增强自噬的乙型脑炎重组DNA疫苗促进BALB/c小鼠树突状细胞功能

目的:探讨自噬介导的乙型脑炎重组DNA疫苗对BALB/c小鼠体内树突状细胞(dendritic cells,DCs)免疫相关功能的影响。方法:取健康BALB/c雌性小鼠,随机分配成5组,分别用pcDNA3.1(＋)空载体、pJME质粒、pJME-LC3重组质粒肌注免疫小鼠,并使用无菌PBS肌注作为空白对照组,乙型脑炎减...     

高剂量乙型肝炎疫苗对体内血树突状细胞的影响

给6例健康志愿者,各单次皮下接种基因工程乙型肝炎疫苗,剂量为60μg。在接种前和随后25 d内的不同天数,先后多次取肝素抗凝静脉血,及时用全血三色荧光染色法,通过流式细胞仪检测血中表达CD11c+、CD123+和CD80+三型树突状细胞(DC)数的动态变化。结果表明,接种3d后,三型DC数均见上升趋势,2周后达峰值,至25 d后,仍维持较高水平。提示皮下接种高剂量乙型肝炎疫苗可激发DC表达受检的三种分化抗原,表明高剂量乙型肝炎疫苗具有可能增强体内DC参与对HBV特异性细胞免疫应答。     

树突状细胞靶向性DNA疫苗的抗肿瘤免疫效应

目的:构建含OVA-Fc融合基因并对树突状细胞具有靶向性的DNA疫苗,评价其在肿瘤治疗中的作用。方法:构建真核表达载体OVA-Fc-pcDNA3.1,以脂质体转染法将其导入CHO细胞,用流式细胞术和ELISA法检测融合蛋白OVA-Fc的表达。建立E.G7-OVA荷瘤小鼠模型,用51Cr释放实验测定免疫小鼠脾脏细胞毒性T淋巴细胞(CTL)的抗肿瘤活性。通过观察荷瘤小鼠肿瘤的体积和生存期评价该肿瘤疫苗的疗效。结果:酶切鉴定和序列测定证明,真核表达载体OVA-Fc-pcDNA3.1构建正确。用流式细胞术和ELISA法均表明,转染的CHO细胞能表达OVA-Fc融合蛋白。OVA-Fc能激发CTL的杀伤活性,发挥抗肿瘤作用,从而减缓肿瘤的生长,延长荷瘤小鼠的生存期。结论:含OVA-Fc-pcDNA3.1的树突状细胞靶向性DNA疫苗能在体内有效地激发抗瘤免疫应答,为进一步开展临床实验奠定了基础。     

水通道蛋白5基因对小鼠树突状细胞功能影响的研究

目的 探讨水通道蛋白5(AQP5)基因的表达对小鼠骨髓来源的树突状细胞(bonemarrow-derived dendritic cells,BMDC)分化成熟的影响.方法 分离培养AQP5基因敲除(AQP5-/-)的小鼠及野生型小鼠的BMDC,采用LPS诱导其成熟,采用FACS检测DC表型和内吞作用的变化,采用3H-TdR掺入法检测其对异种淋巴细胞的刺激能力.结果 与野生型小鼠相比,AQP5基因敲除后DC表面共刺激分子CD40、CD80、CD86的表达下降;其内吞能力下降;野生型小鼠来源的DC在与颗粒性蛋白质接触30 min后仍可继续上升,而AQP5-/- 来源的DC在相互作用30 min后内吞能力达到高峰;野生型小鼠来源的DC对异种淋巴细胞的刺激能力远大于AQP5-/-小鼠.结论 AQP5介导的跨膜水转运对于DC功能的正常发挥具有重要意义.其具体信号途径有待进一步研究.     

负载滋养层细胞抗原对小鼠树突状细胞表型及功能的影响

目的 研究负载滋养层细胞抗原对小鼠髓源性树突状细胞(DC)分化成熟过程的影响,获得致耐受性DC.方法 体外使用粒细胞巨细胞集落刺激因子(GM-CSF)诱导小鼠骨髓细胞定向分化、经LPS刺激获得成熟DC;通过外胎盘锥组织块培养法获得滋养层细胞,制备可溶性抗原,加入DC培养体系.流式细胞术检测DC表面共刺激分子及MHC-Ⅱ的表达,ELISA法检测DC分泌IL-10和IL-12的浓度,混合淋巴细胞培养评估 DC刺激同种T细胞增殖、活化的功能.结果 成熟DC表型为CD40high CD80highCD86highMHC-Ⅱhigh,分泌大量的IL-12和极少量的IL-10 ,体外能有效刺激T细胞的增殖;负载滋养层细胞抗原的DC表型为CD40midCD80lo wCD86lowMHC-Ⅱlow,在分泌大量IL-12的同时IL-10也明显升高,不能有效刺激T细胞增殖,并使T细胞分泌细胞因子呈现明显Th2偏倚.结论 负载滋养层细胞抗原后的DC表面共刺激分子及MHC-Ⅱ表达降低,刺激T细胞增殖能力下降;其自分泌和促使T细胞旁分泌的细胞因子呈现Th2偏倚,是一种耐受性DC.     

Sel1L 对小鼠骨髓源树突状细胞的影响

目的:研究Sel1L(Suppressor/ enhancer of Lin-12-like)基因对小鼠骨髓源树突状细胞(BMDCs)分化及其功能的影响。方法:利用Cre-Loxp 重组系统构建Sel1L 基因敲除小鼠模型,用ELISA 和实时荧光定量法检测BMDCs 分泌细胞因子IL-6、IL-12 的表达;用免疫印迹法(Western bolt)检测BMDCs 细胞中Sel1L蛋白的表达;用流式细胞术检测BMDCs 细胞CFSE、细胞表面分子CD80、CD86、MHC-Ⅰ、MHC-Ⅱ及其对特异性CD4⁺ T 细胞抗原提呈能力的影响。结果:Sel1L的缺失抑制BMDC诱导分化过程中的增殖效率,上调DCs 分泌因子的能力和MHC-Ⅰ的表达,减少MHC-Ⅱ的表达,并抑制BMDCs 细胞对OVA323-339 抗原特异性T 细胞的增殖。结论:Sel1L 缺失可以抑制小鼠骨髓源树突状细胞的分化,下调DC 对OVA 特异性的CD4⁺ T细胞的抗原提呈功能。     

ICAM-1基因表达的调节

细胞间粘附分子-1(ICAM-1)是免疫球蛋白超家族的成员之一,是存在于多种细胞表面的诱导性细胞粘附糖蛋白.致炎因子、细胞应激、感染等主要通过激活ICAM-1基因的转录,增加ICAM-1的表达.NF-κB在激活ICAM-1启动子中起主要作用,其通过特异性的蛋白-蛋白的相互作用,同其它转录因子和共激活因子一起参与ICAM-1的表达调节.调节ICAM-1表达的主要细胞内信号通路包括PKC,MAPK,JAK/STAT等.     

树突状细胞靶向性DNA疫苗的构建及其在CHO细胞的表达

目的 构建含OVA-Fc融合基因的真核表达质粒，证明其能在真核细胞CHO细胞表达。方法通过PCR从OVA-pcDNA3．1质粒中扩增出全长的小鼠OVA cDNA，酶切后克隆到含有小鼠IgG2a Fc cDNA的真核载体pMIgV，然后将OVA-Fc酶切后亚克隆入pcDNA3．1，构建真核表达载体OVA-Fc-pcDNA3．1；脂质体转染法将其导入CHO细胞，流式细胞仪、Western blot、ELISA法检测融合蛋白OVA-Fc的表达。结果酶切鉴定和序列测定证明真核表达载体OVA-Fc-pcDNA3．1构建正确；流式细胞术、Western blot、ELISA法均检测到转染的CHO细胞能表达OVA-Fc融合蛋白。结论OVA-Fc-pcDNA3．1真核表达质粒构建正确并能在真核细胞中表达，该质粒的成功构建与表达为进一步将其作为DNA疫苗治疗肿瘤、哮喘等疾病奠定了基础。     

BTLA-HVEM通路阻断对树突状细胞功能影响的体内外研究

目的 探讨阻断BTLA-HVEM(B/T淋巴细胞弱化因子疱疹病毒进入介质)通路对树突状细胞功能的影响和相关免疫学机制.方法 构建小鼠BTLA胞外功能区的真核表达载体psBTLA,转染CHO细胞;HSP70-TC-1肿瘤抗原肽刺激小鼠骨髓来源DCs,流式细胞仪检测处理后DCs表面BTLA、HVEM的表达,同时给予转染了psBTLA质粒的CHO细胞的培养上清处理后,检测DCs表面B7-1的表达,ELISA检测上清中IL-12的分泌;处理后的DCs刺激脾细胞,检测淋巴细胞增殖和细胞因子分泌;检测psBTLA体内转染对宫颈癌细胞系TC-1成瘤小鼠DCs表达B7-1和肿瘤生长的影响.结果 成功构建小鼠BTLA胞外段的真核表达载体psBTLA,获得了稳定转染psBTLA的CHO细胞,在其培养上清检测到BTLA胞外段(sBTLA)的表达.DCs经抗原肽刺激后BTLA、HVEM表达均上调,加入含sBTLA的上清处理后上调B7-1,上清中分泌的IL-12增加,与脾细胞共培养时促进细胞增殖和IL-2、IFN-γ的分泌;体内基因转染psBTLA促进DCs表达B7-1以及抑制肿瘤生长.结论 通过sBTLA阻断BTLA-HVEM共抑制通路,可以进一步促进DCs的功能,更好地激活淋巴细胞,促进抗肿瘤免疫应答.     

树突状细胞肿瘤疫苗的研究进展

树突状细胞(DC)是目前发现的功能最强大的专职抗原递呈细胞(APC),能激活静息T细胞并使其增殖,产生抗原特异性细胞毒性T淋巴细胞(CTL),从而发挥抗肿瘤免疫效应.近年来,人们在肿瘤疫苗的研究中,采用多种策略制备DC肿瘤疫苗,尝试通过改变机体免疫系统对肿瘤的免疫状态来加强机体的免疫功能,进而清除肿瘤细胞,而对正常组织几乎无伤害,因此有很好的临床应用前景.     

乙脑病毒prME与E蛋白编码基因重组构建的DNA免疫试验研究

目的　研究乙脑病毒prME、E蛋白的体外表达特点 ,比较不同重组质粒所进行的DNA免疫效率。方法　分别将乙脑病毒 (JaGAr 0 1株 )prME、E蛋白编码基因 (2 0 0 1bp、15 0 0bp)构建于融合FLAG标记基因的真核表达载体pcDNA(FLAG) 3上 (pJME、pJE) ,脂质体法将二重组质粒转染于HepG2及COS 1细胞系 ;采用不同抗体系统 (anti FLAG、anti E) ,经Westernblot法检测乙脑病毒prME[相对分子质量 (Mr)约 72× 10 3]与E(Mr 约 5 4× 10 3)蛋白表达水平 ;将质粒肌注BALB c鼠 ,二次免疫后 3周 ,腹腔注射乙脑病毒 (10 5PFU 10 0 μl)作为病毒攻击并观察 2 1d。 80 %空斑减少中和实验法检测中和抗体滴度变化。结果　anti FLAG可检测到由质粒pJME、pJE编码的相应蛋白产物在转染细胞内表达 ,同时在pJME转染的细胞内检测到一个新的 11× 10 3的低Mr 蛋白。anti E仅在pJME转染的细胞内检测到E蛋白。肌注pJME可产生高水平中和抗体滴度以及诱导有效的保护性免疫 ,效果等同于普通灭活JE疫苗 ,但优于pJE。结论　pJME的体外表达水平优于pJE。DNA免疫实验结果与prME、E蛋白体外表达特点相一致     

乙脑病毒prME蛋白与BALB/c鼠IgG Fc段编码基因联合构建DNA免疫研究

目的 研究IgG Fc编码基因对流行性乙型脑炎(Japanese encephalitis,JE)DNA疫苗免疫增强效应的影响.方法 巢式RT-PCR法从BALB/c鼠脾组织获取IgG Fc段编码基因,用限制性内切酶从含流行性乙型脑炎病毒(Japanese encephalitis virus,JEV)prME蛋白基因重组子获取prME蛋白基因,分别插入同-真核表达载体pcDNA3.1(+)不同酶切位点,构建蘑组子pJME/IgG Fc并经酶切及DNA测序分析.脂质体法将pJMrY/IgG Fc转染CHO细胞.免疫荧光、Western blot法检测转染的CHO细胞中融合蛋白分布与表达.将pJME/IgG Fc肌注免疫BALB/c鼠,检测小鼠脾特异性细胞毒T细胞(CTL)杀伤活性和中和抗体滴度.结果 pJME/IgG Fc经BamH Ⅰ/EcoR Ⅰ和BamH Ⅰ/Not Ⅰ酶切释出的插入子大小,(2001 bp,2730 bp)分别与预期结果相符合.所编码的融合蛋白相对分子质量(Mr)为101×103,主要分布于胞浆,少最分布于胞膜,pJME/IgG Fc转染CHO细胞经32次传代仍可表达融合蛋白.pJME/IgGFc免疫组中和抗体滴度与CTL活性较pJME及灭活疫苗组均升高(P<0.05).结论 pJME/IgG Fc成功构建,转染的CHO细胞可稳定表达融合蛋白,IgG Fc段编码基因能够增强JEV DNA疫苗的细胞和体液免疫应答.     

丙型肝炎病毒核心蛋白DNA免疫初步研究

目的　观察HCV核心蛋白基因的DNA免疫效果。方法　将HCV核心蛋白基因插入真核表达载体pcDNA3.1( ) ,构建重组质粒pcDNA3.1 c。在证明该重组质粒可在哺乳动物COS7细胞中表达的基础上 ,用重组质粒 10 0 μg免疫小鼠 ,同时设立空白质粒组和PBS组两组对照 ,初次免疫后 4周、8周各进行一次加强免疫。小鼠体液免疫反应和T淋巴细胞增殖检测分别采用间接免疫荧光法和MTT法。结果　pcDNA3.1 c可在COS7细胞内表达HCV核心抗原 ,接种于Balb c小鼠能有效诱导体液和细胞免疫应答。结论　重组质粒pcDNA3.1 c对于丙型肝炎防治具有潜在价值     

Modulation of dendritic cell maturation and function by the Tax protein of human T cell leukemia virus type 1

Human T cell leukemia virus type 1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is characterized by the generation of an intense CTL cell response directed against the viral transactivator protein Tax. In addition, patients diagnosed with HAM/TSP exhibit rapid activation and maturation of dendritic cells (DC), likely contributing to the robust, Tax-specific CTL response. In this study, extracellular Tax has been shown to induce maturation and functional alterations in human monocyte-derived DC, critical observations being confirmed in freshly isolated myeloid DC. Tax was shown to promote the production of proinflammatory cytokines and chemokines involved in the DC activation process in a dose- and time-dependent manner. Furthermore, Tax induced the expression of DC activation (CD40, CD80, and CD86) and maturation (CD83) markers and enhanced the T cell proliferation capability of DC. Heat inactivation of Tax resulted in abrogation of these effects, indicating a requirement for the native structure of Tax, which was found to bind efficiently to the DC membrane and was internalized within a few hours, suggesting that extracellular Tax may possess an intracellular mechanism of action subsequent to entry. Finally, inhibitors of cellular signaling pathways, NF-kappaB, protein kinase, tyrosine kinase, and phospholipase C, were shown to inhibit Tax-mediated DC activation. This is the first study reporting the immunomodulatory effects of extracellular Tax in the DC compartment. These results suggest that DC, once exposed to Tax by uptake from the extracellular environment, can undergo activation, providing constant antigen presentation and costimulation to T cells, leading to the intense T cell proliferation and inflammatory responses underlying HAM/TSP.     

热休克蛋白60对小鼠树突状细胞功能影响体外研究

目的：探讨动脉粥样硬化中重要炎性物质——热休克蛋白60（HSP60）体外对小鼠树突状细胞（mDC）功能影响.方法：小鼠骨髓提取DC,体外培养成熟后与两种浓度mHSP60孵育,动态观察DC突起改变;流式细胞仪检测孵育前后mDC表面标志改变;MLR测定孵育前后mDC刺激功能变化;ELISA法测定MLR上清液中细胞因子浓度.结果：孵育后,mDC突起增加明显;CD11c^＋、CD80及CD86表型显著增加;淋巴细胞刺激功能明显增强;分泌细胞因子IL-12、IFN-γ增加（P＜0.01）而IL-4增加不明显（P＞0.05）,IFN-γ/IL-4比值升高.结论：mHSP60体外可以促进mDC功能,作用呈剂量依赖性.     

热休克蛋白对树突状细胞成熟的影响研究

目的:探讨热休克蛋白(HSP)对树突状细胞(DCs)成熟的影响,同时对其形态学动态变化进行研究。方法:从肝癌组织中提取HSP(gp96 )抗原肽复合物;肝癌细胞进行热休克处理诱导其表面表达HSP,再用DiI荧光标记;将两种HSP与DCs混合培养,动态观察DCs捕获抗原和成熟过程中的形态学变化。流式细胞仪检测不同情况下热休克蛋白对DCs成熟表型的影响。结果:使用DiI标记的方法良好地显示了DCs摄取抗原的形态学变化;DCs通过直接接触、包绕、形成囊泡和伸出伪足且末端形成囊泡4种方式摄取抗原;热休克蛋白可促进DCs成熟表型的表达。结论:DiI是适合DCs形态学研究的良好的荧光标记物;DCs捕获抗原有4种不同方式;不同热休克蛋白均可促进DCs成熟,但提取的热休克蛋白作用更显著。     

Elimination of HIV-1 plasmid DNA from virus samples obtained from transfection by calcium-phosphate co-precipitation

Molecular genetics is a powerful tool to analyze the replication cycle of human immunodeficiency virus type 1 (HIV-1). Culture fluids obtained from HIV-1 plasmid-transfected cells by calcium-phosphate co-precipitation were treated with ethyleneglycol bis (beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) and DNase I to obtain HIV-1 stocks virtually free of input plasmid DNAs. Even after amplification by polymerase chain reaction (PCR), no plasmid DNA was detected in cells following infection with EGTA/DNase I-treated virus samples. This method is particularly useful for the examination of the early replication phase of HIV-1 by PCR.     

Effect of interleukin-10 on dendritic cell maturation and function

The main function of dendritic cells (DC) is to induce the differentiation of naive T lymphocytes into helper cells producing a large array of lymphokines, including interleukin (IL)-2; interferon-γ (IFN-γ), IL-4, IL-5 and IL-10. The potent immunostimulatory properties of DC develop during a process of maturation that occurs spontaneously in vitro. Since IL-10 has been shown to inhibit Th1 responses, we determined its effect on DC maturation and accessory function. Our data show that DC that have undergone maturation in vitro in the presence of IL-10, have an impaired capacity to induce a Th1-type response in vivo, leading to the development of Th2 lymphocytes. Their inability to promote the synthesis of IFN-γ seems to correlate with a decreased production of IL-12, an heterodimeric cytokine necessary for optimal generation of Th1-type cells. These results suggest that IL-10 skews the Th1/Th2 balance to Th2 in vivo by selectively blocking IL-12 synthesis by the antigen-presenting cells that play a role of adjuvant of the primary immune response. The cytokines present in the environment at the presentation step may, therefore, determine the class of the immune response induced by DC in vivo, i.e. Th0 Th1 and/or Th2.     

Enhancement of the immunogenicity of an infectious bronchitis virus DNA vaccine by a bicistronic plasmid encoding nucleocapsid protein and interleukin-2

A DNA vaccine against infectious bronchitis virus (IBV) can induce specific humoral and cell-mediated immunity. However, compared to conventional vaccines, DNA vaccines usually induce poor antibody responses. To develop a more potent IBV DNA vaccine formulations, a monocistronic vector encoding the nucleocapsid protein of IBV and a bicistronic vector separately encoding the nucleocapsid protein and immune-stimulatory interleukin-2 were constructed. When the DNA vaccines were administered to the quadriceps muscle of chickens, the induced humoral and cellular responses were evaluated. There was a significant difference in ELISA antibody levels elicited by either monocistronic or bicistronic DNA vaccines. The percentage of CD3+, CD3+CD8+ and CD3+CD4+ subgroups of peripheral blood T-lymphocytes in chickens immunized with bicistronic DNA vaccine were higher than those in chickens immunized with monocistronic DNA vaccine. When chickens were challenged with a virulent strain of IBV, the protective efficacy could be enhanced significantly after immunization with bicistronic DNA vaccine. These results demonstrated that bicistronic DNA vaccine is an effective approach to increase IBV DNA vaccine immunogenicity.     

Immunization with a plasmid DNA expressing rinderpest virus hemagglutinin protein protects rabbits from lethal challenge

Summary.  Rinderpest virus haemagglutinin (H) protein was expressed in eukaryotic cells and the plasmid encoding H gene was used for immunization of rabbits. The immunized rabbits were completely protected from lethal challenge with virulent lapinized rinderpest virus. Similar results were also observed in rabbits vaccinated with tissue culture rinderpest (TCRP) vaccine. About 80% of control rabbits vaccinated with empty vector (mock-vaccinated) died after challenge. The thermal reactions and clinical symptoms were less intense in H-DNA vaccinated rabbits as compared to the other two groups. Marked lymphopenia was observed in TCRP and mock-vaccinated rabbits after challenge, however, the H-DNA vaccinated rabbits showed lymphocytosis following challenge. The ap-pearance of lymphocytosis in H-DNA vaccinated rabbits is an interesting finding which needs further investigation. Received June 27, 2001 Accepted November 8, 2001