pJAK2 polypeptide,an antagonist of suppressors of cytokine signaling-1,can enhance the antitumor effect of dendritic cells

目的:探讨细胞因子信号转导抑制因子-1(SuPPrESSOrS Of CyTOkInE SIgnAlIng1,SOCS1)拮抗物PJAk2多肽(氨基酸序列号为1001-1013)参与树突状细胞(dEndrITIC CEllS,dCS)的体外诱导培养后对dCS抗肿瘤作用的影响。方法:采集健康人外周血,离心获得单个核细胞,用重组人粒细胞巨噬细胞集落刺激因子(rECOMbInAnT HuMAn grAnulOCyTE-MACrOPHAgE COlOny STIMulATIng fACTOr,rHgM-CSf)及重组人白细胞介素-4(rECOMbInAnT HuMAn InTErlEukIn-4,rHIl-4)诱导dCS,第5天分为4组:单纯dCS培养(对照)组、抗原负载(lySATE-dCS)组、多肽修饰(PJAk2-dCS)组和抗原与多肽共培养(lySATE+PJAk2-dCS)组,第6天各组加入肿瘤坏死因子-α(TuMOr nECrOSIS fACTOr-AlPHA,Tnf-α)促成熟。倒置显微镜下观察dCS形态;fCM法检测dCS表型;乳酸脱氢酶(lACTATE dEHydrOgEnASE,ldH)细胞毒实验检测各组细胞毒性T淋巴细胞(CyTOTOXIC T lyMPHOCyTE,CTl)对胃癌细胞bgC-823的靶向杀伤作用;ElISA法检测白细胞介素-12(InTErlEukIn-12,Il-12)和γ干扰素(InTErfErOn-γ,Ifn-γ)的水平。结果:与未加入诱导剂组相比,各组均成功诱导出成熟dCS,均高表达Cd80、Cd83、Cd86和人类白细胞dr抗原(HuMAn lEukOCyTE AnTIgEn dr,HlA-dr),但以lySATE+PJAk2-dCS组的表达水平最高。在10:1~30:1的效靶比范围内,CTl杀伤作用与效靶比呈正相关。当效靶比为30:1时,对照组的CTl杀伤率达(19.77±2.34)%,低于其他3组(P<0.01),而lySATE+PJAk2-dCS组较lySATE-dCS组及PJAk2-dCS组都高(P<0.05)。lySATE+PJAk2-dCS组培养上清液中Il-12及Ifn-γ的分泌水平明显高于对照组(P<0.01)。结论:SOCS1拮抗物PJAk2多肽(1001-1013)可增强dCS对胃癌细胞的抗原递呈及特异性抗肿瘤作用。Objective:To investigate the effect of pJAK2 polypeptide,an antagonist of SOCS1(suppressors of cytokine signaling 1),on antitumor effect of in vitro cultivation-induced DCs(dendritic cells).Methods:Peripheral blood was collected from the healthy volunteers,and the PBMCs(peripheral blood mononuclear cells)were isolated.DCs were induced by rhGM-CSF(recombinant human granulocyte-macrophage colony-stimulating factor)and rhIL-4(recombinant human interleukin-4).On the fifth day,DCs were divided into four groups:control group,Lysate-DCs group,pJAK2-DCs group,and Lysate + pJAK2 DCs group.On the sixth day,TNF-α(tumor necrosis factor-alpha)was added into each group.The morphological features of DCs were observed under an inverted microscope;the phenotypes were detected by FCM(flow cytometry);the killing effect of CTLs(cytotoxic T lymphocytes)on gastric cancer BGC-823 cells was evaluated by LDH(lactate dehydrogenase)cytotoxicity test;the concentrations of IL-12(interleukin-12)and IFN-γ(interferon-γ)were detected by ELISA(enzyme-linked immuno sorbent assay).Results:Mature DCs presented typically morphological and phenotypic features;the DCs in Lysate + pJAK2-DCs group had the highest expression levels of CD80,CD83,CD86 and HLA-DR(human leukocyte antigen DR).When the ratio of effectors to target cells ranged from 10:1 to 30:1,the killing activity of CTLs had a positive correlation with the ratio.When the ratio of effectors to target cells was 30:1,the killing activity of CTLs in the control group was(19.77±2.34)%,which was lowest as compared with the other groups(P < 0.01),meanwhile the killing activity of CTLs in Lysate + pJAK2-DCs group was higher than those in Lysate-DCs and pJAK2-DCs groups(P < 0.05).The levels of IL-12 and IFN-γ secretion in Lysate + pJAK2-DCs group were apparently higher than those in the control group(P < 0.01).Conclusion:An antagonist of SOCS1,pJAK2 polypeptide,can enhance the ability of antigen presentation and specific antitumor effect of DCs on gastric cancer cells.南京军区医学科技创新课题资助项目(编号:10MA068); 福建省自然科学基金面上项目(编号:2010D013); 厦门市科技计划创新项目(编号:3502z20104014

Effect of Helicobacter pylori on the function of peripheral blood monocyte-derived dendritic cells in gastric cancer patients

分析HP阳性和HP阴性的胃癌患者外周血单核细胞来源树突状细胞(MOdCS)功能的差异性及其临床意义。方法:用尿素14C呼气试验对解放军第一七四医院2011年1月至2012年10月收治的84例胃癌患者进行HP感染状态鉴定,分别采集HP阳性和阴性胃癌患者外周血,分离外周血单个核细胞,采用经典方法(rHgM-CSf、rHIl-4联合rHTnf-α)诱导产生dCS,采用流式细胞仪检测dCS表型,采用ldH释放法检测dCS致敏T细胞对胃癌细胞的毒性杀伤作用,采用ElISA方法检测细胞因子Il-12、Ifn-γ的分泌水平。结果:两组MOdCS成熟过程形态变化无差异,HP阳性组MOdCS表面标记分子Cd1A、Cd80、Cd83、Cd86和HlA-dr平均表达百分率均高于HP阴性组,其中Cd80、Cd83、Cd86的表达水平差异有统计学意义,Cd1A、HlA-dr差异无统计学意义。HP阳性组dCS致敏T淋巴细胞对胃癌细胞杀伤率和Il-12、Ifn-γ的分泌水平均高于HP阴性组(P<0.05)。结论:HP感染状态不影响胃癌患者外周血MOdCS成熟过程形态变化,HP感染的胃癌患者MOdCS成熟和活化水平更高。Objective:This study aimed to compare and analyze the functional differences between peripheral blood monocyte-derived dendritic cells(DCs) of Helicobacter pylori-positive and H.pylori-negative patients with gastric cancer.Methods:H.pylori infection was detected in 84 patients with gastric cancer in our hospital from January 2011 to October 2012 by the14C-urea breath test.DCs were generated from monocytes isolated by an adherent method from the two groups of patients and cultured in the presence of rhIL-4,rhGM-CSF,and rhTNF-α.Furthermore,the expression of surface marker molecules was determined by fluorescence-activated cell sorting analysis.The cytotoxicity of DCs pulsed T cells against gastric carcinoma cell was assessed by the lactate dehydrogenase-releasing assay.The secretion of IL-12 and IFN-γ in the supernatant was determined by enzyme-linked immunosorbent assay.Results:No difference was observed in the morphological change of the maturation process.The mean expression of CD1a,CD80,CD83,CD86,and HLA-DR molecules in DCs of H.pylori-infected patients was higher than that in DCs of H.pylori-negative group,and the differences were statistically significant except for CD1a and HLA-DR.The cytotoxicity activities,IL-12 release,and IFN-γ release in the H.pylori-positive group were significantly higher than those in the H.pylori-negative group(P<0.05).Conclusion:H.pylori infection has no effect on the morphological change of the maturation process of monocyte-derived DCs.These data clearly demonstrate that monocyte-derived DCs of H.pylori-infected patients with gastric cancer can induce stronger maturation and activation than those of H.pylori-negative patients.南京军区医学科技创新课题(编号:10MA068); 福建省自然科学基金资助项目(编号:2010D013); 厦门市科技计划创新项目(编号:3502z20104014)资助~