肿瘤诱导树突状细胞凋亡研究进展

树突状细胞作为最重要的抗原递呈细胞,在抗肿瘤免疫中起重要作用.恶性肿瘤可以通过分泌细胞因子、蛋白质、神经酰胺和神经节苷脂引起树突状细胞功能缺陷及数量减少,还可以诱导树突状细胞发生凋亡.因此,肿瘤相关抗原不能有效传递给T细胞,激活抗肿瘤免疫应答.利用某些蛋白和细胞因子对树突状细胞进行预处理,可以减少肿瘤诱导凋亡的发生,基因技术可以增强树突状细胞耐受凋亡的能力.现综述恶性肿瘤诱导树突状细胞凋亡的研究进展.     

Fusion vaccine therapy by IL-2-gene-transduced dendritic cells and tumor cells

We evaluated the usefulness of fusion vaccine prepared from IL-2-gene-transduced splenic dendritic cells (DCs) and fibrosarcoma tumor cells (QRsP) in treating of lung metastasis. The IL-2 or LacZ gene was transferred into spleen-derived DCs using an adenoviral vector. Irradiated QRsP tumor cells were fused with IL-2 gene transduced DCs (fusion/IL-2) or LacZ gene transduced DCs (fusion/LacZ) by polyethyleneglycol. These fusion cells expressed major histocompatibility complex (MHC) class I and MHC class II, CD86, CD11c and CD8alpha. IFN-gamma and cytotoxic T lymphocyte (CTL) activity of splenic lymphocytes in mice vaccinated with fusion cells increased significantly as compared with those of DC or tumor cells vaccinated mice. CTL levels in fusion/IL-2-vaccinated mice were higher than that in fusion/LacZ-vaccinated mice. The number of lung metastasis in the fusion/IL-2 or fusion/LacZ-vaccineatd mice was significantly lower than that in mice vaccinated with DCs, tumor or PBS. The introduction of the IL-2 gene into fusion cells produced more potent therapeutic effects. Our results suggest that the fusion cells prepared from IL-2 gene transduced spleen derived DCs and tumor cells have the ability to induce therapeutic effect against lung metastasis.     

树突状细胞与肾癌融合细胞疫苗诱导抗肿瘤免疫

目的探讨自体树突状细胞(DC)与肾癌融合细胞疫苗诱导特异性抗肿瘤免疫应答。方法利用肿瘤细胞纯化技术,从手术切除的肾癌组织中分离出高纯度的肾癌细胞,用10%胎牛血清(FCS)的RPMI-1640进行原代培养。分离外周血单核细胞(PBMCs),在含重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)和白细胞介素4(rhIL-4)的培养条件下,诱导分化为DC。用细胞融合技术将DC与肾癌细胞融合制备DC与肾癌融合细胞疫苗。用流式细胞仪检测肾癌细胞、DC和融合细胞的表型;用噻唑盐(MTT)比色法检测融合细胞刺激自体T细胞的增殖能力,乳酸脱氢酶(LDH)释放的细胞毒实验检测融合细胞刺激的细胞毒性T淋巴细胞(CTLs)的杀瘤活性。结果DC高表达主要组织相融性复合体(MHC)Ⅰ类分子、MHCⅡ类分子和共刺激分子(CD86,CD80);原代肾癌细胞高表达一种高分子量的糖蛋白(MUC-1);自体DC与肾癌融合细胞疫苗同时表达肾癌细胞和DC细胞的表面抗原,并能有效刺激自体T细胞增殖,诱导产生的CTLs对自体肾癌细胞具有显著的杀伤活性。结论自体DC与肾癌融合细胞疫苗能诱导特异性抗自体肾癌细胞的免疫应答,为自体DC与肾癌融合细胞疫苗在治疗肾癌的临床应用中提供了实验依据。     

Monocyte differentiation induced by co-culture with tumor cells involves RGD-dependent cell adhesion to extracellular matrix

Macrophages that infiltrate tumor tissues, or tumor-associated macrophages (TAMs), affect the malignant behaviors of tumor cells. In this study, we attempted to induce monocytes to differentiate into TAM-like cells producing matrix metalloproteinases (MMPs) by co-culture with tumor cells. When human monocytes were co-cultured for 3-7 days with tumor cell lines, monocytes differentiated to produce MMP-9, accompanied by morphological changes. The in vitro cell invasion of MKN1 human gastric carcinoma cells into Matrigel membranes was promoted in the presence of differentiated monocytes, and the enhancement of cell invasion by differentiated monocytes was correlated with their MMP-9 productivity. The addition of an RGD (Arg-Gly-Asp) peptide to the culture significantly inhibited monocyte differentiation. The MMP-9 production from monocytes was diminished by the depletion of fibronectin from the conditioned media with gelatin-Sepharose, and potentiated by culturing them in fibronectin-coated plates. These results suggest that cell adhesion to the extracellular matrix plays a crucial role in monocyte differentiation into TAM-like cells.     

THE HUMORAL ANTITUMOR RESPONSES INDUCED BY IL-4 GENE-MODIFIED TUMOR VACCINE

ＴＨＥＨＵＭＯＲＡＬＡＮＴＩＴＵＭＯＲＲＥＳＰＯＮＳＥＳＩＮＤＵＣＥＤＢＹＩＬ４ＧＥＮＥＭＯＤＩＦＩＥＤＴＵＭＯＲＶＡＣＣＩＮＥ１ＹｕＹｉｚｈｉ于益芝ＣａｏＸｕｅｔａｏ２曹雪涛ＺｈａｎｇＭｉｎｇｈｕｉ张明徽ＬｅｉＨｏｎｇ雷虹ＴａｏＱｕｎ陶群Ｄｅｐ...     

基因修饰树突状细胞介导肿瘤治疗的进展与展望

研究表明,肿瘤的发生、发展与患者体内的T淋巴细胞功能缺陷有关,特别是晚期肿瘤患者,其体内往往存在体液免疫和细胞免疫功能的普遍低下,且其低下程度与其临床分期密切相关。进一步的研究发现,导致免疫功能低下的根本原因在于机体对成熟树突状细胞(Dendritic cell,DC)的功能下调     

Vaccination of fusion cells of rat dendritic and carcinoma cells prevents tumor growth in vivo

Several reports on immunotherapy using dendritic cells-based vaccine have been published. We investigated findings using fusion cells (FCs) generated from rat dendritic cells and a syngeneic hepatic cancer cell line with regard to inducing anti-tumor immunity. Vaccination of rats using FCs protected against growth of the subcutaneously implanted tumor in vivo and induced infiltration of CD8(+) T cells into the tumor. At the site of CD8(+) T cell infiltration, there were apoptotic tumor cells. T cells from spleen of FCs-vaccinated rats with protective ability against tumor growth included tumor specific cytotoxic CD8(+) T cells restricted to major histocompatibility complex Class I. In addition, adaptive transfer of in vitro re-stimulated splenic T cells with FCs was effective in preventing tumor growth and in vivo vaccinations of rats with FCs after resection of the subcutaneous implanted tumor inhibited local tumor recurrences. Immunotherapy using FCs appears to be an effective method if used in combination with surgical or other anti-cancer therapies.     

树突状细胞与热休克处理抗原联合诱导抗肿瘤免疫的研究

目的建立体外培养与扩增脐血树突状细胞(DC)的方法,并利用DC与热休克处理的肿瘤抗原联合诱导产生一种高效特异的抗肿瘤免疫.方法用GM-CSF和IL-4联合刺激诱导脐血单个核细胞分化为DC,再用加热处理的肿瘤抗原负载,与同源的淋巴因子活化的杀伤(LAK)细胞共培养.用电镜观察DC形态,CD1a和HLA-DR试剂盒检测DC细胞表面CD1a和HLA-DR的表达情况,MTT法测定细胞杀伤活性.结果在体外,DC-LAK对同源的SPC-A-1细胞具有相对特异的杀伤作用,LAK和DC-L的作用不具有明显特异性,DC-LAK的细胞毒作用最强;加热抗原组作用明显优于未加热处理组.结论将DC与热休克处理的肿瘤抗原联合,可以诱导产生一种高效并具有相对特异性的抗肿瘤免疫活性细胞.     

HIFU治疗后肿瘤抗原对树突状细胞的活化及其抗肿瘤效应

目的：探讨HIFU治疗小鼠H22抑制性肝癌后产生的肿瘤抗原对机体抗肿瘤免疫功能增强的机制。方法：正常小鼠骨髓中提取骨髓细胞,在rmIL-4、rmGM-CSF奈件下培养7d,制备小鼠骨髓树突状细胞,用HIFU治疗小鼠移植性肝癌后产生的肿瘤抗原活化树突状细胞,再用活化后的树突状细胞激活T淋巴细胞为细胞毒性T细胞,用MTT法检测CTL在体外特异性杀伤肿瘤靶细胞的能力。结果：B16肿瘤HSP70-肽复合物组和H22肿瘤HSP70-肽复合物组的脾淋巴细胞的增殖率均高于阴性对照组、H22肿瘤粗提物组和HIFU后H22肿瘤粗提物组,P〈0．001;但两组之间差异无统计学意义,P〉0．05。H22肿瘤HSP70-肽复合物组CTL对H22肿瘤细胞的杀伤率为70．0％,明显高于阴性对照组、H22肿瘤粗提物组和HIFU后H22肿瘤粗提物组（P〈0．001）,但对非靶细胞B16肿瘤的杀伤率与上述各组的差异无统计学意义;B16肿瘤HSP70-肽复合物组CTL对B16肿瘤细胞的杀伤率为78．5％,对H22细胞的杀伤率为21．4％,表明CTL对肿瘤细胞的杀伤作用具有特异性。结论：HIFU治疗后坏死肿瘤组织中的HSP70-肽复合物作为肿瘤疫苗,通过活化DC和刺激T淋巴细胞增殖为CTL,发挥特异性抗肿瘤免疫功能。     

封闭B7-H1分子对肿瘤浸润树突状细胞介导T细胞免疫功能的影响

目的:研究肿瘤浸润树突状细胞（tumorinfiltrating dendritic cell,TIDC）及脾脏树突状细胞（splenic dendritic cell,SDC）表面B7H1、B71、B72分子的表达情况;探讨封闭TIDC及SDC表面B7H1分子对其介导T细胞免疫功能的影响。方法： CD11c磁珠阳性分选法提取荷瘤小鼠的TIDC及SDC,流式细胞术检测其表面B7H1、B71、B72分子的表达情况。TIDC及SDC作为刺激细胞,脾脏T细胞作为反应细胞行混合淋巴细胞反应,同时加入B7H1抗体或其对照抗体,XTT比色法检测T细胞增殖指数,ELISA法检测T细胞分泌IL10的量。〖HT5W〗结果：〖HT5"SS〗B71及B72分子在TIDC表面的表达水平显著低于SDC（P<001）;B7H1分子在TIDC及SDC表面皆中度表达,表达水平无明显差异（P>0.05）。TIDC刺激T细胞增殖能力显著低于SDC,且诱导T细胞分泌更多的IL10。封闭DC表面B7H1分子后,TIDC刺激T细胞增殖能力显著提高（P<0.01）,且诱导T细胞分泌IL10的量明显减少（P<0.01）;SDC刺激T细胞增殖能力及诱导T细胞分泌IL10的量无明显变化（P>0.05）。结论：封闭DC表面的B7H1分子能显著提高TIDC活化T细胞的能力,可能解除TIDC介导的肿瘤免疫抑制     

膀胱肿瘤抗原致敏的树突状细胞诱导T淋巴细胞抗肿瘤效应的研究

目的：研究经膀胱肿瘤抗原致敏后的树突状细胞（Dc）对T淋巴细胞的激活、增殖作用及对T24膀胱肿瘤细胞的抑癌效应。方法：分离健康供血者外周血单个核细胞及T淋巴细胞，联合应用粒／巨噬细胞集落刺激因子（GM—CSF）及白介素-4（IL-4）从单个核细胞中培养出Dc，以人膀胱癌细胞系T24肿瘤细胞裂解物刺激Dc，检测经膀胱肿瘤抗原致敏后的DC对T淋巴细胞的细胞增殖动力学影响并用M1rr显色法测定致敏Dc诱导的T淋巴细胞对T24膀胱肿瘤细胞体外的抗肿瘤效应。结果：膀胱癌细胞裂解物致敏的Dc可诱导T淋巴细胞强烈的增殖反应，与对照组比较具有显著性差异（P〈0．01）；增殖后的T淋巴细胞对T24膀胱肿瘤细胞有明显的细胞毒作用。结论：经膀胱肿瘤抗原致敏的Dc能诱导产生显著的T淋巴细胞增殖，在体外有明显的抑癌效应。     

CM—CSF及IL—4增强树突状细胞免疫功能

目的 探讨粒／巨噬细胞集落刺激因子（ＧＭ－ＣＳＦ）及白介素－４（ＩＬ－４）对正常成人及大肠癌患者树突状细胞（ＤＣ）表现人白细胞抗原（ＨＬＡ）－ＤＲ及Ｂ７等免疫分子表达及其免疫功能的影响。方法 分离正常成人（ｎ＝１０）及大肠癌患者（ｎ＝１０）外周血ＤＣ,以ＧＭ－ＣＳＦ及ＩＬ－４联合刺激正常成人及大肠癌者ＤＣ,检测ＧＭ－ＣＳＦ及ＩＬ－４联合刺激前后ＤＣ表现ＨＬＡ－ＤＲ及Ｂ７表达水平及ＤＣ免疫功能变化。     

IL-18基因增强肿瘤抗原致敏DC诱导的CTL特异性杀伤肝癌细胞

目的:探讨腺病毒介导的白细胞介素18(IL18）基因转染能否使肿瘤抗原冲击的树突状细胞(dentritic cell,DC) 在体外诱导出更强的抗肝癌免疫反应。方法：携IL18 的重组腺病毒载体感染经肝癌细胞株HepG2冻融抗原致敏的DC(AdIL18HepG2/DC), FACS分析AdIL18HepG2/DC表面分子的表达, ELISA法检测IL18的分泌水平, 3HTdR掺入法检测T淋巴细胞增殖能力, MTT法检测细胞毒性T 淋巴细胞杀伤效应。结果： AdIL18HepG2/DC较未转染DC能高水平地表达CD1a、CD11c、CD80、CD86以及HLADR;较未经IL18转染的DC分泌较高水平的IL18。AdIL18HepG2/DC 能非常有效地刺激自体T 细胞增殖(CPM 值为228 018±1 079),其刺激强度显著强于AdIL18DC、HepG2/DC、AdlzcZ/DC及DC（均P＜0.05）。当靶细胞为HepG2时,AdIL18HepG2/DC诱导的CTL杀伤活性显著高于其他各组（均P＜0.05）,并且其杀伤能力与效应细胞数量成正比。结论： IL18 基因转染且肝癌抗原致敏的DC可以显著增强DC的特异性抗肝癌效应。     

Glycan modification of the tumor antigen gp100 targets DC-SIGN to enhance dendritic cell induced antigen presentation to T cells

Dendritic cells (DC) have gained much interest in the field of anticancer vaccine development because of their central function in immune regulation. However, the clinical application of ex vivo cultured DC has significant disadvantages. A vaccine that targets dendritic cells in vivo and enhances antigen presentation would be of great benefit. Because of its DC-restricted expression pattern, and its function as an antigen uptake receptor, DC-SIGN is an interesting candidate target structure for human immature DC. Here, we studied whether modification of the melanoma differentiation antigen gp100 with DC-SIGN-interacting glycans enhances targeting to human DC. A high-mannose form of gp100, as protein or as tumor lysate, not only interacted specifically with DC through DC-SIGN but also resulted in an enhanced antigen presentation to gp100-specific CD4(+) T cells. Our results indicate that glycan modification of tumor antigens to target C-type lectin receptors, such as DC-SIGN, is a new way to develop in vivo targeting DC strategies that simultaneously enhance the induction of tumor-specific T cells.     

Engineered fusion hybrid vaccine of IL-18 gene-modified tumor cells and dendritic cells induces enhanced antitumor immunity

Dendritic cell (DC)-tumor fusion hybrid vaccines that facilitate antigen presentation represent a novel powerful strategy in cancer immunotherapy. In our study, we investigated the antitumor immunity derived from the vaccination of fusion hybrids between engineered J558/IL-18 myeloma cells secreting Th1 cytokine IL-18 and DCs. DC/J558/IL-18 could secret a higher level of IL-18 than DCs, efficiently expressed J558 tumor antigen P1A, and enhanced ability of allogeneic T cell stimulation when compared to J558/IL-18. Our data showed that the immunization of BALB/c mice with DC/J558/IL-18 hybrids induced the most potent protective immunity against 1 x 10(6) cells with a J558 tumor challenge, compared to those immunized with the mixture of DCs and J558/IL-18, J558/IL-18, or J558. Furthermore, the immunization of mice with engineered DC/J558/IL-18 hybrids elicited stronger NK activity and J558 tumor-specific cytotoxic T lymphocyte (CTL) responses in vitro. In addition, DC/J558/IL-18 tumor cells into syngeneic mice induced a Th1 dominant immune response to J558 and resulted in tumor regression, which indicated that the antitumor effect mediated by DC/J558/IL-18 appeared to be dependent on TH1 cytokine production. These results demonstrate that the engineered fusion hybrid vaccines that combine Th1 gene-modified tumor with DCs may be an attractive strategy for cancer immunotherapy.     

The attenuated hepatocellular carcinoma-specific Listeria vaccine Lmdd-MPFG prevents tumor occurrence through immune regulation of dendritic cells

Immunotherapy is a promising treatment for liver cancer. Here, we tested the ability of the attenuated hepatocellular carcinoma-specific Listeria vaccine (Lmdd-MPFG) to treat hepatocellular carcinoma (HCC) in a mouse model. Immunization with the vaccine caused a strong anti-tumor response, especially in mice reinfused with dendritic cells (DCs). In mice that were also administered DCs, tumor suppression was accompanied by the strongest cytotoxic T lymphocyte response of all treatment groups and by induced differentiation of CD4+ T cells, especially Th17 cells. Additionally, the Lmdd-MPFG vaccine caused maturation of DCs in vitro. We demonstrated the synergistic effect of TLR4 and NLRP3 or NOD1 signaling pathways in LM-induced DC activation. These results suggest that the Lmdd-MPFG vaccine is a feasible strategy for preventing HCC.     

人肺腺癌细胞系SPC-A1 mRNA修饰的树突状细胞体外抗肿癌免疫反应

目的探讨人肺腺癌细胞系SPC-A1mRNA转染的人外周血树突细胞(dendriticcell,DC)疫苗在体外诱导特异性抗肿瘤免疫反应的能力。方法从健康人外周血分离外周血单个核细胞(peripheralbloodmononuclearcells,PBMC),在白细胞介素-4(interleu-kin-4,IL-4)和人粒细胞集落刺激因子(granulocyte-macrophagecolony-stimulatingfactor,GM-CSF)作用下诱导培养成DC。体外扩增、转录的方法获得人肺腺癌细胞系SPC-A1mRNA,并利用脂质体转染的方法导入DC,流式检测转染后DC表面标记,并与淋巴细胞共孵育,激活T淋巴细胞,MTT法检测其对人肺腺癌细胞的特异性杀伤活性。结果转染后的DC特异性表面标志及功能相关分子表达显著升高(CD8680·32±1·60;HLA-DR86·03±2·58;CD8326·97±1·62),诱导的特异性杀伤反应能力与经肿瘤细胞裂解物负载的DC、未经转染的DC及空白淋巴细胞组相比显著提高(80·34±2·71,P<0·01)。结论人肺腺癌细胞系SPC-A1mRNA转染的DC疫苗体外诱导的特异性抗肿瘤免疫能力显著增强。     

Selective repression of retinoic acid target genes by RIP140 during induced tumor cell differentiation of pluripotent human embryonal carcinoma cells

Background  

The use of retinoids as anti-cancer agents has been limited due to resistance and low efficacy. The dynamics of nuclear receptor coregulation are incompletely understood. Cell-and context-specific activities of nuclear receptors may be in part due to distinct coregulator complexes recruited to distinct subsets of target genes. RIP140 (also called NRIP1) is a ligand-dependent corepressor that is inducible with retinoic acid (RA). We had previously shown that RIP140 limits RA induced tumor cell differentiation of embryonal carcinoma; the pluriopotent stem cells of testicular germ cell tumors. This implies that RIP140 represses key genes required for RA-mediated tumor cell differentiation. Identification of these genes would be of considerable interest.     

细胞因子与树突状细胞分化诱导

树突状细胞(DC)是一类非常重要的抗原递呈细胞,在肿瘤、慢性感染和自身免疫疾病等治疗方面应用前景乐观.通过不同细胞因子诱导不同的前体细胞分化以获得可供利用的DC是目前研究热点.现综述GM-CSF、IL-4、IL-3、IL-13、IL-10、TNF-α、G-CSF、SCF、FL、TGF-β等各类细胞因子在DC诱导分化及成熟中的应用和作用机制.