13种肿瘤细胞系中的表达及意义

背景与目的:NKG2D及其配体的相互作用在肿瘤免疫监视中起着非常重要的作用。本研究探讨NKG2D配体在13种肿瘤细胞系中的表达及其意义。方法:采用半定量RT-PCR法检测肿瘤细胞系中NKG2D配体的mRNA表达。应用MTT法检测人NK细胞对肿瘤细胞的杀伤活性,免疫组织化学技术和Western blot法检测肿瘤细胞中MHCⅠ类相关蛋白A(MHC classⅠchain-related A)蛋白表达情况。结果:13种肿瘤细胞系表达不同水平的NKG2D配体mRNA。其中Hep2细胞中MICA强阳性表达,HeLa、HepG2、MDA231、HT29、SGC7901、M21、K562、Jurkat细胞MICA表达阳性,而Caski、PG、HL-60和Raji细胞中MICA mRNA阴性。MICA和MICB的表达水平与NK细胞杀伤活性具有高度相关性(r=0.851,P<0.001;r=0.652,P<0.05)。除ULBP3外,其余ULBP成员的表达水平与NK细胞杀伤均无相关性。结论:在6种人NKG2D配体中,MICA的表达水平与肿瘤细胞对NK细胞杀伤的敏感性关系最为密切,肿瘤细胞MICA的表达水平可能决定着机体NK细胞抗肿瘤免疫应答的强弱。     

γ干扰素对人NK细胞识别功能的负调节作用

目的 探讨γ干扰素(IFN-γ)对人NK细胞识别功能的负调节作用。方法 用MTT法测定人NK细胞系(NK92,NKL)的细胞毒活性及细胞增殖能力;用RT-PCR检测NK细胞受体(NKG2D、NKG2A／B、KIR2DLI、KIR2DSI)及NKG2D的识别配体主要组织相容性复合体Ⅰ类链相关分子A(MICA)的表达。结果 NK细胞系(NK92、NKL)对MICA表达阳性的肿瘤细胞杀伤活性明显高于对MICA表达阴性者;IFN-γ 1000U／ml以上可明显抑制NK细胞对MICA表达阳性肿瘤细胞的细胞毒活性,并轻度抑制NK细胞的增殖,而对MICA表达阴性肿瘤细胞的杀伤活性无明显抑制作用;IFN-γ可抑制NK细胞系活化受体NKG2D的表达,增强抑制性受体NKG2A／B和KIR2DLI的表达。结论 IFN-γ可能通过下调NK细胞活化受体的表达,上调抑制性受体的表达,使NK识别的信号平衡向抑制性方向倾斜,从而对NK细胞功能发挥负调节作用,这种作用可能是NK细胞自我调节功能的表现。     

食管癌中MICA的表达及其意义

  目的  观察食管癌细胞株组织膜MHCⅠ链相关分子A(MHC class Ⅰ chain-related gene A, MICA)及血清中可溶性MICA表达, 探讨食管癌中MICA的表达及其临床意义。  方法  流式细胞术检测膜型MICA(mMICA)及NK细胞表面NKG2D表达, ELISA方法检测乳腺肿瘤患者外周血可溶性MICA(sMICA)的分泌。分组实验分析可溶性MICA(sMICA)对NKG2D表达的影响。  结果  食管细胞膜型MICA在食管癌旁组织无表达, 在31.7%食管癌组织有表达, 表达量为(42.2±3.6)%。可溶性MICA含量在健康成年者为阴性, 食管癌患者中74.67%阳性, 含量为(1977.30±292.67)ng/L。食管癌患者中血清sMICA含量高低与TNM分期呈正相关, 远处转移组比无远处转移组高(P < 0.05), 但和病理类型表达无差异。含可溶性M!CA的血清可明显下调NK细胞NKG2D的表达, 从而降低NK细胞对食管癌细胞株ECA-109的杀伤活性。  结论  食管癌细胞膜表达MICA, 可作为食管肿瘤相关性抗原。而可溶性MICA含量高低与TNM分期呈正相关, 可通过下调NK细胞NKG2D表达介导肿瘤免疫逃逸。      

磁性纳米铁联合多柔比星对Raji细胞的影响

 目的　观察干扰素α（IFN-α）对恶性造血细胞系主要组织相容性复合体（MHC）Ⅰ类链相关蛋白A（MICA）表达的影响。方法　反转录-聚合酶链反应（RT-PCR）法检测肿瘤细胞MICA mRNA的表达,免疫组织化学法检测MICA蛋白的表达,四甲基偶氮唑蓝（MTT）比色法检测人类外周血自然杀伤（NK）细胞对肿瘤细胞的杀伤。结果　人类慢性髓系白血病细胞K562 MICA表达阳性,人类伯基特淋巴瘤细胞Raji MICA表达阴性。Raji和K562细胞分别在1000 U／ml IFN-α诱导24 h（t＝17.016,P＜0.05）和48 h（t＝5.616,P＜0.05）时MICA mRNA开始表达上调。72 h Raji细胞和K562细胞分别在500 U／ml（t＝6.622,P＜0.05）和1000 U／ml（t＝9.071,P＜0.05）诱导条件下MICA mRNA表达开始上调。IFN-α对MICA mRNA表达的上调作用呈一定时间和剂量依赖关系,且蛋白与mRNA表达水平一致。1000 U／ml IFN-α诱导72 h后,Raji细胞（t＝20.016,P＜0.05）和K562细胞（t＝7.969,P＜0.05）对NK细胞杀伤的敏感性明显上调,抗MICA抗体封闭MICA后,NK细胞对Raji细胞杀伤率恢复至诱导前水平（t＝0.393,P＞0.05）,而K562细胞则低于诱导前水平（t＝9.841,P＜0.05）。结论　IFN-α上调了恶性造血细胞中MICA 基因的转录表达,继而增强了NK细胞对其识别与杀伤。     

耐地塞米松B细胞淋巴瘤细胞逃逸同种异体NK细胞的杀伤及其机制

目的:观察地塞米松耐药的人B细胞淋巴瘤细胞系对NK细胞杀伤敏感性的变化,并探讨其作用机制。方法:20μg/ml地塞米松(dexamethasone,DXM)诱导B细胞淋巴瘤细胞系SU-DHL-4(简称SU细胞)发生耐药,建立多药耐药细胞系SU/DXM。流式细胞术分选健康人外周血NK细胞,流式细胞术检测效靶比20∶1时,NK细胞对SU和SU/DXM细胞的杀伤效应。实时定量PCR检测SU和SU/DXM细胞表面NK细胞活化性受体(soluble NK group 2 member D,NKG2D)配体基因[可溶性MHCⅠ类分子相关A/B(MHC classⅠchain-related molecules A/B,MICA/B)及人UL16结合蛋白(UL16 binding protein,ULBP)1、2、3]的表达。结果:成功建立多药耐药细胞系SU/DXM。与SU细胞相比,SU/DXM细胞对NK细胞杀伤的敏感性明显下降[SU细胞为(11.38±3.51)%,SU/DXM细胞为(3.57±4.22)%,P<0.05],细胞表面NKG2D配体基因MICA、MICB、ULBP2 mRNA表达量降低(SU细胞分别为1.014±0.121、1.009±0.092、0.993±0.108,SU/DXM细胞分别为0.017±0.006、0.682±0.063、0.773±0.066,P<0.05或P<0.01)。结论:地塞米松能诱导B细胞淋巴瘤SU细胞发生多药耐药,多药耐药SU/DXM细胞能够抵抗NK细胞的杀伤,其机制可能与NKG2D配体基因表达量下降有关。     

肿瘤生物治疗的新模式:分子靶向-过继性细胞免疫治疗

分子靶向－过继性细胞免疫治疗是分子靶向药物和过继性细胞免疫疗法有机结合的新治疗模式,该模式建立在NK细胞活化性信号通路(NKG2DNKG2DLs) 的生物学特性,尤其是受配体可调控性的理论基础之上。分子靶向药物在其中扮演双重角色,除了药物本身对肿瘤细胞的毒性作用外,还作为“刺激诱导”源,诱导肿瘤细胞表达免疫激活物NKG2DLs（natural killer group 2 member D ligands）,与NK细胞表面NKG2D（natural killer group 2 member D）结合,激活NK细胞的杀伤活性。NKG2D与NKG2DLs是NK细胞主要的活化性受、配体,在机体抗肿瘤免疫中起重要作用。NKG2D主要表达于NK、CD8+T、γδT和活化的巨噬细胞,参与适应性及固有性免疫应答。与NKG2D结合的配体(NKG2DLs)广泛低表达于多种肿瘤细胞,而在正常组织细胞几乎未见表达,靶细胞的NKG2DLs表达水平直接关系到免疫效应细胞（NK、DC、CTL细胞等）对其的杀伤活性。NKG2DLs的转录与表达可受多种因素影响,分子靶向药物可以诱导肿瘤细胞高表达NKG2DLs,NK细胞对高表达NKG2DLs的肿瘤细胞有较高杀伤活性,而对正常组织无杀伤作用,具有杀伤靶向性;NKG2DLs的高表达也增强了肿瘤细胞对其他免疫效应细胞的杀伤敏感性,具有良好的应用前景。分子靶向药物联合过继性细胞免疫治疗将使肿瘤患者获得更好的临床疗效,预示了肿瘤生物治疗新的发展方向——分子靶向-过继性细胞免疫治疗新模式的来临。     

硼替佐米提高耐药K562/ADM细胞对NK细胞杀伤的敏感性及其机制

目的:探讨硼替佐米提高耐药K562/ADM细胞对NK细胞杀伤敏感性的可能机制.方法:流式细胞术和real-time PCR检测硼替佐米处理前后K562/ADM细胞表面MHC Ⅰ类链相关分子A(major histocompatibility complex class Ⅰ chainrelated molecule A,MICA)蛋白和mRNA的表达,LDH释放法检测硼替佐米处理前后K562/ADM细胞对NK细胞的杀伤敏感性.结果:硼替佐米处理后,K562/ADM细胞表面MICA蛋白表达率上升[(17.03 ±4.94)％vs(23.77±5.26)％,P＜0.05];处理后K562/ADM细胞MICA mRNA的表达水平是处理前的(2.03±0.33)倍.效靶比为10∶1、20∶1时,NK细胞对硼替佐米处理后的K562/ADM细胞的杀伤率上升[(23.22±3.03)％、(30.30±0.74)％ vs(33.69±1.28)％、(41.40 ±1.97)％,P＜0.05].结论:硼替佐米提高耐药K562/ADM细胞对NK细胞杀伤的敏感性,其机制可能与硼替佐米上调K562/ADM细胞MICA表达有关.     

HLA-G反义基因增强免疫效应细胞杀伤活性的研究

目的:研究HLA-G反义寡核苷酸逆转肿瘤细胞免疫逃逸,提高免疫效应细胞识别杀伤活性的作用和机制.方法:采用反义核酸技术合成HLA-G反义寡核苷酸(ASODN),硫代化修饰与脂质体形成复合物,转导入高表达HLA-G的绒毛膜癌细胞系JEG-3.采用RT-PCR方法检测HLA-G mRNA表达水平的变化;流式细胞术检测细胞表面HLA-G蛋白表达水平的变化;MTT法检测:NK杀伤活性、CD3AK杀伤、增殖活性的变化;ELISA方法探讨ASODN调节CD3AK细胞因子IFN-γ产生的变化.结果:HLA-G ASODN可显著抑制HLA-G mRNA和蛋白水平的表达,逆转可溶型HLA-G分子对NK细胞杀伤活性的抑制作用;可部分逆转HLA-G分子对CD3AK细胞产生IFN-γ的抑制作用,并增强CD3AK的增殖活性,增强JEG-3细胞对CD3AK的杀伤敏感性.结论:HLA-G ASODN通过抑制肿瘤细胞HLA-G mRNA和蛋白水平的表达,增强NK,CD3AK的杀伤作用和机制,发挥逆转肿瘤细胞免疫逃逸作用.     

细胞因子组合体外扩增人NK细胞的研究

目的：探讨细胞因子IL-2、IL-12、IL-15和IL-18组合对体外扩增人外周血来源的NK细胞受体表达及杀伤肿瘤细胞能力。方法：NK细胞的培养分为cIK组、IL2＋II,15组和IL-2＋IL-12＋IL-15＋IL-18组;流式细胞术检测培养的细胞表型及NK细胞受体表达;LDH法检测不同效靶比NK细胞对不同肿瘤细胞株的杀伤作用。结果：外周血淋巴细胞经细胞因子IL-2＋IL广12＋IL-15＋IL-18组合作用下,17d后NK细胞（CD3-CD56＋）比例上升至80％以上,NK细胞数扩增〉1000倍,明显高于其他两组,F=37．154,P〈0．001。NK细胞活化标志CD69。‘分子增加（t-21．271,P〈0．001）,NK细胞活化性受体（NKG2D、NKp30、NKp44和NKp46）均有不同程度上调（P值均〈0．05）,而NK细胞抑制性受体CDl58b（t=3．416,P=0．021）和CDl59a（t=4．209,P=0．018）不同程度下调,T淋巴细胞、B淋巴细胞（CD19＋）、单核巨噬细胞（CD14＋）、调节性T细胞（T—reg）等均显著减少,P〈0．001。对扩增的NK细胞杀伤敏感的肿瘤细胞株均高表达MHCI类相关蛋白A（majorhistocompatibility complexclass Ichainrelated moleculesA,MICA）,MICA表达分别为K562（46．2±3．2）％、MCF-7（56．5±4．7）％、HTC-8（52．5±4．1）％和Eca-109（36．5±2．5）％,而对NK细胞抵抗的细胞株均低表达或不表达MICA分子,分别为Raji0、MDA-MB-435s0和HT-29（1．2±0．8）％。结论：细胞因子IL-2＋IL-12＋IL-15＋IL-18组合能有效的扩增外周血来源NK细胞,上调其活化性受体,下调抑制性受体,其数量及功能均满足临床治疗需要。     

沉默HIG2促进NK细胞对喉癌细胞杀伤敏感性的机制探讨

目的:探讨缺氧诱导基因2（HIG2）在喉癌细胞逃逸NK细胞免疫杀伤中的作用机制。方法:免疫组织化学法检测喉癌组织和癌旁组织中HIG2的表达;Western blot检测HIG2、NKG2D配体（MICA、MICB、ULBP1、ULBP2和ULBP3）的蛋白表达;Transwell实验检测Hep-2细胞的侵袭;流式细胞术检测Hep-2细胞的凋亡、CD3-CD56+的NK细胞纯度、NKG2D受体表达及Hep-2细胞NKG2D配体的表达;乳酸脱氢酶释放法检测不同效靶比时,NK细胞对Hep-2细胞的杀伤活性。结果:HIG2在喉癌组织中高表达。CD3-CD56+的NK细胞纯度大于90%。沉默HIG2抑制Hep-2细胞侵袭,促进细胞凋亡,提高NKG2D配体的表达,增强NK细胞对Hep-2细胞的杀伤敏感性。NKG2D抗体抑制NK细胞NKG2D受体表达,减弱NK细胞对沉默HIG2 的Hep-2细胞的杀伤活性。结论:沉默HIG2可抑制喉癌细胞侵袭,促进细胞凋亡,上调NKG2D配体表达,增强NK细胞对喉癌细胞的杀伤敏感性。     

Regulation Roles of MICA and NKG2D in Human Renal Cancer Cells

Objective: Our aim was to investigation the roles of MHC class I chain-related gene A(MICA) and naturalkiller cell group 2D(NKG2D) in human renal cancer cells. Materials and Methods: The expression of membraneMICA (mMICA) on renal cells and NKG2D on NK cells were detected by flow cytometry (FCM); the contentof sMICA were detected by enzyme linked immunosorbent assay (ELISA) and the distribution of mMICA onrenal tumor tissues by immunohistochemistry; the interaction between MICA and NKG2D was observed byantibody closed method. Results: Our results showed that the expression of mMICA in renal cancer tissues wassignificantly higher than in controls, where the soluble MICA was not expressed. Cytotoxic activity of NK cellswas significantly reduced after exposure to NKG2D and MICA antibodies (P<0.05), and serum containing sMICAcan obviously lower the function of NKG2D (P<0.05). Conclusions: The interaction of mMICA and NKG2Dplay important roles in mediation of cytotoxicity of NK cells in RCC. On the other hand, sMICA may mediatetumor immune escape through down- regulated NKG2D expression.     

MICA triggering signal for NK cell tumor lysis is counteracted by HLA-G1-mediated inhibitory signal

MICA, a highly glycosylated membrane-anchored cell-surface MHC Class I-related chain, has recently been reported to activate NK cell cytolytic responses in epithelial tumors. Tumor cells may escape from NK lysis by counteracting NK cytotoxicity activating signals with inhibitory ones. Among the molecules that mediate an NK inhibitory signal, HLA-G1, a non-classical MHC Class I antigen, is of particular interest. HLA-G1 is ectopically expressed in various tumors, including melanoma and constitutes the major NK inhibitory ligand in the M8 melanoma cell line when coexpressed with HLA-A, -B, -C and -E molecules. We have evaluated the balance between 2 powerful signals that affect NK cell tumor lysis, one inhibitory and the other one activating, respectively HLA-G1 and MICA. For this purpose, we transfected the M8 melanoma cell line, which spontaneously expresses MICA, with HLA-G1 cDNA, using it as a target for the NKL effector. We carried out cytotoxicity assays, using antibodies that disrupt interactions between the MICA and HLA-G1 ligands and their respective NK effector counterparts, the NKG2D activating and ILT2 inhibitory receptors. Results showed that 1) MICA expressed in the M8 melanoma cell line triggered NK cell tumor lysis and 2) HLA-G1 coexpression mediated the inhibition of NK cytotoxicity by mitigating the MICA activating signal. HLA-G1 expression in a tumor cell line in which MICA is switched on would therefore appear to be a powerful way to turn off NK cells, supporting the emerging idea that the balance between positive and negative NK cytolysis signals critically influences tumor progression.     

5-Fluorouracil and Interleukin-2 Immunochemotherapy Enhances Immunogenicity of Non-Small Cell Lung Cancer A549 Cells through Upregulation of NKG2D Ligands

Background: The aim of this study was to investigate the anti-cancer effects and mechanisms ofimmunochemotherapy of 5-fluorouracil (5-FU) and interleukin-2 (IL-2) on non-small cell lung cancer (NSCLC)A549 cells. Materials and Methods: In order to detect whether 5-FU+IL-2 could effectively inhibit tumorgrowth in vivo, we established an A549-bearing nude mouse model. The cytotoxicity of natural killer (NK) cellswas evaluated using a standard chromium release assay. To evaluate the relevance of NK cells in 5-FU+IL-2-mediated tumor inhibitory effects, we depleted NK cells in A549-bearing mice by injecting anti-asialo-GM-1antibodies. Effects of 5-FU+IL-2 on the expression and promoter activity of NKG2D ligands (MICA/MICB) inA549 cells in vitro were also assessed. Results: In A549-bearing nude mice, combination therapy significantlyinhibited tumor growth in comparison with monotherapy with 5-FU or IL-2 and enhanced the recognitionand lysis of tumor cells by NK cells. Further study of mechanisms showed that NK cells played a vital role inthe anticancer immune response of 5-FU+IL-2 immunochemotherapy. In addition, the combination therapysynergistically stimulated the expression and promoter activity of MICA/MICB. Conclusions: 5-FU and IL-2immunochemotherapy significantly inhibited tumor growth and activated NK cytotoxicity in vivo, and theseeffects were partly impaired after depleting NK cells in tumor-bearing mice. Combination treatment of 5-FUand IL-2 upregulated the expression and the promoter activity of MICA/MICB in A549 cells, which enhancedthe recognition of A549 cells by NK cells. All of the data indicated that immunochemotherapy of 5-FU and IL-2may provide a new treatment option for patients with lung cancer     

丙戊酸钠上调人肾癌细胞MICA/B的表达并增强自然杀伤细胞的抗肿瘤活性

目的:观察丙戊酸钠(valproic acid,VPA)对人肾癌细胞主要组织相容性复合体Ⅰ类相关链A/B (major histocompatibility complex class Ⅰ -related chain A/B,MICA/B)表达的影响,比较肾癌细胞经VPA处理前后自然杀伤( natural liller,NK)细胞对其杀伤作用的差异.方法:用0.5～8.0 mmol/L VPA分别处理人肾癌786-O和ACHN细胞后,应用FCM检测不同浓度VPA对细胞活力的影响.选择4.0 mmol/L VPA处理肾癌细胞,通过实时荧光定量PCR和FCM分别检测肾癌细胞MICA/B mRNA和蛋白的表达水平；钙黄绿素释放法和FCM分别检测VPA处理肾癌细胞后NK细胞对其的杀伤作用以及NK细胞的脱颗粒行为.结果:8.0 mmol/L VPA处理肾癌细胞48 h后,对肾癌细胞活力的影响大于其他处理组(P＜0.05); 4.0 mmol/L VPA处理组肾癌细胞MICA/BmRNA和蛋白的表达水平均明显高于未经VPA处理的对照组(P＜0.05); 4.0 mmol/L VPA处理组NK细胞对肾癌细胞的杀伤率明显高于未经VPA处理的对照组(P＜0.05),且杀伤率的升高可被抗NKG2D (natural-killer group 2,member D)抗体特异性拮抗；肾癌细胞经4.0 mmol/L VPA处理后,其激活NK细胞的脱颗粒作用明显强于未经VPA处理的对照组(P＜0.05).结论:VPA可以明显上调肾癌细胞MICA/B的表达,增强NK细胞对其的杀伤活性.     

干扰素α对恶性造血细胞系主要组织相容性复合体Ⅰ类链相关蛋白A表达的上调作用

目的观察干扰素a（IFN-a）对恶性造血细胞系主要组织相容性复合体（MHC）I类链相关蛋白A（MICA）表达的影响。方法反转录-聚合酶链反应（RT-PCR）法检测人类慢性髓系白血病细胞K562及人类伯基特淋巴瘤细胞Raji细胞MICAmRNA的表达,免疫组织化学法检测MICA蛋白的表达,四甲基偶氮唑蓝（MTF）比色法检测人类外周血自然杀伤（NK）细胞对肿瘤细胞的杀伤。结果K562细胞MICA表达阳性,Raji细胞MICA表达阴性。Raji和K562细胞分别在1000U／ml IFN-a诱导24h和48h时MICAmRNA开始表达上调（t=17．016,P〈0．05;t=5．616,P〈0．05）。72hRaji细胞和K562细胞分别在500U／ml和1000U／ml诱导条件下MICAmRNA表达开始上调（t=6．622,P〈0．05;t=9．071,P〈0．05）。IFN—Ot对MICAmRNA表达的上调作用呈一定时间和剂量依赖关系,且蛋白与mRNA表达水平一致。1000U／ml IFN—a诱导72h后,Raji细胞和K562细胞对NK细胞杀伤的敏感性明显上调（t=20．016,P〈0．05;t=7．969,P〈0．05）,抗MICA抗体封闭MICA后,NK细胞对Raji细胞杀伤率恢复至诱导前水平（t=0．393,P〉0．05）,而K562细胞则低于诱导前水平（t=9．841,P〈0．05）。结论IFN—a上调了恶性造血细胞中MICA基因的转录表达,继而增强了NK细胞对其识别与杀伤。