Induction of HLA-G expression in a melanoma cell line OCM-1A following the treatment with 5-aza-2＇-deoxycytidine

The non-classical HLA class Ⅰ antigen HLA-G is an immune modulator which inhibits the functions of T cells, NK cells, and the Dendritic cells （DC）. As a result, HLA-G expression in malignant cells may provide them with a mechanism to escape the immune surveillance. In melanoma, HLA-G antigen expression has been found in 30% of surgically removed lesions but in less than 1% of established cell lines. One possible mechanism underlying the differential HLA-G expression in vivo and in vitro is that the HLA-G gene is epigenetically repressed in melanoma cells in vitro. To test this hypothesis, we treated the HLA-G negative melanoma cell line OCM-1A with the DNA methyltransferase inhibitor 5-aza-2＇-deoxycytidine （5-AC） and analyzed whether HLA-G expression can be restored. Our data strongly suggest that HLA-G is silenced as a result of CpG hypermethylation within a 5＇ regulatory region encompassing 220 bp upstream of the start codon. After treatment, HLA-G mRNA expression was dramatically increased. Western blot and flow cytometry showed that HLA-G protein was induced. Interestingly, HLA-G cell surface expression on the 5-AC treated OCM-1A cells is much less than that on the HLA-G positive JEG-3 cells while a similar amount of total HLA-G was observed. Possible mechanisms for the difference were analyzed in the study such as cell cold-treatment, peptide loading and antigen processing machinery components （APM） as well as β2 microglobulin （β2-m） expression. Data revealed that the APM component calreticulin might be involved in the lower HLA-G surface expression on OCM-1A cells. Taken together, our results indicated that DNA methylation is an important epigenetic mechanism by which HLA-G antigen expression is modulated in melanoma cells in vitro. Furthermore, to the first time, we hypothesized that the deficiency of calreticulin might be involved in the low HLA-G surface expression on the 5-AC treated OCM-1A cells.     

Residues Met^76 and Gin^79 in HLA-G α1 domain involved in KIR2DIA recognition

Human leukocyte antigen-G (HLA-G) has long been speculated as a beneficial factor for a successful pregnancy for its restricted expression on fetal-matemal extravillous cytotrophoblasts and its capability of modulating uterine natural killer cell (uNK) function such as cytotoxicity and cytokine production through NK cell receptors. HLA class I α1 domain is an important killer cell Ig-like receptor (KIR) recognition site and the Met^76 and Gln^79 are unique to HLA-Gin this region. NK cell receptor KIR2DL4 is a specific receptor for HLA-G, yet the recognition site on HLA-G remains unknown. In this study, retroviral transduction was applied to express the wild type HLA-G (HLA-wtG), mutant HLA-G(HLA-mG) on the chronic myelogenous leukemia cell line K562 cells and KIR2DL4 molecule on NK-92 cells,respectively. KIR2DL4-IgG Fc fusion protein was generated to determine the binding specificity between KIR2DL4 and HLA-G. Our results showed that residue Met76, Gln79 mutated to Ala^76.79 in the α1 domain of HLA-G protein could affect the binding affinity between KIR2DL4 and HLA-G, meanwhile, the KIR2DL4 transfected NK-92 cells (NK-92-2DL4) showed a considerably different cytolysis ability against the HLA-wtG and HLA-mG transfected K562 targets.Taken together, our data indicated that residue Met^76 and Gin^79 in HLA-G α1 domain plays a critical role in the recognition of KIR2DL4, which could be an explanation for the isoforms of HLA-G, all containing the α1 domain, with the potential to regulate NK functions.     

Inhibition of the activating signals in NK92 cells by recombinant GST-sHLA-G1α chain

The soluble HLA-G1 (sHLA-G1) isoform was found to be secreted by trophoblast cells at the materno-fetal interface,which suggests that it may act as an immunomodulator during pregnancy. In this paper, we reported that GST-sHLA-G1α chain could bind to its receptor ILT-2 on NK92 cells and then the latter recruited Src homology 2 domaincontaining tyrosine phosphatase-1 (SHP-1), which consequently dephosphorylated some important protein tyrosine kinases and blocked the activation of downstream molecules such as MEK and ERK so that the cytotoxicity of natural killer (NK) cells was inhibited. These results indicated that GST-sHLA-G1α chain might be exploited in new immunotherapy strategies aiming at inducing immunotolerance during allograft, xenograft and autoimmune situations. In addition,we found that modification of O-linked β-N-acetylglucosamine (O-GlcNAc) was involved in NK cells‘ activating and inhibitory signals. This may provide a novel molecular target for inducing immunotolerance but needs further study.     

HLA-G在肿瘤组织中表达情况研究进展

人类白细胞抗原G(human leukocyte antigen,HLA-G)属于非经典HLA-I类分子,在多种肿瘤细胞上均有表达。从结构上可以将HLA-G分为7种亚型:膜结合型HLA-G1-HLA-G4和可溶型HLA-G5-HLA-G7。研究表明,HLA-G1和HLA-G5具有明确的生物学活性也是研究较为深入的两种亚型,他们可以与T淋巴细胞、B淋巴细胞和NK细胞表面的ILT2/CD85j/LILRB1,ILT4/CD85d/LILRB2,KIR2DL4/CD158d受体结合而发挥免疫抑制功能。目前,HLA-G分子可以在肝癌、肾癌、肺癌、胃癌、食道癌、鼻咽癌、卵巢癌、乳腺癌、宫颈癌、直肠癌和血液肿瘤中表达。本文从HLA-G分子的结构和功能出发,综述了HLA-G分子在上述肿瘤中表达的情况,旨在分析HLA-G在各种肿瘤组织中表达的特点以及临床意义,为临床早期诊断和治疗肿瘤提供参考。     

HLA-G参与骨髓间充质干细胞的抑制作用机制探讨

目的：探讨人骨髓间充质干细胞（MSC）在活体肾移植供受者间的混合淋巴细胞培养（MLR）中的作用机制,证明人白细胞抗原（HLA）-Ⅰ类分子HLA-G参与了MSC的免疫调节作用。方法：从人骨髓中分离培养MSC,采用形态学观察及流式细胞术分析鉴定后,加入活体肾移植供受者间的MLR,观察MSC的HLA-G表达及对T细胞增殖的影响,淋巴细胞增殖实验采用MTT法。结果：MSC细胞表面和胞浆内均表达HLA-G,流式细胞术分析细胞表面HLA-G平均表达率为37.3%,胞浆内HLA-G平均表达率为65.1%,ELISA法检测细胞培养上清中可溶性HLA-G含量为18.9ng/mL。将MSC和培养上清液加入活体肾移植供受者间的MLR体系中,均使得T细胞抑制率增高;而加入HLA-G特异性抗体,MSC的抑制率降低。结论：MSC表面和胞浆内均有HLA-G表达,在其培养上清中检测到由MSC分泌的可溶性HLA-G5;MSC表达和分泌的HLA-G是其发挥抑制功能的关键因素之一。为MSC在预防器官移植术后排斥反应的临床应用提供了理论依据。     

HLA-G在生殖系统癌中的检测:免疫组织化学研究

为检测生殖系统癌有无HLA-G表达,采用免疫组化LDP法对223例生殖系统癌手术切除标本进行了鼠抗HLA-G单克隆抗体染色,观察了HLA-G在乳腺癌（n=100),卵巢癌（n=30),子宫颈癌（n=30),子宫内膜癌（n=40),前列腺癌（n=20)和睾丸胚胎癌（n=3)中的表达与分布。结果发现,除睾丸胚胎癌外,生殖系统其余部位癌标本可见到40-57.5%的HLA-G阳性表达,HLA-G阳性反应物在癌细胞内呈颗粒状及均质状,主要分布于细胞浆,结果提示,HLA-G表达可能是肿瘤生物学中的普遍现象,生殖系统癌细胞在发生和发展过程中其基因表达有可能出现反分化现象,开始表达HLA-G,从而使癌细胞产生免疫耐受,逃逸宿主的免疫监视。     

人白细胞抗原G在结直肠癌中的表达及意义

目的：在蛋白水平检测人白细胞抗原G（HLA-G）在结直肠癌组织中的表达,探讨HLA-G分子在结直肠癌不同分级和不同分期中的表达差异及在肿瘤逃逸中作用。方法：用免疫组织化学法检测结直肠癌和癌旁正常结直肠组织HLA-G的表达情况,用SPSS13.0软件、Kruskal-Wallis test进行分析。结果：HLA-G分子在结直肠癌组织中的表达率为42.3%（41/97）,在癌旁正常结直肠组织的表达率为0（0/20）;HLA-G分子表达与结直肠癌临床TNM分期（P〈0.05）和组织学分级（P〈0.01）相关。结论：HLA-G分子在结直肠癌组织中表达上调,且与结直肠癌的侵袭性生长密切相关;HLA-G分子可能下调宿主对肿瘤细胞的免疫应答反应,使肿瘤细胞逃避机体的免疫监视。     

HLA-G蛋白及基因在子宫腺肌病中的表达

目的:探讨人类白细胞抗原G(human leucocyte antigen-G,HLA-G)在子宫腺肌病(adenomyosis,AM)患者在位、异位内膜中的表达及其生物意义,为AM的病因研究和治疗展望提供依据.方法:采用免疫组化和RT-PCR法检测36例AM患者和30例正常子宫内膜在位、异位内膜HLA-G蛋白及基因表达情况.结果:AM在位、异位内膜HLA-G蛋白及基因表达量显著高于正常子宫内膜(P<0.05);AM在位与异住内膜HLA-G蛋白表达量无显著差异(P>0.05);AM在位、异位内膜腺体细胞HLA-G蛋白的表达量呈正相关(r=0.948,P<0.05);AM在位、异位子宫内膜间质细胞HLA-G蛋白的表达量也呈正相关(r=0.863,P<0.05).结论:HLA-G在AM在位、异位内膜中呈高表达,参与了AM的发病机制,HLA-G阳性的子宫内膜更容易逃避免疫细胞的攻击而浸润肌层生长.     

HLA-G5与器官移植排斥反应的关系研究

目的:探讨HLA-G5表达与器官移植排斥之间的关系。方法:应用酶联免疫方法检测移植术后2个月以上的50例肝移植受者和60例肾移植受者外周血HLA-G5的表达情况。结果:肝、肾移植受者中HLA-G5阳性表达受者的急慢性排斥反应发生率明显低于阴性表达受者。结论:HLA-G5的表达与肝、肾移植受者排斥反应发生率的降低密切相关。     

高等植物的PCD研究进展(一)

植物细胞程序性死亡(programmed cell death,PCD)已成为当前生物学的研究热点之一。植物PCD普遍存在于植物器官和个体生长发育过程及与环境相互作用过程中,具有重要的生物学意义。在高等植物生长发育过程中,根冠细胞、导管细胞、绒毡层细胞、胚乳细胞、胚柄细胞、糊粉细胞、大孢子细胞、助细胞和反足细胞等细胞在一定程度上均发生了PCD。另外,衰老也涉及PCD。本文综述了最近几年来与发育有关的PCD研究进展,主要包括高等植物细胞死亡的形式、起因及其PCD的形态、生化特征及高等植物营养器官(根、茎和叶)和生殖器官(花、果实和种子)在其生长发育过程中的PCD。文章最后还对植物PCD的进化和生物学意义作了进一步的讨论。 Abstract：Plant programmed cell death(PCD),the details of which are becoming a focus of intensive research in biology, is a ubiquitous phenomenon and plays an improtant biological role in the develpoment of organs and whole organisms and in interactions with the environment.During higher plant development,root cap cells,tracheary elements(TEs),tapetalcells,endosperm cells,suspensor cells,aleurone cells,megaspore cells,help cells and antipodal cells,etc.undergo PCD to some degree.In addition,senescence also involves PCD.This paper mainly reviewed PCD research progress in higher plant development in recent years,including forms and causes of cell death and PCD morphological and biochemical features in higher plants;PCD in development of nutritive organs(root ,stem and leaf) and reproductive organs(flower ,fruit and seed),evolution and biological rloes of plant PCD were further discussed in the paper.     

JEG-3细胞分泌孕激素与HLA-G mRNA表达的相关性研究

目的:探讨JEG-3细胞自身分泌孕激素与其HLA-G mRNA表达的相关性,为进行滋养叶细胞HLA-G分子表达的调控研究提供线索和依据.方法:培养JEG-3细胞,在不同时点(1,2,3,4,6,12,24h)收集细胞和培养上清液,分别采用实时定量PCR方法和EIA方法测定JEG-3细胞的HLA-G mRNA的表达及孕激素的浓度.结果:JEG-3细胞培养上清液中孕激素浓度与JEG-3细胞HLA-G mRNA表达的强度随时间进程具有相似变化趋势,经Spearman等级相关分析,JEG-3细胞自身孕激素的分泌与其HLA-G mRNA的表达量呈显著正相关(r=0.964,P＜0.05).结论:JEG-3细胞孕激素的分泌与其HLA-GmRNA的表达量具有高度相关性,进一步体外研究孕激素对滋养叶细胞HLA-G分子表达的调节作用时,应考虑到JEG-3细胞自身孕激素分泌的影响.     

Effects of adeno-associated virus (AAV) of transforming growth factors β_1 and β_3 (TGFβ_(1,3)) on promoting synthesis of glycosaminoglycan and collagen type II of dedifferentiated nucleus pulposus (NP) cells

The effects of AAV-TGFβ1 and AAV-TGFβ3 on promoting synthesis of glycosaminoglycan and collagen type II of dedifferentiated rabbit lumbar disc NP cells were studied in this work. The rabbit lumbar disc NP cells were isolated and cultured. The earlier and later dedifferentiated NP cells were established by subculture. The AAV transfection efficiency to dedifferentiated NP cells was analyzed with AAV-EGFP in vitro. After dedifferentiated NP cells were transfected by AAV-TGFβ1 or AAV-TGFβ3, their biological effects on promoting synthesis of glycosaminoglycan or collagen type II were detected and compared by the methods of 35S incorporation or immunoblotting. The experimental results showed that AAV could transfect efficiently the earlier dedifferentiated NP cells, but its transfection rate was shown to be at a low level to the later dedifferentiated NP cells. Both AAV-TGFβ1 and AAV-TGFβ3 could promote the earlier dedifferentiated NP cells to synthesize glycosaminoglycan and collagen type II, and the effect of AAV-TGFβ1 was better than that of AAV-TGFβ3. For the later dedifferentiated NP cells, the AAV-TGFβ3 could promote their synthesis, but AAV-TGFβ1 could slightly inhibit their synthesis. Therefore, AAV-TGFβ1 and AAV-TGFβ3 could be used for the earlier dedifferentiated NP cells, and the TGFβ3 could be used as the objective gene for the later dedifferentiated NP cells.     

Effects of adeno-associated virus (AAV) of transforming growth factors β1 and β3 (TGFβ1,3) on promoting synthesis of glycosaminoglycan and collagen type II of de-differentiated nucleus pulposus (NP) cells

The effects of AAV-TGFβ1 and AAV-TGFβ3 on promoting synthesis of glycosaminoglycan and collagen type Ⅱ of dedifferentiated rabbit lumbar disc NP cells were studied in this work. The rabbit lumbar disc NP cells were isolated and cultured. The earlier and later dedifferentiated NP cells were established by subculture. The AAV transfection efficiency to dedifferentiated NP cells was analyzed with AAV-EGFP in vitro. After dedifferentiated NP cells were transfected by AAV-TGFβ1 or AAV-TGFβ3, their biological effects on promoting synthesis of glycosaminoglycan or collagen type Ⅱ were detected and compared by the methods of 35S incorporation or immunoblotting. The experimental results showed that AAV could transfect efficiently the earlier dedifferentiated NP cells, but its transfection rate was shown to be at a low level to the later dedifferentiated NP cells. Both AAV-TGFβ1 and AAV-TGFβ3 could promote the earlier dedifferentiated NP cells to synthesize glycosaminoglycan and collagen type Ⅱ, and the effect of AAV-TGFβ1 was better than that of AAV-TGFβ3. For the later dedifferentiated NP cells, the AAV-TGFβ3 could promote their synthesis, but AAV-TGFβ1 could slightly inhibit their synthesis. Therefore, AAV-TGFβ1 and AAV-TGFβ3 could be used for the earlier dedifferentiated NP cells, and the TGFβ3 could be used as the objective gene for the later dedifferentiated NP cells.     

Induction of HLA-G expression in a melanoma cell line OCM-1A following the treatment with 5-aza-2''''-deoxycytidine

~~Induction of HLA-G expression in a melanoma cell line OCM-1A following the treatment with 5-aza-2'-deoxycytidine@Chien Chung CHANG$Department of Immunology,Roswell Park Cancer Institute,Buffalo.NY 14263,USA @Soldano FERRONE$Department of Immunology,R…     

血清sCD44v6、HLA-G、G-17、Hp-IgG联合检测在胃癌与癌前病变筛查中的临床价值探讨

摘要 目的：探讨血清可溶性CD44变体6（sCD44v6）、人类白细胞抗原-G（HLA-G）、胃泌素-17（G-17）、幽门螺杆菌-免疫球蛋白G（Hp-IgG）联合检测在胃癌与癌前病变筛查中的临床价值。方法：选择2019年2月至2022年2月期间首都医科大学附属北京朝阳医院消化内科收治的121例胃癌癌前病变患者（癌前病变组）和125例胃癌患者（胃癌组）,另选择同期120例健康体检者作为对照组,检测并比较三组间血清sCD44v6、HLA-G、G-17水平以及Hp-IgG阳性率。根据Hp-IgG阳性情况,将胃癌组进一步分为Hp-IgG阳性组与Hp-IgG阴性组,比较两组间血清sCD44v6、HLA-G、G-17水平。应用Spearman秩相关分析胃癌组血清sCD44v6、HLA-G、G-17水平与Hp-IgG阳性率的相关性,应用受试者工作特征（ROC）曲线分析sCD44v6、HLA-G、G-17、Hp-IgG单独鉴别和联合四项指标鉴别胃癌癌前病变与胃癌的价值。结果：胃癌组血清sCD44v6、HLA-G、G-17水平以及Hp-IgG阳性率高于癌前病变组、对照组（P＜0.05）,癌前病变组血清sCD44v6、HLA-G、G-17水平以及Hp-IgG阳性率高于对照组（P＜0.05）,胃癌组中Hp-IgG阳性组sCD44v6、HLA-G、G-17水平高于Hp-IgG阴性组（P＜0.05）。胃癌组血清sCD44v6、HLA-G、G-17水平与Hp-IgG阳性率呈正相关（rs=0.536、0.492、0.512,P＜0.05）。联合sCD44v6、HLA-G、G-17、Hp-IgG鉴别胃癌癌前病变以及胃癌的曲线下面积为0.863,高于各指标单独鉴别。结论：血清sCD44v6、HLA-G、G-17水平以及Hp-IgG阳性率在胃癌患者与胃癌癌前病变患者中存在明显差异,胃癌患者血清sCD44v6、HLA-G、G-17水平与Hp-IgG阳性有关,联合四项指标检测在胃癌与癌前病变筛查中具有较高的临床价值。     

The associated regulators and signal pathway in rIL-16／CD4 mediated growth regulation in Jurkat cells

IL-16 is a ligand and chemotactic factor for CD4 T cells. IL-16 inhibits the CD3 mediated lymphocyte activation and proliferation. The effects of IL-16 on the target cells are dependent on the cell type, the presence of co-activators etc. To understand the regulation function and mechanism of IL-16 on target cells, we used a 130 a.a. recombinant IL-16 to study its effects on the growth of Jurkat T leukemia cells in vitro. We found that the rIL-16 stimulated the proliferation of Jurkat cells at low dose (10^-9M), but inhibited the growth of the cells at higher concentration (10^-5M). Results showed that 10^-5 M of rIL-16 treatment induced an enhanced apoptosis in Jurkat cells. The treatment blocked the expression of FasL, but up-regulated the c-myc and Bid expression in the cells. Pre-treatment of PKC inhibitor or MEK1 inhibitor markedly increased or decreased the rIL-16 induced growth-inhibiting effects on Jurkat cells, respectively.The results suggested that the rIL-16 might be a regulator for the growth or apoptosis of Jurkat cells at a dose-dependent manner. The growth-inhibiting effects of rIL-16 might be Fas/FasL independent, but,associated with the activation of PKC, up-regulated expression of c-Myc and Bid, and the participation of the ERK signal pathway in Jurkat cells.