树突细胞联合细胞因子诱导的杀伤细胞对急性白血病细胞的体外杀伤作用

目的:研究细胞因子诱导的杀伤细胞(CIK)与同源树突状细胞(DC)共培养后CIK细胞的表型、增殖活性的变化,及抗急性白血病细胞活性.方法:正常人外周血单个核细胞诱导DC和CIK细胞,将DC与CIK共培养,以CIK细胞单独培养为对照.用台盼蓝活细胞计数计算细胞扩增倍数,MTT法测定杀伤活性,流式细胞术分析免疫表型.结果:DC-CIK细胞增殖能力明显高于CIK细胞(P＜0.05); DC、CIK细胞共培养后,CD3+ CD8+、CD3+ CD56+双阳性细胞比率较同条件下CIK细胞组显著增多(P＜0.05);在2.5∶1-20∶1的效靶比范围内,DC-CIK共培养物对AML细胞的杀伤率显著高于CIK细胞(P＜0.05),且杀伤率与效靶比呈正相关.结论:DC-CIK细胞的增殖能力、对AML细胞的杀伤活性均高于CIK细胞,为DC-CIK细胞免疫治疗提供了实验和理论依据.     

细胞凋亡基因的发现

细胞凋亡基因的发现李雨民,孙元明（中国医学科学院放射医学研究所天津３００１９２）细胞凋亡是一种具有特征性的形态变化和生化改变的细胞死亡过程,机体启动这一机制以清除无用和有害的细胞。近来细胞凋亡基因的研究取得了很大进展,８０年代以来Ｈｏｒｖｉｔｚ等在研...     

蛋白细胞

蛋白细胞(albuminous cells)是裸子植物韧皮部中唯一与筛胞相连的一种特殊的薄壁组织细胞,其结构和功能相当于被子植物的伴胞。蛋白细胞最早是由 Strasburger(1891)发现并命名的,因此又有人称之为Strasburger 细胞。根据 Stras-     

细胞内吞的研究进展

细胞内吞(endocytosis)是细胞从周围环境中摄取各种物质的过程,是细胞生理代谢的一个极为重要的现象。几乎所有的真核细胞都通过内吞作用摄取细胞外基质中的大分子。70年代中期,Steinman等人发现了细胞膜系就再循环现象,Goldstein等人第一次较为完整地描述了由受体介导的低密度脂蛋白(Low densitylipoprotein,简称LDL)内吞过程,这两项成果使细胞内吞成为细胞生物学研究领域中的     

细胞粘附与树突状细胞迁移机制

树突状细胞（dendritic cell,DC)是迄今已知功能最强的抗原递呈细胞,DC体内迁移机制与其分化成熟。表型转换及生物学功能发挥密切相关。粘附分子及其介导的细胞粘附参与了DC的迁移机制。深入研究DC迁移的分子基础。有助于进一步阐明DC的生物学特性和功能。也可以通过DC进行免疫调控用于临床疾病防治提供重要理论基础。     

细胞外间质

细胞外间质由四大家族组成,胶原蛋白,蛋白多糖。弹性蛋白和细胞外间质糖蛋白。细胞外间质成分不仅仅是细胞的惰性支持物,它具有活性的生物功能,例如细胞粘附及迁移,甚至涉及基因表达。细胞外间质研究是一个十分活跃的生物学领域。     

谈谈植物的栓质细胞和硅质细胞

在禾本科植物的茎、叶表皮中有两种特殊的细胞——栓质细胞和硅质细胞。因为它们的形态特点与茎叶表皮的其他细胞有很大差别,学生了解的不多,故笔者根据有关文献及个人实验观察,对此作一简介。栓质细胞、硅质细胞分布的特点除禾本科植物外,在单子叶植物的其他一些科如莎草科、鸭跖草科、姜科等植物中的茎叶表皮上也有栓质细胞和硅质细胞的分布。紫露草属(Gibasis)的叶表皮上的硅质细胞为等径四边形,主要分布于叶的两面,并陷于表皮细胞以下。在不同的种中,硅质细胞的分布     

细胞凋亡与线粒体细胞色素c

１细胞色素ｃ在细胞凋亡中的作用Ｋｅｒｒ于１９７２年提出了细胞凋亡（ａｐｏｐｔｏｓｉｓ）,随后发现细胞凋亡现象普遍存在于各种生物中,与细胞的生长发育,免疫调节以及许多病变如肿瘤有直接关系。与细胞坏死不同,细胞凋亡是一个相对主动的过程,伴随着一系列形态上...     

虚拟细胞

虚拟细胞是20世纪末在国外刚刚兴起的一种细胞生物学研究方法,主要是通过计算机建立人工细胞模型,模拟细胞内外环境,从而进行生物学的研究和探索。虚拟细胞包含了多门学科前沿的研究成果,具有十分重要的意义。现从其发展历程、应用及其对生物学工作者的关系等方面进行综述。     

Human cytokine-induced killer cells have enhanced in vitro cytolytic activity via non-viral interleukin-2 gene transfer

Modulation of the immune system by genetically modified immunological effector cells is of potential therapeutic value in the treatment of malignancies. Interleukin-2 (IL-2) is a crucial cytokine which induces potent antitumor response. Cytokine-induced killer cells (CIK) have been described as highly efficient cytotoxic effector cells capable of lysing tumor cell targets and are capable of recognizing these cells in a non-MHC restricted fashion. Dendritic cells (DC) are the major antigen presenting cells. This study evaluated the antitumor effect of CIK cells which were non-virally transfected with IL-2 and co-cultured with pulsed and unpulsed DC. Human CIK cells generated from peripheral blood were transfected in vitro with plasmid encoding for the human IL-2. Transfection involved a combination of electrical parameters and a specific solution to deliver plasmid directly to the cell nucleus by using the Nucleofector(R) electroporation system. Nucleofection resulted in the production of IL-2 with a mean of 478.5 pg/106 cells (range of 107.6-1079.3 pg /106 cells/24 h) compared to mock transfected CIK cells (31 pg/106 cells) (P = 0.05). After co-culturing with DC their functional ability was assessed in vitro by a cytotoxicity assay. On comparison with non-transfected CIK cells co-cultured with DCs (36.5 +/- 5.3 %), transfected CIK cells co-cultured with DC had a significantly higher lytic activity of 58.5 +/- 3.2% (P = 0.03) against Dan G cells, a human pancreatic carcinoma cell line.     

负载Her-2多肽的DCIK细胞对乳腺癌细胞杀伤作用研究

本文观察利用树突状细胞(DC)呈递肿瘤抗原(Her-2多肽)的特性提高DCIK细胞对乳腺癌细胞的杀伤活性。提取外周血来源的有核细胞诱导分离出细胞因子诱导的杀伤细胞(CIK)和树突状细胞(DC),DC负载Her-2多肽后和CIK细胞共培养产生DCIK细胞,并鉴定其HLA基因型。分析三株肿瘤细胞(MDA-MB-231、SK-BR-3、MCF-7)HLA基因型和Her-2蛋白表达情况。用细胞毒试验(CCK-8法)测定DCIK细胞的对三株Her2表达不同的乳腺癌细胞株的杀伤活性。结果表明DCIK细胞对MDA-MB-31、SK-BR-3、MCF-7的杀伤率(效靶比10:1)分别为50.38%±3.25%、52.19%±3.25%、47.09%±2.41%。而负载Her-2多肽的DCIK细胞对MDA-MB-231、SK-BR-3、MCF-7的杀伤率分别为76.30%±1.74%(P<0.001)、55.70%±3.05%(P=0.0143)、47.67%±2.40%(P=0.6972)。实验证明负载Her-2多肽的DCIK细胞能显著提高对Her-2( )的乳腺癌细胞的杀伤作用,为乳腺癌患者进行过继免疫治疗提供了理论依据。     

Sunitinib Indirectly Enhanced Anti-Tumor Cytotoxicity of Cytokine-Induced Killer Cells and CD3+CD56+ Subset through the Co-Culturing Dendritic Cells

Cytokine-induced killer (CIK) cells have reached clinical trials for leukemia and solid tumors. Their anti-tumor cytotoxicity had earlier been shown to be intensified after the co-culture with dendritic cells (DCs). We observed markedly enhanced anti-tumor cytotoxicity activity of CIK cells after the co-culture with sunitinib-pretreated DCs over that of untreated DCs. This cytotoxicity was reliant upon DC modulation by sunitinib because the direct exposure of CIK cells to sunitinib had no significant effect. Sunitinib promoted Th1-inducing and pro-inflammatory phenotypes (IL-12, IFN-γ and IL-6) in DCs at the expense of Th2 inducing phenotype (IL-13) and regulatory phenotype (PD-L1, IDO). Sunitinib-treated DCs subsequently induced the upregulation of Th1 phenotypic markers (IFN-γ and T-bet) and the downregulation of the Th2 signature (GATA-3) and the Th17 marker (RORC) on the CD3⁺CD56⁺ subset of CIK cells. It concluded that sunitinib-pretreated DCs drove the CD3⁺CD56⁺ subset toward Th1 phenotype with increased anti-tumor cytotoxicity.     

抗原负载DCs诱导的CIK细胞对B16黑色素瘤抑制作用的形态学观察

目的:探讨抗原负载树突状细胞(dentritic cells,DCs)诱导的CIK(cytokine induced killer)细胞对B16黑色素瘤的抑瘤作用。方法:分离、培养DC和CIK细胞,取部分DC进行肿瘤抗原负载,将其与CIK细胞按1:10的比例共培养3d,即为抗原负载的DC-CIK。建立B16黑色素瘤小鼠模型,分别于瘤周围皮下注射经Brdu标记的CIK、DC-CIK、抗原负载DC-CIK。按注射细胞进行分组,测量注射前后各组小鼠的瘤体积,计算抑瘤率,比较其抑瘤作用。应用免疫组化方法和透射电镜观察抗原负载DC-CIK细胞在皮肤中的分布及杀伤肿瘤细胞的形态学表现。结果:抗原负载DC诱导的CIK(细胞组抑瘤率(86.57%)高于CIK细胞组(33.34%,P<0.05)和DC-CIK细胞组(61.08%,P<0.05);光镜下抗原负载DC-CIK细胞主要分布在皮下组织,癌组织周围,特别是癌巢周边。透射电镜下抗原负载DC-CIK细胞体积大,核有切迹,细胞质内细胞器丰富,粗面内质网扩张。细胞表面有突起,与肿瘤细胞密切接触。大量肿瘤细胞凋亡、坏死。结论:CIK细胞经抗原负载DC诱导后抑瘤作用明显强于单纯CIK细胞和DC-CIK细胞。     

Generation of functional dendritic cells for potential use in the treatment of acute lymphoblastic leukemia

Immunotherapy of malignant diseases mediated by dendritic cells (DC) pulsed with tumor antigens ex vivo is a promising new tool in the individual treatment of malignant diseases. The present study focuses on the problem of how to optimize in vitro culture conditions and induce the maturation of DC with the capacity to induce antitumor immunity toward leukemic cells. DC were generated from peripheral mononuclear cells by co-cultivation with granulocyte/macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4). Tumor antigens were added for 2 h after 7 days in culture. Irradiated leukemic blasts, blast lysate, apoptotic cells from the Jurkat cell line (T ALL) and their lysate were used in various concentrations for antigen pulsing. Harvested DC were phenotyped by flow cytometry, and viability was assessed using trypan blue exclusion (Annexin test). After the cells had been pulsed with tumor antigens and co-cultured with autologous lymphocytes, the production of interferon-gamma (IFN-gamma) and IL-12 was analyzed, and lymphocyte proliferative response and cytotoxicity against the target tumor cell line were assessed. The cultivation of monocytes under the described conditions led to the expression of surface markers typical of DC (i.e. CD83, CD86, HLA-DR, CD11c and CD40). Pulsation by antigens from leukemic cells further increased the cell populations expressing these markers. Antigen pulsation decreased the viability of generated DC depending on the increase in concentration of tumor antigens. Pulsed DC-lymphocyte interaction increased the proliferative response of lymphocytes and IFN-gamma production depending on the type of tumor antigens used for pulsation. The highest proliferative response was detected with DC pulsed with Jurkat cell-line lysate. Similarly to the proliferation assay, cytotoxic testing showed the highest efficiency of DC pulsed with Jurkat cell-line lysate in killing the target malignant cells. Our results show that an appropriate antigen concentration used for DC pulsing is one of the crucial factors in an effective treatment strategy, as high concentrations of tumor antigens induce apoptosis of DC, thereby rendering them non-functional. Under optimal conditions, pulsation by lysate from leukemic blasts induced the maturation of DC and led to an increase in the proliferation of autologous lymphocytes, to the production of Th1-cytokines and to the induction of cytotoxicity toward the leukemic cell line. These results are encouraging for the possible application of pulsed DC in the therapy of acute lymphoblastic leukemia.     

树突状细胞与CIK细胞共培养诱生的DCCIK细胞群对肿瘤的杀伤作用

由树突状细胞(DC)与细胞因子诱导的同源杀伤细胞(CIK)的共培养诱生的细胞群(DCCIK)对肿瘤细胞的细胞毒活性的研究。DCCIK细胞体外杀伤肿瘤靶细胞A549(MTT法),效靶比为10∶1、5∶1时杀伤率分别为61%、52%。DCCIK细胞诱导培养3周后,效靶比为10∶1、5∶1时杀伤率分别为64%和56%。数据亦表明DCCIK细胞对靶细胞的杀伤优于CIK细胞。动物体内实验分荷瘤A549、BEL7404和A375三组,每组分(A)DCCIK 化疗、(B)单用化疗。治疗20天、35天后测量各组肿瘤消失率。结果显示:DCCIK 化疗的抑瘤效果明显好于单纯化疗。提示DCCIK细胞有临床应用前景。     

MAGE-3原核表达载体的构建和表达

通过RT-PCR扩增957bp的MAGE-3全长编码序列,将该片段克隆至Pgex-4T-2原核表达载体,转化大肠杆菌BL-21,经IPTG诱导表达,并经12%SDSPAGE凝胶电泳,考马斯亮蓝染色及Western blot鉴定,证明了目的基因的有效表达,目的蛋白高达细菌总蛋白的32%。表达产物经Glutathione Sepharose 4B 纯化后,每100mL菌液最终可获得3mg的目的蛋白,蛋白纯度在90%以上。纯化的GST-MAGE-3蛋白在体外冲击树突状细胞,能诱导特异性CTL杀伤肿瘤细胞活性。     

Comparative analysis of cytotoxic T lymphocyte response induced by dendritic cells loaded with hepatocellular carcinoma -derived RNA or cell lysate

The choice of the tumor antigen preparation used for dendritic cell (DC) loading is important for optimizing DC vaccines. In the present study, we compared DCs pulsed with hepatocellular carcinoma (HCC) total RNA or cell lysates for their capacity to activate T cells. We showed here that HCC total RNA pulsed-DCs induced effector T lymphocyte responses which showed higher killing ability to HCC cell lines, as well as higher frequency of IFN-γ producing of CD4+ and CD8+ T cells when compared with lysate pulsed-DCs. Both of RNA and lysate loading did not influence the changes of mature DC phenotype and the capacity of inducing T cell proliferation. However, HCC lysate loading significantly inhibited the production of inflammatory cytokines IL-12p70, IFN-γ and enhanced the secretion of anti-inflammatory cytokines IL-10 of mature DCs. Our results indicated that DCs loaded with HCC RNA are superior to that loaded with lysate in priming anti-HCC CTL response, suggesting that total RNA may be a better choice for DCs-based HCC immunotherapy.     

The apoptotic and proliferative fate of cytokine-induced killer cells after redirection to tumor cells with bispecific Ab

BACKGROUND: Cytokine-induced killer (CIK) cells are ex vivo expanded T cells with co-expression of CD3 and CD56 and NK activity. They have recently been evaluated in a phase I/II clinical trial against malignant lymphoma. Bispecific Ab (bsAb) redirect CIK cells to tumor targets, thus enhancing their cytotoxicity. While bsAb may improve T-cell mediated anti-tumor activity, little is known about the fate of effector cells upon redirection to tumor targets using a bsAb. METHODS: Using ex vivo-activated CIK cells, Her2/neu expressing breast and ovarian cell lines and a F(ab')2 Her2/neu x CD3 bsAb, we investigated the anti-tumor activity and the proliferative and apoptotic outcome of CIK cells. RESULTS: When redirected to tumor targets with bsAb, there was a significant increase in anti-tumor activity as well as an increase in both CIK cell proliferation and apoptosis. The addition of agonistic Ab against CD28 did not significantly increase proliferation or apoptosis of CIK cells redirected to CD80- and CD86- tumor targets. To attempt to reduce T-cell apoptosis, we incubated CIK cells in the presence of the pan-caspase inhibitor z-VAD-fmk, which led to a partial reduction in T-cell apoptosis without increasing cellular cytotoxicity. DISCUSSION: bsAb are effective in redirecting activated T cells to tumor targets and such redirection leads to both T-cell proliferation and apoptosis that are not altered by co-stimulation through CD28. Effector cell apoptosis can be reduced by using a caspase inhibitor but this does not increase CIK cell cytotoxicity.     

Dendritic cells efficiently acquire and present antigen derived from lung cancer cells and induce antigen-specific T-cell responses

Active immunotherapy of cancer requires the availability of a source of tumor antigens. To date, no such antigen associated with lung cancer has been identified. We have therefore investigated the ability of dendritic cells (DC) to capture whole irradiated human lung tumor cells and to present a defined surrogate antigen derived from the ingested tumor cells. We also describe an in vitro system using a modified human adenocarcinoma cell line (A549-M1) that expresses the well-characterized, immunogenic influenza M1 matrix protein as a surrogate tumor antigen. Peripheral blood monocyte-derived DC, when co-cultured with sub-lethally irradiated A549 cells or primary lung tumor cells derived from surgical resection of non-small cell carcinoma (NSCLC), efficiently ingested the tumor cells as determined by flow cytometry analysis and confocal microscopic examination. More importantly, DC loaded with irradiated A549-M1 cells efficiently processed and presented tumor cell-derived M1 antigen to T cells and elicited antigen-specific immune responses that included IFNgamma release from an M1-specific T-cell line, expansion of M1 peptide-specific Vbeta17+ and CD8+ peripheral T cells and generation of M1-specific cytotoxic T lymphocytes (CTL). We also compared DC loaded with irradiated tumor cells to those loaded with tumor cell lysate or killed tumor cells and found that irradiated lung tumor cells as a source of tumor antigen for DC loading is superior to tumor cell lysate or killed tumor cells in efficient induction of antigen-specific T-cell responses. Our results demonstrate the feasibility of using lung tumor cell-loaded DC to induce immune responses against lung cancer-associated antigens and support ongoing efforts to develop a DC-based lung cancer vaccine.