膜表达的HLA-G1对同种反应性PBMC分泌Th1/Th2型细胞因子的影响

目的研究人脐静脉内皮细胞系(ECV304)表达的HLA-G1对同种异体外周血单个核细胞(PBMC)Th1/Th2型细胞因子分泌的影响。方法采用脂质体介导的基因转染技术将pcDNA3-HLA-G1转入ECV304,以间接免疫荧光技术在蛋白质水平上检测HLA-G1分子在ECV304上的表达;以表达HLA-G1的ECV304作为刺激细胞,灭活后,与健康人PBMC共同培养,用ELISA检测上清液中Th1/Th2型细胞因子的浓度,观察HLA-G1对同种异体抗原激活的PBMC分泌Th1/Th2型细胞因子的影响。结果与转染pcDNA3空质粒的对照组相比,HLA-G1能使PBMC的IL-10分泌增加(P<0.05),而IL-2,IFN-γI、L-4分泌无明显影响。结论HLA-G1能引起由同种异体抗原激活的PBMC分泌IL-10增加,提示HLA-G1有可能使Th1/Th2型细胞因子向Th2型偏移。     

同种异体NK细胞对人脐静脉内皮细胞ECV304的杀伤活性差异及分子机制探讨

目的 探讨供者KIR分子表达差异对NK细胞杀伤人脐血内皮细胞系ECV304活性的影响,并观察参与杀伤的活化信号通路.方法 RT-PCR及流式细胞仪检测ECV304细胞NKG2D配体MICA/B、ULBP1-3表达,PCR-SSP法行HLA-A、B、Cw分型.自8例健康供者分离外周血NK细胞,流式细胞仪检测KIR2DL1的表达率,LDH释放法测定NK细胞在效靶比20∶1时对ECV304细胞的杀伤活性及anti-KIR2DL1mAb对NK细胞杀伤活性的影响.结果 ECV30细胞在mRNA水平表达MICA/B、ULBP1-3,但在膜蛋白水平均不表达.HLA分型表明,ECV304表达KIR2DL1的配体,而不表达KIR2DL2/3、KIR3DL1的配体.8例健康供者NK细胞KIR2DL1表达率有较大差异,对ECV304细胞的杀伤活性也有不同,双变量相关分析示个体KIR2DL1表达率与NK细胞对ECV304的杀伤率存在负相关(rS=-0.994,P=0.000).anti-KIR2DL1 mAb明显增强NK细胞对ECV304的杀伤活性(t=-4.860,P=0.002).结论 NK细胞对ECV304细胞的杀伤分子机制主要为HLA-KIR信号系统错配,目前已知的NKG2D配体MICMB、ULBP1-3并不参与,这有助于临床活体器官移植时在遗传指导下选择供体.     

肝癌Hep3B细胞HLA-G过表达对NK细胞体外杀伤作用的影响

目的: 探讨肝癌Hep3B细胞白细胞抗原-G(HLA-G)过表达对NK细胞体外杀伤作用的影响。方法: 实验分为正常对照组(无干预措施, Hep3B组)、空载体对照组(转染空质粒, Hep3B-GFP组)和实验组(转染pEGFP-C1-HLA-G质粒,Hep3B-HLA-G组)。构建HLA-G真核表达质粒,转染至Hep3B细胞,经G418筛选,建立稳定过表达HLA-G的肝癌Hep3B细胞。Real-time PCR及Western blotting检测肝癌Hep3B细胞HLA-G mRNA及蛋白质水平的表达。分析肝癌Hep3B细胞HLA-G过表达及HLA-ABC抗体封闭靶细胞相应位点对NK细胞体外杀伤作用的影响。结果: 经酶切及测序鉴定证实, 插入序列与设计的序列相符。Real-time PCR和Western blotting结果均表明,转染重组质粒的Hep3B细胞HLA-G过表达(P<0.01)。NK细胞对转染pEGFP-C1-HLA-G的Hep3B细胞杀伤作用明显降低(P<0.01),HLA-ABC单抗封闭靶细胞相应位点后,NK细胞对Hep3B、Hep3B-GFP和Hep3B HLA-G细胞的杀伤均增加(P<0.01)。结论: pEGFP-C1-HLA-G表达载体可有效增加HLA-G表达,HLA-G过表达可以削弱NK细胞对肝癌细胞的杀伤效应。HLA-ABC单抗封闭靶细胞相应位点后,NK细胞对靶细胞的杀伤能力普遍有所提高。     

表达HLA—G1的K562细胞抵抗外周血NK细胞杀伤作用的研究

目的 研究HLA－G1分子对NK细胞杀伤活性的影响。方法 供助脂质体介导的DNA转染技术,将本室构建的真核南闰pcDNA3-HLA-G1转染人K562细胞;通过G418筛选,获得克隆化细胞株K562－G1;应用RT－PCR及流式细胞术分别在RNA水平和蛋白质水平检测HLA－G1的表达;最后,应用MTT比色法检测转染细胞对不同个体外周血NK细胞杀伤活性的抑制效应。结果 与转染了空质粒的对照组相比,外周血NK细胞对K562－G1的杀伤率降低32.43%（P＜0.01）。结论 靶细胞表达HLA－G2分子可明显抑制NK细胞的杀伤效应。     

HIV-1 Nef通过Erk/Mapk途径调节内皮细胞黏附分子ICAM-1的表达

目的:研究HIV-1Nef基因对内皮细胞ECV304细胞ICAM-1表达的影响及其信号途径。方法:选用Nef基因稳定表达细胞株ECV304-Nef和对照细胞株ECV304pcDNA3.1(+),应用Westernblot分析ECV304-Nef细胞ERK的磷酸化水平,利用ERK磷酸化抑制剂通过Westernblot、流式细胞术分析ECV304-Nef细胞ICAM-1的表达水平与信号分子ERK磷酸化的相关性。结果:Westernblot显示ECV304-Nef细胞ICAM-1和p-ERK蛋白的表达水平均高于对照组;流式细胞术检测结果表明ECV304-Nef细胞和对照组ICAM-1阳性细胞百分率分别为(35.3±2.2)%和(12.5±0.8)%(P0.01)。加入p-ERK抑制剂PD98059后,ECV304-Nef细胞p-ERK水平被显著抑制,ICAM-1降至对照组水平,ECV304-Nef细胞和对照组细胞ICAM-1阳性细胞百分率分别为(11.4±1.1)%和(10.4±1.5)%(P0.05)。结论:HIV-1Nef基因上调血管内皮细胞细胞黏附分子ICAM-1的表达与ERK信号分子磷酸化有关,为HIV-1感染的致病机制及临床治疗提供实验基础。     

接触抑制下调CD112和CD155在ECV304细胞上的表达伴有对NK细胞杀伤的抵抗

目的：研究细胞培养接触抑制条件下，ECV304细胞表面CD112和CD155表达的变化及其对NK细胞杀伤敏感性的变化。方法：以ECV304细胞接触抑制为模型，应用免疫荧光染色和流式细胞术分析，观察CD112和CD155分子在对数生长状态和发生接触抑制状态ECV304细胞上表达情况的变化；采用51Cr-4h释放试验，观察NK细胞对2种状态ECV304细胞杀伤活性的改变。结果：CD112和CD155分子在ECV304细胞发生接触抑制时表达水平明显低于对数生长期细胞。NK细胞对发生接触抑制的ECV304细胞的杀伤率比对数生长期的细胞有所下降。结论：接触抑制会使ECV304细胞对NK细胞杀伤有一定抵抗，同时伴有CD112和CD155分子表达的下调，两者间是否存在因果关系值得进一步深入探讨。     

膜结合型HLA-G1～G4分子的克隆表达及对NK细胞杀伤功能的影响

目的 探讨膜结合型HLA-G1～G4异构体的表达及对NK细胞杀伤功能的影响.方法 通过基因克隆及转染,分别建立稳定表达HLA-G1～G4抗原的人绒癌JAR细胞株.采用RTPCR、流式细胞术、Western blot及免疫细胞化学法分析、鉴定转染细胞中HLA-G的mRNA及蛋白表达.通过加载HLA-G高亲和性KIPAQFYIL抗原肽,观察对HLA-G表达的影响.LDH释放法检测HLA-G1～G4表达对NK细胞杀伤活性的影响.结果 RT-PCR、Western blot及免疫细胞化学结果显示,HLA-G1～G4/pVITRO2-mcs重组质粒成功转染HLA-G表达阴性的人绒癌JAR细胞株.FACS分析显示HLA-G1抗原能在JAR-HLA-G1细胞株表面表达,HLA-G2～G4抗原不能有效到达细胞表面.体外杀伤试验发现表达HLA-G1～G4抗原的细胞均能抑制NK细胞的杀伤活性(P＜0.05);加载HLA-G高亲和性KIPAQFYIL抗原肽对HLA-G表达无明显影响,对NK细胞杀伤抑制程度也未见明显改变.结论 HLA-G1～G4能够明显抑制NK细胞的杀伤活性,提示不同膜结合型HLA-G异构体分子均能作为免疫耐受分子,具备免疫调节功能.     

HLA-G在卵巢癌组织中的表达及对NK细胞杀伤活性的影响

通过免疫组织化学法(IHC)检测卵巢癌组织中HLA-G的表达;克隆编码全长HLA-G1cDNA,将该基因通过真核表达载体pVITRO2-mcs转染至HLA-G阴性的HO-8910、OVCAR-3细胞株,采用RT-PCR、流式细胞术(FACS)及Westernblot鉴定、分析转染细胞中HLA-G的mRNA及蛋白质表达;LDH释放法检测卵巢癌细胞表达HLA-G后对NK细胞杀伤功能的影响。结果显示,66.7%(22/33)卵巢癌组织表达HLA-G分子,而正常组织未见其表达;基因转染后,HLA-G在HO-8910及OVCAR-3细胞表面获得稳定表达。细胞毒实验结果显示,HLA-G能显著抑制NK-92对卵巢癌细胞的杀伤活性(P<0.01);HLA-G特异性抗体87G阻断后,能明显恢复NK-92细胞对HO-8910-G、OVCAR-3-G的杀伤功能。本研究结果提示,卵巢癌细胞通过表达HLA-G分子抑制NK细胞的杀伤活性,在卵巢癌细胞逃避宿主的免疫监视中起重要作用。     

HIV-1Nef调节内皮细胞黏附分子ICAM-1的表达

目的研究HIV-1的调节基因Nef对ECV304细胞ICAM-1表达的影响,从而为分析HIV-1感染引起内皮细胞生物学活性的变化,以及为阐明Nef参与HIV-1致病的分子机制奠定基础。方法应用本实验室已经建立保存的HIV-1Nef基因在内皮细胞的稳定表达细胞株ECV304-Nef和其阴性对照细胞株ECV304 pcDNA 3.1(+),通过RT-PCR、实时定量PCR(real-time PCR)、Western blot、FCM和细胞黏附试验分析ECV304-Nef细胞ICAM-1的表达水平。结果 RT-PCR、real-time PCR结果显示ECV304-Nef细胞ICAM-1 mRNA表达水平明显升高,为对照组的(4.3±0.2)倍;Western blot结果示ECV304-Nef细胞I-CAM-1蛋白的表达水平高于对照组;FCM分析显示ECV304-Nef细胞和对照组细胞ICAM-1阳性细胞百分率分别为(35.3±2.2)%和(12.5±0.8)%(P0.01),两组间ICAM-1表达有显著差异。细胞黏附实验观察到ECV304-Nef细胞黏附的Jurkat细胞数明显多于对照组,荧光仪定量分析结果显示ECV304-Nef细胞黏附的Jurkat细胞的荧光强度值显著高与对照组(P0.05)。结论本实验证实了HIV-1Nef基因可以上调血管内皮细胞细胞黏附分子ICAM-1的表达。     

K562细胞转染HLA-B分子对NK细胞杀伤功能的影响

为了研究不同HLA B分子对NK细胞杀伤活性的影响 ,我们分别构建pcDNA3 HLA B 390 5 2、B 2 70 4、B 5 1 0 2 2基因真核表达载体 ;借助脂质体将各质粒转染入K5 6 2细胞 ,经G4 1 8筛选 ,分别获得阳性表达细胞株 ;并应用LDH法检测转染细胞对不同个体外周血NK细胞杀伤活性的抑制效应。结果显示 :与转染了空质粒的对照组相比 ,外周血NK细胞对K5 6 2 B39的杀伤率无明显影响 ,而对K5 6 2 B2 7,K5 6 2 B5 1的杀伤率降低。当使用针对NK细胞受体KIR3DL1的单抗DX9封闭NK细胞后 ,此抑制效应大部分消失。提示靶细胞表达HLA Bw4分子可明显抑制NK细胞的杀伤效应 ,而表达HLA Bw6分子对NK细胞杀伤功能无明显影响     

针对HLA-G的siRNA的载体构建与表达及效应检测

目的构建针对HLA-G的siRNA的表达载体,研究其对NK细胞杀伤效应的影响。方法构建针对HLA-G的siRNA真核表达质粒pSuppressor-U6-neo-HLA-G,转染JEG-3滋养细胞系。采用RT-PCR、Western blot检测转染的JEG-3细胞中HLA-G的表达水平。将NK-92MI细胞(效应细胞)与滋养细胞(靶细胞)共同培养,以LDH释放法观察不同效靶比例时NK-92MI细胞对靶细胞的杀伤效应。结果NK-92MI细胞对转染针对HLA-G的siRNA的JEG-3细胞的杀伤作用较未转染细胞明显升高(P<0.001)。结论针对HLA-G的SiRNA增加NK细胞对滋养细胞的杀伤,HLA-G具有保护滋养细胞免受NK细胞杀伤的作用。     

HLA-G3蛋白能够抑制多克隆NK细胞的杀伤功能

目的 研究HLA－G3蛋白对NK细胞杀伤功能的抑制作用。方法 用RT－PCR方法扩增HLA－G3和HLA－G1的cDNA，并将其克隆到真核表达载体pcDNA3中，采用脂质体转染法将其转入K562细胞，用免疫荧光法确定其在细胞表面表达，细胞毒试验研究其对NK细胞杀伤功能的抑制作用。结果 HLA－G3蛋白和HLA－G1表达在细胞表面，以PBL细胞为效应细胞的细胞毒实验表明K562－G1细胞和K562－G3细胞的细胞裂解率都比K562细胞裂解率低。结论 HLA－G3蛋白能够抑制多克隆NK细胞的杀伤功能。     

转染PF4 cDNA的GRC-1细胞培养液对于ECV-304细胞生长及VEGF表达的影响

目的：构建真核表达载体pcDNA3-PF4-SS，检测稳定转染后的GRC-1细胞的培养上清对于血管内皮细胞ECV304的生长以及转染细胞中血管内皮生长因子(VEGF)表达的影响。方法：构建真核表达载体pcDNA3-PF4-SS，并经Bgl Ⅱ／Bam HI双酶切鉴定.通过脂质体介导，以该载体稳定转染GRC-1细胞，转染细胞中VEGF因子的表达情况用免疫组化染色法检测，转染细胞的培养上清对于ECV304细胞生长的影响用MTT法检测。结果：经Bgl Ⅱ／BamH I双酶切证实，hPF4 cD—NA已成功地克隆到真核表达载体pcDNA3-CD34-SS中。RT—PCR法检测证实，hPF4 cDNA已成功地转染GRC-1细胞。免疫组化染色检测显示，转染PF4基因的GRC—1细胞的胞浆及胞膜均有VEGF表达，但较转染前明显减弱。细胞计数及MTT法显示，转染后GRC-1细胞的培养上清对ECV304细胞的生长具有抑制作用，吸光度值(A)明显降低。结论：构建了真核表达载体pcDNA3-PF4-SS，并稳定转染GRC-1细胞。转染细胞的培养上清对ECV304细胞的生长及VEGF的表达，均具有明显的抑制作用，为探讨抗肿瘤生长机制和肿瘤疫苗的研制奠定了一定的基础。     

Porcine aortic endothelial cells transfected with HLA-G are partially protected from xenogeneic human NK cytotoxicity

In this study we tested whether the expression of HLA-G protects porcine endothelial cells (PEC) from the lysis mediated by human natural killer (NK) cells. Because HLA-E is not present in PEC, this model provides an ideal tool to study the direct role of HLA-G in NK inhibition. Immortalized porcine aortic endothelial cells (PED) were stably transfected with a vector coding for the HLA-G1 protein and surface expression was demonstrated by flow cytometry analysis. Although the adhesion of human NK cells to PED was not compromised by HLA-G, the expression of HLA-G partially protected PED from the lysis mediated by polyclonal NK lines derived from different donors. A decrease of the surface expression of HLA-G on PED corresponded to a loss of the capacity of PED to inhibit NK cytotoxicity, indicating that the surface density of HLA-G molecules must exceed a certain threshold to protect target cells. In summary, these data show that HLA-G, independent from the presence of HLA-E, can only partially and inefficiently protect PED from human NK cell-mediated cytotoxicity. Because ILT-2/LIR-1 expression did not correlate with HLA-G mediated inhibition, we hypothesize that other yet unidentified receptors expressed by peripheral blood NK cells are involved in the recognition of HLA-G.     

β2-Microglobulin-free HLA-G activates natural killer cells by increasing cytotoxicity and proinflammatory cytokine production

Human leukocyte antigen-G (HLA-G) is a nonclassical HLA class-I molecule and plays a role in tissue specific immunoregulation. Many studies have addressed functional aspects of β2-microglobulin (β2m)-associated HLA-G1. β2m-free HLA-G has been found in human placental cytotrophoblasts and pancreatic β cells although its function remains unclear. In the present study, we investigated the function of β2m-free HLA-G by transfecting HLA-G1 and -G3 into human β2m deficient rat pancreatic β cell carcinoma (BRIN-BD11) cells. RT-PCR and western blots studies confirmed high expression of HLA-G1 and -G3 in -G1 and -G3 transfectants, respectively. HLA-G1 and -G3 were detected mainly in intracellular compartments of BRIN-BD11 transductants by confocal fluorescent microscopy and flow cytometry. Functional analysis revealed that β2m-free HLA-G promoted xenogeneic cytotoxic lysis of BRIN-BD11 cells by natural killer (NK) cells and increased production of IL-1β, TNF-α, and IFN-γ. Stimulation of cytotoxic lysis was impaired by blocking the MAPK and DNA-PKcs pathways in NK cells. Importantly, treatment with 33mAb, a KLR2DL4 receptor agonist, induced NK-mediated cytotoxic lysis of BRIN-BD11 cells transfected with a mock vector. Our data suggest that β2m-free HLA-G activates NK cells via engagement of KLR2DL4 receptors.     

TLR-4信号转导通路介导层流切应力诱导血管内皮细胞IL-8基因表达的研究

目的: 观察TLR-4信号转导通路在层流低切应力诱导血管内皮细胞IL-8基因表达中的作用。方法: 设计突变引物, 用RT-PCR技术从血管内皮细胞扩增出胞内区段缺失突变TLR4 cDNA, 用PCR技术从其DNA中扩增出IL-8上游调控序列(IL-8USCS), 分别克隆于真核表达质粒pcDNA3及绿色荧光增强蛋白报告基因pEGFP1质粒,构建出重组TLR-4缺失突变基因真核表达质粒pcDNA3-mTLR4和IL-8报告基因表达质粒pEGFP1-IL8USCS。用pEGFP1-IL8USCS转染或pcDNA3-mTLR4和pEGFP1-IL8USCS共转染ECV304细胞, 用4.2 dyne/cm²层流切应力刺激3 h, 流式细胞仪观察荧光蛋白表达强度变化。细胞裂解物IκB免疫印迹和NF-κB p65免疫荧光细胞化学染色检测IκB的磷酸化及降解和NF-κB的活化。结果: 用pEGFP1-IL8USCS转染细胞, 经层流切应力刺激3 h后荧光蛋白表达增强(1.06:2.71), 同样用pcDNA3-mTLR4和pEGFP1-IL8USCS共转染细胞, 层流切应力刺激3 h后荧光蛋白表达未明显增强。细胞裂解物免疫印迹显示, 刺激10 min时磷酸化IκB即显著增强, 1 h后磷酸化IκB印迹强度几乎降到基线水平, 而IκB随刺激时间延长而逐渐降低, 1 h后几乎测不到IκB。NF-κB p65免疫荧光细胞化学染色显示, 切应力刺激0.5 h, 胞核即出现阳性反应, 刺激1.5 h、2 h后, 胞核呈强阳性染色。结论: 本研究提示, TLR4/NF-κB信号转导通路可能介导层流切应力诱导血管内皮细胞IL-8基因的表达。     

The possible use of HLA-G1 and G3 in the inhibition of NK cell-mediated swine endothelial cell lysis.

The splicing isoform of HLA-G that is expressed in xenogeneic cells, and its effect on NK-mediated direct cytotoxicity was examined, using stable Chinese hamster ovary (CHO) cell or swine endothelial cell (SEC) transfectants. cDNAs of HLA-G (G1 and G3) and human beta2-microglobulin were prepared and subcloned into the expression vector, pCXN. The transfected HLA-G1 was easily expressed on SEC, and co-transfection with human beta2-microglobulin led to an enhanced level of HLA-G1 expression, as evidenced by flow cytometry. The expressed HLA-G1 significantly suppressed NK-mediated SEC cell lysis, which is an in vitro delayed-type rejection model of a xenograft. On the other hand, the swine leucocyte antigen (SLA) class I molecules could be up-regulated as the result of the transfection of human beta2-microglobulin, but did not down-regulate human NK-mediated SEC lysis. The HLA-G3 was not expressed on CHO and SEC in contrast to HLA-G1, as the result of the transfection. The gene introduction of HLA-G3 in SEC showed no protective effect from human NK cells. However, indirect evidence demonstrated that HLA-G3 transfection resulted in HLA-E expression, but not itself, when transfected to the human cell line, 721.221, thus providing some insight into its natural function in human cells. The present findings suggest that the expression of HLA-G1 on the cell surface could serve as a new approach to overcoming NK-mediated immunity to xenografts.