siRNA沉默树突状细胞恒定链以增强其抗肿瘤免疫效果

背景：利用基因修饰树突状细胞（DCs）增强抗肿瘤免疫反应已成为一种有效的策略。RNA干扰（RNAi）能够通过转录后的基因沉默机制特异地降解靶RNA。在本研究中,我们应用DCs恒定链（Ii）特异的小干扰RNA（siRNA）转染DCs后,观察其抗肿瘤效果。 方法：通过WesternBlot验证siRNA沉默效果;利用CCK-8试剂盒检测Ii siRNA转染DCs后其刺激淋巴细胞增殖的能力。此外,通过非放射性cytotox 96® 细胞杀伤检测试剂盒检测被激活细胞的体外杀伤效果。成瘤时间和肿瘤的大小被用作评价Ii siRNA转染DCs体内抗肿瘤效果的可靠指标。为了探讨Ii siRNA转染DCs抗肿瘤的机制,我们还进行了流式细胞仪的检测。 结果：DCs转染Ii siRNA后,其Ii的表达被明显抑制。体外细胞杀伤实验发现,Ii siRNA处理组的T细胞杀伤活力显著增高(P<0.05 )。此外,我们还发现,无论在小鼠接种肿瘤之前还是在接种肿瘤之后给小鼠免疫共转染Ii siRNA和内源性肿瘤抗原的DCs,均能明显抑制肿瘤的生长。通过流式细胞仪检测可能参与体内、外抗肿瘤免疫应答的免疫细胞,结果显示CD4＋和CD8＋T细胞均被明显激活(P<0.05)。 结论：通过siRNA沉默DCs的Ii链可能是一种行之有效的增强抗肿瘤免疫的方法。     

Induction of T-cell immunity against leukemia by dendritic cells pulsed with total RNA isolated from leukemia cells

Objectives To assess the feasibility and efficacy of eliciting leukemia-specific T-cell responses in syngeneic mice in vitro and in vivo using dendritic cells (DCs) pulsed with total RNA from leukemia cells.Methods DCs generated from bone marrow culture in vitro in the presence of combined cytokines were pulsed with cellular total RNA isolated from cultured L615 cells by cationic lipid 1,2-dioleoyloxy-3-(trimethylammonium) propane (DOTAP). T-cell responses were evaluated by in vitro proliferation, and cytotoxicity assay. And in vivo immune protection and proghosis of mice with leukemia were studied.Results DCs pulsed with total RNA isolated from cultured L615 cells (DCs/RNA) were remarkably effective in stimulating L615-specific T-cell response in vitro, but did not cross-react with other leukemia cells from syngeneic mice. Vaccination of naive mice with viable DCs/RNA vaccine was able to partly protect from challenge with a lethal dose of live L615 cells, leading to low leukemia incidence and overall survival prolongation. Statistically significant survival was also observed in a low lethal dose of L615-bearing mice that received treatment using viable DCs∕RNA vaccine alone, suggesting that systemic administration of IL-2 could enhance the anti-tumor efficacy of leukemia RNA/DCs vaccine.Conclusions These data support the use of DCs/RNA vaccine as a feasible and effective route to elicit leukemia immunity against unidentified leukemia-associated antigens for treatment of leukemia-bearing animals.     

沉默树突状细胞恒定链对其活力和分化成熟的影响

目的观察siRNA沉默树突状细胞(DCs)的恒定链(Ii)转染后对DCs活力及分化成熟的影响。方法从小鼠骨髓分离骨髓前体细胞,细胞经100ng/mL粒细胞-巨噬细胞集落刺激因子(GM-CSF)和100ng/mL白细胞介素-4(IL-4)诱导培养6d后,转染针对DCsIi链特异的小分子干扰RNA(IisiRNA),转染后加用(或不加)50ng/mL肿瘤坏死因子-α(TNF-α)继续诱导成熟48h,分别通过Western blotting检测沉默效果,光镜观察DCs的活力和形态,流式细胞仪检测细胞的凋亡和表面分子标志(MHCⅡ、CD86、CD40),ELISA检测细胞IL-12p70的释放。结果IisiRNA能够明显抑制DCsIi的表达,转染IisiRNA后对DCs的活力、形态、细胞表型以及细胞因子IL-12p70的分泌均没有影响。结论通过siRNA沉默DCs的Ii链不会影响DCs的活力及分化成熟,该策略能够用于进一步的DCs肿瘤疫苗的研究。     

Induction of T-cell immunity against Epstein-Barr virus-associated tumors by means of adenovirally transduced dendritic cells

Background Dendritic cells (DCs) are the most powerful antigen-presenting cells to induce specific T-cell immunity, which plays an important role in the body‘s anti-tumor responses. In this study, we assessed the feasibility and efficacy of inducing T-cell immunity against Epstein-Barr virus (EBV)- associated tumors in vivo using dendritic cells transfected with EBV latent membrane 2A (LMP2A) recombinant adenovirus.Methods Cytokine-activated bone marrow-derived DCs transfected with EBV LMP2A recombin antadenovirus were infused into BALB/c mice. Splenic cytotoxic T-cell responses were evaluated by cytotoxicity and interferon-γ production assays, in vivo immune protection was then assessed in the mice tumor models implanted with tumor cells expressing EBV LMP2A. Results DCs transfected with EBV LMP2A recombinant adenovirus could strongly induce EBV LMP2A-specific cytotoxic T-cell responses and upregulate interferon-y production in vivo. Vaccination using these DCs led to prolongation of overall survival rates in the mice tumor models and retarded tumor growth.Conclusions The results suggest that DCs transfected with EBV LMP2A recombinant adenovirus can serve as a feasible and effective tool for eliciting LMP2A-specific cytotoxic T-cell responses against EBV LMP2A in vivo in the treatment of EBV-associated tumors.     

Enhancing Antitumor by Immunization with Fusion of Dendritic Cells and Engineered Tumor Cell

Antitumor immune responses are consideredto be primarily mediated by T cells.Based on theprevious data that genetically engineered tumorcells can be used to be against tumor as a source ofwhole- cell antigens,and as well as the secretion ofcytokines and,or costimulatory molecules,manyof the immunotherapeutic approaches developed totreat tumor in animal model or clinical trials[1— 3 ] .However,tumor cells are poor antigen- presentingcells (APCs) ,because of the lack of costimulatorymolecule e…     

小鼠乳腺癌实验动物模型中树突状细胞的功能检测及意义

目的观察在肿瘤生长过程中DCs的功能是否存在变化，并探讨其意义。方法建立小鼠乳腺癌动物模型，用FACS分析荷瘤早、晚期时间点肿瘤浸润淋巴细胞中DCs的比例及其MHC-Ⅱ类分子和协同刺激分子CD80、CD86的表达；用ELISA法检测DCs的细胞因子IL-4、IL-10、IFN-γ和TNF-α的分泌情况；观察DCs对CD4^＋T细胞增殖及对CD8^＋T细胞杀伤功能的影响。结果与荷瘤早期相比，荷瘤晚期肿瘤浸润淋巴细胞中DCs的比例从11．76±2．3％增加到17．45±3．2％（p〈0．05）；晚期DCs下调表达MHC分子及协同刺激分子CD86，而上调表达IL-4、IL-10；晚期DCs刺激CD4^＋T细胞增值的能力明显减弱，并且对CD8^＋T细胞的杀伤能力的刺激作用也显著降低。结论随着荷瘤时间的延长，DCs的成熟度及其对T淋巴细胞的免疫刺激作用逐渐降低，这可能与肿瘤的免疫逃逸密切相关。     

尤文肉瘤细胞总RNA修饰的树突状细胞的体外培养及其鉴定

目的:探讨树突状细胞(DC)经尤文肉瘤细胞总RNA加载后的体外RNA表达情况. 方法:采用分离尤文肉瘤患者外周血单核细胞体外诱导DC,用流式细胞仪检测DC成熟后的表面标志,Trizol法提取患者尤文肉瘤组织总RNA,用总RNA转染DC,用RT-PCR方法检测肿瘤总RNA加载的DC的表达. 结果:经尤文肉瘤总RNA加载的DC表面标记发生明显变化,DC的特异性表面标志如CD40由32.24%增高至72.32%,CD83由17.39%增高至30.97%,而代表单核细胞的CD14则由26.37%下降至10.46%,标志其抗原呈递功能显著增强. RT-PCR测定显示尤文肉瘤中的特异性序列EWS-FLI1可以和引物特异性结合,并且最佳结合温度为54℃. 结论:尤文肉瘤细胞总RNA转染的DC可以呈递肿瘤特异性抗原,并特异性的表达其特有序列EWS-FLI1.     

Comparison of antileukemic immunity mediated by dendritic cells derived from multidrug resistant leukemia K562/A02 cells and sensitive K562 cells

Dendritic cells (DCs), as the most potent antigen presenting cells in vivo, are central to generating specific immune responses and can be used as important vectors in antitumour immunity. DCs loaded with tumour antigens can induce specific antitumour response in cytotoxic T lymphocytes (CTL) . P-glycoprotein ( P-gp), a product of mdrl gene, widely overexpressed in many multidrug resistant (MDR) tumour cells, is a main mechanism that makes tumour cells escape from death induced by chemotherapeutic agents.     

树突状细胞对胃粘膜免疫的调节作用研究

目的 通过研究胃粘膜中Ｓ－１００＾＋,ＨＬＡ－ＤＲ＾＋,ＣＤ４＾＋和ＣＤ８＾＋产物的表达情况,以了解树突状细胞（ＤＣ）在胃粘膜免疫中的作用。方法 胃癌（１７６例）、慢性萎缩性胃炎（４１例）、不典型增生（１５例）、肠上皮化生（２７例）和正常对照组（１０例）共２６９例,采用ＨＥ染色、Ｓ－Ｐ免疫酶标抗体组化染色法与图像分析技术相结合,观察胃粘膜中Ｓ－１００＾＋,ＨＬＡ－ＤＲ＾＋细胞数量明显多于正常胃粘膜     

Immunity of Unloaded Dendritic Cells in Lung Melanoma of Mice

In order to investigate the immunity of unloaded dendritic cells(DCs) derived from murine bone marrow to preexisting lung melanoma metastases of mice,MO5 were intravenously in-jected to induce lung metastases in syngeneic C57BL/6 mice.Unloaded GM-CSF DCs,PBS and DCs SIINFEKEL were subcutaneously injected into the mice,which were divided as experimental group,negative control group and positive control group respectively.Monoclonal antibody was used to deplete NK or T cells separately.The immunity-inhibitory effects on the lung melanoma were ob-served and the corresponding effector cells were examined.It was found that in the experimental and positive groups,the regression was induced in metastatic nodules in the lungs of tumor-bearing mice,but abrogated by treatment with anti-asialo-GM1 but not anti-CD8.It was concluded that the unloaded DCs could suppress the lung melanoma metastases to some extent,which was mediated by NK cells,and could be used as a potent therapeutic agents for lung tumor.     

Tanshinone ⅡA Inhibits Dendritic Cell-mediated Adaptive Immunity: Potential Role in Anti-atherosclerotic Activity

Objective：Antigen-presenting cells such as monocytes and dendritic cells（DCs） stimulate T-ceil proliferation and activation during adaptive immunity.This cellular interaction plays a role in the growth of atherosclerotic plaques.Tanshinone ⅡA（TSN） had been shown to decrease the growth of atherosclerotic lesions.We therefore investigated the ability of TSN to inhibit human monocyte-derived DCs and their T-cellstimulatory capacity.Methods：DCs derived from human monocytes cultured with recombinant human interieukin（IL）-4 and recombinant human granulocyte-macrophage colony-stimulating factor were co-cultured with TSN and lipopolysaccharide for 48 h.Phosphate-buffered saline was used as a negative control.Activation markers and the capacity of DCs for endocytosis were measured by flow cytometry,and proinflammatory cytokines were measured by enzyme-linked immunosorbent assays.DCs were co-cultured with lymphocytes to measure T-cell proliferation and IL-2 secretion by mixed lymphocyte reactions.Results：TSN dose-dependently attenuated DC expression of costimulatory molecules（CD86）,and decreased expression of major histocompatibility complex class I（human loukocyte antigen-DR） and adhesion molecules（CD54）.Moreover,TSN reduced secretion of the proinflammatory cytokines IL-12 and IL-1 by human DCs,and restored the capacity for endocytosis.Finally,TSN-preincubated DCs showed a reduced capacity to stimulate T-cell proliferation and cytokine secretion.Conclusions：TSN inhibits DC maturation and decreases the expression of proinflammatory cytokines,while impairing their capacity to stimulate T-cell proliferation and cytokine secretion.These effects may contribute to the influence of TSN on the progression of atherosclerotic lesions.     

胶质瘤RNA致敏的人脐血树突状细胞诱导细胞毒T淋巴细胞对肿瘤细胞的杀伤作用

目的:制备人脐血来源的树突状细胞(DCs),用胶质瘤RNA诱导其成熟,并观察DCs诱导细胞毒T淋巴细胞(CTL)体外杀伤肿瘤细胞的活性.方法:以rhGM-CSF、IL-4诱导人脐血单个核细胞向DCs分化并鉴定.提取胶质瘤RNA与DCs混合,使DCs致敏,未致敏的DCs为对照组.观察细胞形态并检测致敏前后DCs的表型.将DCs与外周血T细胞共培养,以胶质瘤细胞为靶细胞,采用MTT比色法检测T细胞抗肿瘤活性差异.结果:人脐血单个核细胞于诱导第5 d呈现典型的DCs形态;经rhGM-CSF、IL-4诱导培养后,CD86、CD11c、CD54、MHC-Ⅰ、MHC-Ⅱ类分子表达与培养前比较均明显增高(P＜0.01).负载抗原后,MHC-Ⅰ和CD54表达与负载抗原前相比进一步增高(P＜0.05).以DCs诱导活化的T细胞与胶质瘤细胞共培养可使胶质瘤细胞的生长受到抑制,并最终导致其死亡;杀伤率与未负载抗原的对照组相比差异有统计学意义(P＜0.01).结论:人脐血单个核细胞在细胞因子的诱导下扩增并分化为DCs.经抗原致敏后,能增强淋巴细胞对相应肿瘤细胞的体外杀伤效应.     

同池分隔培养NS1骨髓瘤细胞对树突状细胞的影响

目的 :观察同一孔池中培养 ,NS1骨髓瘤细胞对树突状细胞 (DC)的影响。方法 :用不同孔径微小池分隔法 ,同池培养骨髓瘤NS1与骨髓DC。分别设为①无微小池DC孔 ,② 3μm微小池DC孔 ,③ 3μm微小池DC +NS1孔 ,④ 8μm微小池DC孔 ,⑤ 8μm微小池DC +NS1孔各 3孔。每个培养孔底均有生长 3周的DC ,铺满孔底 ,每孔5× 10 5,仅③、⑤孔微小池内加入NS11× 10 4,每 2d换 1次培养液 ,同池共育 4 0d ,观测DC数目和形态改变 ;共育 7d时 ,做体外细胞毒性T淋巴细胞杀伤活性检测。结果 :与NS1骨髓瘤细胞同一孔池中培养的DC的数目减少 ,形态改变 ,抗原递呈功能下降。结论 :同一孔池中培养 ,NS1骨髓瘤细胞抑制DC的增殖和功能。     

辅助性T细胞17与肿瘤免疫

 辅助性T细胞（Th17细胞）是近年新定义的一类CD4⁺效应T细胞,其与肿瘤的发生、发展密切相关。在不同的免疫背景下,Th17细胞表型存在可塑性。这种可塑性与Th17细胞及其相关细胞因子在肿瘤免疫中作用的双向性密切相关。进一步明确Th17细胞表型重塑过程中所需的各种细胞因子的结构及功能,将有助于提高肿瘤免疫治疗的疗效。     

树突状细胞在胃癌组织中的分布特征及与预后的关系

目的探讨S-100蛋白阳性肿瘤浸润树突状细胞(tumor infiltrating dendritic cells,TIDC)在胃癌组织中的分布及意义.方法将S-100蛋白抗体作为TIDC的特异性标记物,对30例胃癌标本进行免疫组化染色,对TIDC进行定量计数分析.结果胃癌患者TIDC高度浸润率随肿瘤TNM分期趋向晚期而显著降低(P＜0.05);淋巴结转移阳性组TIDC高度浸润率显著低于阴性组(P＜0.01);不同部位、不同分化程度和不同浸润程度的病例间无显著差异(P＞0.05).结论胃癌组织中TIDC浸润情况改变,可能是导致机体细胞免疫功能低下的重要原因之一.检测胃癌组织内TIDC浸润程度对判断胃癌预后有一定意义.     

黄芪多糖应用于人外周血源性树突状细胞体外培养的实验研究

目的 探讨黄芪多糖对人外周血源性树突状细胞(DC)成熟的影响.方法 从健康人外周血中分离获得PBMC,体外采用多种细胞因子(TNFα、IL-4、GM-CSF)联合诱导,获得了分化与功能相对成熟的DC.并采用流式细胞仪检测技术,观察了不同浓度黄芪多糖(终浓度为50、100、200 mg/L)对DC表面分子表达以及DC与T细胞在体外混合培养体系中活细胞相互作用过程的干预作用.结果 LPS组、50mg/L组、100mg/L组的细胞呈悬浮生长,可见部分细胞聚集成簇,细胞表面可见数量不等、形态不一的树突样突起,扫描电镜下细胞呈不规则形,表面粗糙,胞体有形态不一的突起,α-萘酸酯酶非特异性酯酶染色呈阴性,200 mg/L组细胞大量凋亡崩解.培养6 d后实验组的DC表面CD86(TCD86*LPS=9.1302、TCD86*50 mg/L=6.4024、TCD86*100 mg/L=8.3165,P<0.001)、HLA-DR(THLA-DR*LPS=6.3118、THLA-DR*100mg/L=7.0752,P<0.001;THLA-DR*50 mg/L=3.5797,P<0.01)等表型表达上调,与对照组比较差异有显著性意义.LPS组和100mg/L组的CD14等表达下调,与对照组比较差异有显著性意义(TCD14*LPS=8.5709、TCD14*100=8.6103,P<0.001),50mg/L组的CD14表达下调,但与对照组比较差异无显著性意义(TCD14*50 mg/L=1.7918,P>0.05).结论 研究初步表明,适当剂量黄芪多糖可以上调DC膜表面与抗原递呈相关的HLA-DR、CD86等共刺激分子的高表达,对促进DC的分化与成熟,有着显著的影响,并增加DC的免疫活性.     

垂体腺苷酸环化酶激活多肽调节LPS激活的树突状细胞免疫功能

目的:研究垂体腺苷酸环化酶激活多肽(pituitary adenylate cyclase-activating polypeptide,PACAP)能否调节小鼠骨髓起源的LPS激活的树突状细胞(dendritic cell,DC)的免疫功能。方法:联合应用rmGM-CSF和rmIL-4自C57BL/6小鼠骨髓细胞制备DC,以LPS和(或)PACAP刺激DC;收集被激活的DC及其上清液行ELISA和流式细胞术分析;自DC提取总RNA行RT-PCR和RNase分析。结果:PACAP能明显抑制LPS激活的DC分泌细胞因子IL-2、IL-12和TNF-α(P<0.05,P<0.01,P<0.01),以及LPS激活的DC分泌趋化因子MIP-2(P<0.01),但抑制细胞因子IL-6,趋化因子MIP-1α和MIP-1β的作用并不明显(P>0.05)。结论:PACAP对LPS激活的DC的免疫功能具有负性调节作用。     

In vitro induction of specific anti-tumoral immunity against laryngeal carcinoma by using human interleukin-12 gene-transfected dendritic cells

Background  Objective evaluation of the antitumor effect of interleukin-12 (IL-12) gene-transfected dendritic cell (DC) vaccine on laryngeal carcinoma requires in vivo and in vitro tests. The aim of this study was to investigate the function of IL-12 gene transfected DC at initiating specific immune response to laryngeal carcinoma in vitro．

Methods  Recombinant adenovirus with IL-12 gene was constructed. DCs were isolated from the peripheral blood of patients with laryngeal carcinoma, pulsed with tumor lysate of laryngeal carcinoma cells (DC⁺Ag), and transfected with IL-12 (DC-IL-12⁺Ag). The cells pheotypes including CD83, CD86 and HLA-DR on surface of DCs were assayed by flow cytometry (FCM). The concentration of IL-12 in culture supernatant of DCs and interferon γ (IFN-γ) in culture supernatant of T cells cocultured with DCs were quantified by ELISA. Methyl thiazolys tetrazolium (MTT) was used to evaluate proliferation of autologous T lymphocytes and activation of cytotoxic T lymphocytes (CTL) stimulated by IL-12-transfected DCs pulsed with tumor lysate against laryngeal carcinoma cells.

Results  The recombinant adenovirus expressing IL-12 gene was constructed successfully. Gene-transfected DC plused with tumor lysate with IL-12 (DC-IL-12⁺Ag) expressed higher level of CD83, CD86 and produced higher level of IL-12 than untransfected DCs (DC⁺Ag) (CD83: (60.2±1.8)% vs. (50.7±1.2)%, P <0.05; CD86: (88.9±2.1)% vs. (78.2±3.9)%, P <0.05; IL-12: (262.5±3.0) ng/L vs. (103.8±5.1) ng/L, P <0.05). The proliferation of autologous T lymphocytes and production of IFN-γ stimulated by DC transfected with IL-12 were more obviously than untransfected DCs. Cytotoxicity of CTL stimulated by IL-12-transfected DC pulsed with tumor lysate against laryngeal carcinoma cells were significantly stronger than stimulated by untransfected DC.

Conclusion  It is a promising approach for IL-12-transfected DC pulsed with tumor lysate to increase the antitumoral effect.