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

Influence of Helicobacter pylori infection on autologous DC-CIK maintenance therapy in the advanced gastric cancer patients

目的:对比幽门螺杆菌(HElICObACTEr PylOrI,HP)感染阴性和阳性的晚期胃癌患者接受自体树突状细胞联合细胞因子诱导的杀伤(dEndrITIC CEllS-CyTOkInE InduCEd kIllEr,dC-CIk)细胞维持治疗的疗效差异。方法:收集2010年6月至2012年6月中国人民解放军第174医院肿瘤中心收治的72例晚期胃癌患者,年龄29~90岁,中位年龄56岁,通过胃镜检查进行胃癌确诊并检测HP,分为HP阳性组(45例)及HP阴性组(27例),在接受手术或/和放化疗后,接受2疗程自体dC-CIk维持治疗,比较两组外周血培养的dC分化成熟情况及临床疗效差异。结果:两组dC成熟过程形态变化无差异;HP阳性组dC表面分子Cd83、Cd86表达显著高于HP阴性组(P0.05)。HP阳性组治疗后生活质量评分(kPS)、外周血T细胞亚群(Cd3+、Cd4+、Cd8+)比例较治疗前显著提高(P0.05)。HP阳性组治疗后kPS评分、CEA和CA199表达以及Cd3+和Cd4+T细胞数量等的改善均优于HP阴性组(P0.05)。共随访2年,阳性组患者中位生存期为12.64个月,长于阴性组的11.42个月(P 0.05).Levels of CD83 and CD86 on the surface of DCs were significantly higher in the Hp-positive group than those in the Hp-negative group( P 0.05).In the Hp-positive group,the quality of life KPS score and levels of T lymphocyte markers( CD3+,CD4+and CD8+) were significantly increased( P 0.05).Hp-positive patients had better KPS score,higher levels of CEA and CA199,and more abundant CD3+CD4+T cells than Hp-negative patients( P 0.05).At the end of 2-years follow-up,the median survival time was 12.64 months in the HP-positive group and 11.42 months in the HP-negative group( P < 0.05).Conclusion: Hp infection may help stabilize the tumor size,improve the quality of life,and prolong the survival time in patients with advanced gastric cancer undergoing maintenance therapy with DC-CIK cells.南京军区医学科技创新课题资助(No.10MA068); 厦门市科技计划创新项目资助(No.3502z20134026;No.3502z20144034)~

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~

Purification and Partial Characterization of the Glutathione S-transferase Isozymes from the mussel,Perna viridis

以翡翠贻贝(PErnA VIrIdIS)消化腺为材料,经gST rAP--(TM)ff柱亲和层析,分离纯化得到总谷胱甘肽硫转移酶。而后经dEAE离子交换层析得到三个洗脱峰M_1、M_2、M_3,分别占总蛋白含量的77%,16%,2.9%,对其进行SdS-PAgE分析,结果表明M_1可能是由分子量为25 kd的蛋白质亚基组成的同型二聚体,M_2、M_3可能为异型二聚体,M_2由25 kd和23kd两个亚基组成,M_3由27kd和23kd两个亚基组成。以1-氯-2,4-二硝基苯(Cdnb)、3,4-二氯硝基苯(dCnb)、4-硝基氯化苄(nbC)、利尿酸(ETHA)4种底物对M_1、M_2、M_3进行动力学分析,发现M_1、M_2、M_3分别对ETHA、dCnb、nbC亲和力更好,kM分别为1.08MMOl/l、1.51 MMOl/l、0.89MMOl/l,VMAX分别为54.9μMOl/MIn/Mg,40.3μMOl/MIn/Mg,19.4μMOl/MIn/Mg。Total glutathione S-transferases were purified from digestive gland of mussel(Perna viridis) by GST rap FF.Then three glutathione S-transferase isozymes(M_1,M_2,M_3) were purified and separated with ion exchange chromatography.SDS-PAGE analysis of glutathione S-transferase isozymes(M_1,M_2,M_3) indicated that M_1 may be a single homodimer and the molecular weight of M_1 is 25 kD,while M_2 and M_3 may be two heterodimers,M_2 showed two bands and the molecular weight of the subunits are 25 kD and 23 kD respectively,M_3also showed two bands and the molecular weight of the subunits are 27 kD and 23 kD respectively.Enzymatic kinetic analysis of M_1,M_2,M_3 using l-chloro-2,4-dinitrobenzene(CDNB ),3,4-dichloronitrobenzene(DCNB),4-nitrobenzyl chloride (NBC) and ethacrynic acid(ETHA)as substrates has a better affinity to ETHA,DCNB and NBC respectively, K_m are 1.08 mmol/L,1.51 mmol/L 1,0.89 mmol/L respectively and V_(max) are 54.9μmol/min/mg,40.3μmol/ (min·mg--(-1)),19.4μmol/(min·mg--(-1)) respectively.福建省自然科学基金(2008J0138)资

Experimental Study on New Material of Large Molecules Organic Silicon on Efficiency of Rainwater Catchments

将高分子有机硅材料喷施在夯实的坡面上,在坡表面形成一层致密的防水膜,减少土壤入渗,增加径流量,从而提高了坡面的集流效率,并减少土壤的侵蚀量,是一种简单、方便的集雨方式。本试验以一种新型有机硅材料作为对象,采用室内渗透和室外人工模拟降雨试验,探讨其应用于集雨工程的可能性,并确定其最佳配比和最适宜用量。研究结果表明:硅水最优配比(体积比,文中所有硅水配比均指体积比)在1∶5～1∶7,适宜用量为150ml/m2。当雨强在1.2～1.5mm/min,前期含水率17%左右,干容重为1.3g/cm3左右,其集流效率可以达到70%以上,是对照夯实坡面集流效率的2～3倍,而侵蚀量只是对照坡面侵蚀量的1/5。新型有机硅材料具有集流效率高、成本低、施工简单等优点,在集雨工程中具有广阔的应用前景

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将壳聚糖溶液分散于葵花籽油中,以Span-80为乳化剂,搅拌形成油包水乳液,加入香草醛的丙酮溶液进行交联,得到微囊.扫描电镜显示微囊呈圆整的球形,表面致密,内部有空隙.FT-IR光谱和XRD光谱证实壳聚糖的氨基与香草醛的醛基的交联作用和丙酮的脱水作用是壳聚糖液滴固化成形的原因.以萘普生为模型药物制备了载药微囊.结果表明,壳聚糖的脱乙酰度增加,粘均分子量减小,香草醛的用量增加,则微囊的产量、包封率和载药率增加,而药物的释放速率减小.脱乙酰度为96.3%、粘均分子量60.5万的壳聚糖,以醛基/氨基为3.75:1(摩尔比)与香草醛交联制得的载有萘普生的微囊,在含有0.5g/L溶菌酶的模拟结肠环境的释放介质中在10h内可完全释放药物

NMR、FTIR及电位滴定法测定壳聚糖脱乙酰度

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使用微波间歇法可快速制备高脱乙酰度和高粘均分子量的壳聚糖.单因素实验确定的工艺条件为微波功率800W,将45%(&omega;)的氢氧化钠溶液与250～380&mu;m的20g甲壳素粉以8:1的体积比混合,在100℃下反应10min,洗涤、微波干燥后在相同条件下再反应1次,共20min,可制得脱乙酰度为94.5%、粘均分子量1.48&times;106的白色壳聚糖粉末.其他制备条件相同,使用电加热法间歇处理甲壳素粉3次,反应共5h,可以得到脱乙酰度为96.2%、粘均分子量为3.8&times;105的褐色壳聚糖粉末.微波间歇法所制壳聚糖的结晶度高,内部有规整的有序结构,用它制备的膜致密,性能优于用电加热法所制壳聚糖制备的膜