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1.
[目的]从人外周血分离、纯化、扩增树突状细胞(DC),并对其形态学和免疫学特性进行初步探讨.[方法]从人外周血分离DC前体细胞(主要为CD14 细胞)用重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)和重组人白细胞介素-4(rhIL-4)联合培养,诱导扩增成熟DC.观察DC形态、分析DC表型、核型及检测DC激发同种异体淋巴细胞增殖能力.[结果]分离的DC前体经rhGM-CSF和rhIL-4共同培养1周后,可获得大量成熟DC,扩增了24.5倍,纯度达90%以上.DC高表达分化抗原CD86、CD40、HLA-DR、CD83、CDIa,能强烈激活同种异体T淋巴细胞增殖.[结论]人外周血CD14 细胞经体外诱导培养,可以生成大量功能成熟的DC,从而为进一步开展DC的基础研究和临床应用打下基础. 相似文献
2.
人外周血树突状细胞体外稳定、高效培养的新方法 总被引:1,自引:0,他引:1
目的:建立稳定、高效的人外周血树突状细胞(dendritic cells,DC)体外培养的新方法。方法:离心获取人外周血白膜层细胞,裂解去除红细胞后获得全部白细胞,贴壁培养1 h获得单核细胞,加入重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)1 000 U/ml 重组人白细胞介素-4(rhIL-4)500 U/ml,体外培养7 d获得DC,以流式细胞术分析表型,体外混合淋巴细胞反应检测细胞抗原提呈功能。结果:从100 ml全血分离获得具有较好活力的贴壁单核细胞仅需2 h;部分肿瘤患者应用传统分离方法不能获得单个核细胞,用新方法仍能从患者外周血稳定地获得足够数量的单核细胞。新方法培养生成DC的数量为传统方法培养生成DC数量的1.8~2.6倍;其中40%~85%为CD1a ,表达高水平的HLA-DR、CD80、CD86,体外可以强烈刺激同种T细胞增殖。结论:建立了更为稳定的、得率更高的DC体外扩增培养的新方法。 相似文献
3.
人外周血树突状细胞的体外诱导和鉴定 总被引:1,自引:0,他引:1
目的:建立从人外周血体外诱导扩增树突状细胞(DC)的方法:方法:从正常人外周血F1细胞分离单个核细胞,经三种细胞因子——重组人粒细胞一巨噬细胞集落刺激因子(rh GM—CSF)、重组人白细胞介素4(rh IL-4)和重组人肿瘤坏死因子(rh TNFα)联合诱导培养,10天后收获细胞,利用光学显微镜、透射电镜观察其外部形态和细胞超做结构,流式细胞仪检测其细胞表面抗原,自体混合淋巴细胞反应检测其刺激T细胞的增殖活性:结果:培养收获了高纯度的DC,这些细胞表面有大量的不规则突起,核也呈不规则状,核仁小,核膜异染色质聚集,含有丰富的细胞器,如核糖体、内质网、溶酶体等;细胞表面高水平表达HLA—DR、CD1α、CD80、CD83和CD86;自体混合淋巴细胞反应表明诱导所得的DC具有很强的激发同种T细胞增殖的能力。结论:人外周血单个核细胞经细胞因子诱导培养可以得到大量的成熟DC。 相似文献
4.
目的 探索体外扩增宫颈癌患者外周血来源的成熟树突状细胞(denelritic cell,DC)的方法,并鉴定DC的形态、结构、表型及生物学活性。方法 淋巴细胞分离液分离宫颈癌患者外周血,得到DC前体细胞(PBMC),以(3M-CSF、IL-4、TNF-α诱导培养。用光镜和透射电镜观察形态结构,流式细胞仪检测细胞表面特异性分子,MTT法测定DC刺激T细胞增殖的活性。结果 联合应用细胞因子可诱导扩增出源自宫颈癌患者外周血的成熟DC,具有典型形态特征,较高表达HLA-DR、CD1a、CD80,能强烈激发同种异体T细胞增殖反应。结论 宫颈癌患者外周血体外培养可成功获得成熟的DC,为开展宫颈癌临床免疫治疗奠定了基础。 相似文献
5.
目的 探讨体外诱导培养较高纯度和成熟度的树突状细胞(DC)的方法.方法 密度梯度离心分离人类外周血单核细胞,直接贴壁法收集前体细胞,含人类血清及细胞因子rhGM-CSF和rhIL-4完全RPMI1640培养基,37 ℃、5%CO2孵育培养;第3天全量换液并添加细胞因子,第5天加入rhTNF-α促进成熟,第8天收获悬浮细胞.同时进行形态学和细胞表型分析.结果 镜下表现为典型的DC形态特征;流式分析细胞表型CD1a阳性表达从第5天的(18.69±8.73)%增加到第8天的(78.07±9.43)%,CD83表达从(14.74±4.06)%增加到(46.82±14.15)%,第5天CD80表达(9.82±4.61)%,第8天CD80表达(60.11±20.50)%;40 ml外周血约得(3.12±1.30)×106个DC.结论 该方法可以体外诱导培养出较大数量和较高纯度的成熟DC,并且培养体系成熟,操作性和可重复性强. 相似文献
6.
树突状细胞的诱导培养及对胃癌细胞株MKN45的体外杀伤效应的初步研究 总被引:3,自引:0,他引:3
目的 探讨树突状细胞(DC)体外培养的方法及CpG ODN1826刺激DC对胃癌细胞MKN45的杀伤效应.方法 分离正常人外周血DC,培养至第5天,实验分为4组,A组[粒细胞-巨噬细胞集落刺激因子(GM-CSF)+白细胞介素-4(IL-4)]、B组[GM-CSF+IL-4+肿瘤坏死因子-α(TNF-α)]、C组(nonCpG ODN)和D组(CpG ODN 1826).自外周血单核细胞(PBMC)诱导分离DC,流式细胞仪检测DC表面标志,MTT法测定其刺激对同种异体淋巴细胞增殖的影响及对胃癌细胞的杀伤效应.结果 体外培养至第10天,D组和B组均出现大量典型的DC形态.倒置显微镜下见细胞呈树突状、伪足状突起.流式细胞仪检测,D组显著高表达共刺激分子(CD40、CD1a、CD80、CD86)和MHC-Ⅱ类分子,均分别高于其他各组(P<0.05).在体外能强烈刺激同种异体混合淋巴细胞的增殖,显著增强DC杀伤胃癌细胞MKN45的活性.结论 该培养方法可获得较高纯度典型的DC,同时CpG ODN可显著诱导人外周血DC分化成熟,增强DC对胃癌细胞MKN45的杀伤作用. 相似文献
7.
目的:探讨正常人外周血单按细胞、肿瘤患者外周血单按细胞、脐带血CD34^ 细胞、非淋巴细胞白血病细胞来源的树突状细胞性能差异。方法:分别利用正常人及肿瘤患者的外周血分离单个核细胞,黏附贴壁法收集单核细胞,在GM-CSF、IL-4及TNF-α细胞因子组合下诱导树突状细胞;脐带血细胞则利用MACS系统分离纯化CD34^ 细胞作为树突状细胞的前体细胞,培养体系为FL SCF GM-CSF IL-4 TNF-α;非淋巴细胞白血病细胞作为树突状细胞的前体细胞培养条件同CD34^ 细胞。流式细胞仅分析诱导细胞的表型,电镜分析细胞形态。结果:利用4种不同来源的前体细胞在不同细胞因子组合下可分别诱导出DC细胞,细胞CD1a,CD80,HLA-DR的表达较诱导分化前明显上调.细胞形态学表明4种细胞来源的DC均有较典型的DC细胞形态,并均可刺激同种异体T细胞的增生。结论:利用健康人或肿瘤患者外周血可成功诱导出树突状细胞,利用脐血CD34^ 细胞及部分非淋巴细胞白血病原代细胞也可体外诱生出树突状细胞,不同来源的树突状细胞在形态及初步功能上无明显区别。 相似文献
8.
目的:观察脐血、健康人及慢性柱细胞性白血病(CML)患者外周血3种不同来源的树突状细胞(DC)及其抗白血病效应。方法:分离3种来源的单个核细胞(MNC)并在细胞因子(GM-CSF IL-4 TNF-α)作用下诱导培养DC。观察DC形态,流式细胞技术检测DC免疫表型(CD1α、CD80、CD83、HLA-DR);将所得DC经CML细胞可溶性抗原负载后和其同源T细胞混合诱导抗原特异性CTL,MTT法检测其杀伤活性。结果:3种来源MNC在细胞因子作用下均可获得形态典型的DC,且高表达CD1α、CD80、CD83、HLA-DR,经抗原负载后的DC诱导的CTL对CML靶细胞的杀伤活性也较强。结论:脐血、健康人及CML患者外周血均为理想的DC来源;有较好的诱导CTL杀伤白血病细胞效应;应用DC瘤苗免疫治疗白血病必将成为一种崭新的肿瘤免疫治疗方法,有十分诱人的发展前景。 相似文献
9.
人外周血树突状细胞的体外扩增及鉴定 总被引:71,自引:6,他引:71
树突状细胞(DC)作为抗原提呈细胞,在激发T细胞免疫应答及T细胞依赖性抗体生成中起重要作用,骨髓及外周血的CD34造血前体细胞在GM-CSF和TNF-α的作用下,可在体外分化发育,生成树突状细胞.本研究从正常人外周血分离获得单核细胞,加入100ng/ml hGM-CSF、500U/ml hIL-4,体外培养1周后,获得大量高纯度的树突状细胞,其高表达MHC-Ⅰ、MHC-Ⅱ类分子及共刺激分子B7-1和CD40,能强烈激发同种异体T淋巴细胞增殖,培养条件以应用自体血清或胎牛血清为最佳;单独应用hGM-CSF只能生成巨噬细胞;在培养末期加入TNFα可促进DC进一步成熟.本研究为DC的深入研究与临床应用奠定了基础. 相似文献
10.
肺癌患者外周血树突状细胞的体外扩增及鉴定 总被引:2,自引:1,他引:2
目的 探索体外扩增成熟树突状细胞(dendritic cell,DC)的方法,并进一步鉴定Dc的形态及表型,为开展肿瘤临床生物治疗奠定基础。方法 取肺癌患者外周血,经淋巴细胞分离液分离得到DC前体细胞,加入生长因子DCGF诱导DC生成,并加入自体肿瘤相关抗原刺激。收集细胞用透射电镜观察,并用流式细胞仪测定细胞表型。结果 应用该法扩增细胞可以得到大量成熟DC。电镜观察DC具有典型的形态。流式细胞仪细胞表型测定CD80为81.8%,HLA—DR为98.3%,CD86为69.8%,CDla为19.7%,CD14为66.9.1%,CD80和HLA-DR较未成熟DC的表达明显增加。结论 肺癌患者外周血体外培养可成功地获得成熟的DC,进一步为肿瘤临床生物治疗奠定了基础。 相似文献
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12.
Capobianco A Rovere-Querini P Rugarli C Manfredi AA 《International journal of cancer. Journal international du cancer》2006,119(12):2861-2869
Dendritic cells (DCs) and natural killer (NK) cells are key players at the interface between innate resistance and acquired immunity. NK cells can induce DC maturation, a differentiation process whereby DCs respond to a environmental stimulus and acquire the ability of eliciting adaptive immunity. Conversely, maturing DCs promote NK functions in vivo and in vitro. This interplay has important consequences on the immune response to pathogens and possibly to neoplastic cells. Here, we show that B16 melanoma cells actively modulate the interaction between DCs derived from bone marrow precursors and NK/LAK cells propagated from the spleen of C57BL/6 mice. DCs increased in a dose-dependent manner the ability of NK/LAK cells to kill melanoma cells and to produce cytokines. This activatory cross-talk entailed the production of IL-18 by DCs and of IFN-gamma by NK/LAK cells. Melanoma cells were not a passive target of NK activity; they regulated the outcome of the interaction between DCs and NK/LAK cells, inhibiting the in vitro production of cytokines as effectively as the genetic deletion of IL-18 or IFN-gamma. Interference with the NK/DC interaction possibly represents a mechanism used by growing tumors to evade the immune response. 相似文献
13.
背景与目的:目前对前列腺原位癌进展到浸润性癌的机制知之甚少.在前列腺癌中,间质细胞能分泌一定的因子,促进肿瘤细胞的生长和浸润,但是正常的上皮分泌细胞/原位癌细胞和间质细胞被完整的基底细胞层所隔离.本研究探讨前列腺基底上皮细胞分泌的Laminin-5因子对前列腺原位癌细胞侵袭能力的影响.方法:以BPH-1细胞作为前列腺原位癌的体外模型,研究细胞在基底上皮细胞条件培养基(PEC-CM)作用下的粘附、侵袭能力和细胞极性改变情况,并应用免疫沉淀反应和Western blot等技术分析PEC-CM的主要成分.结果:PEC-CM含有粘着蛋白等因子,能促进细胞粘附、极化、侵袭和Akt磷酸化.LY294002和Wortmannin能够部分抑制PEC-CM所激发的细胞侵袭作用,Laminin-5正是PEC-CM中刺激BPH-1细胞侵袭的有效蛋白成分,两者抑制效果比较差异有显著性(P<0.01).用抗体耗竭PEC-CM内的Laminin-5也能有效降低BPH-1细胞的侵袭能力(P<0.01).结论:由前列腺基底细胞分泌的Laminin-5通过PI3K依赖的传导通路,对前列腺癌细胞侵袭能力有促进作用,提示基底细胞在前列腺原位癌向浸润性癌的演变过程中可能具有重要作用. 相似文献
14.
Monica Rieppi Veronica Vergani Carmen Gatto Gerardo Zanetta Paola Allavena Giulia Taraboletti Raffaella Giavazzi 《International journal of cancer. Journal international du cancer》1999,80(2):303-307
The dissemination of ovarian carcinoma cells within the abdominal cavity involves interaction of tumor cells with the peritoneal mesothelium. The aim of our study was to investigate whether mesothelial cells might directly affect the spreading of this tumor by inducing motility and invasiveness of human ovarian carcinoma cells. Serum‐free supernatants of cultured human mesothelial cells [conditioned medium (CM)] induced chemotaxis and invasiveness of the human ovarian carcinoma cell lines SK‐OV‐3, OVCAR‐5 and A2780 in a Boyden chamber. Checkerboard analysis indicated that the stimulated motility was prevalently directional. Most of the chemotactic activity was retained by a heparin affinity column, indicating that the motility factor(s) is a heparin‐binding protein. Using different monoclonal antibodies (MAbs) directed against chemotactic factors that are secreted by mesothelial cells, we found that chemotaxis was partially prevented (64.8% inhibition) by antibodies against fibronectin (FN). CM also induced haptotactic migration of ovarian carcinoma cells, and anti‐FN antibodies significantly inhibited haptotaxis. The presence of FN in the CM was confirmed by Western blot analysis. Our findings suggest that mesothelium plays an active role in inducing the intraperitoneal spread of ovarian carcinoma cells, and point to FN as being one of the main mediators of mesothelium‐induced ovarian carcinoma cell motility. Int. J. Cancer 80:303–307, 1999. © 1999 Wiley‐Liss, Inc. 相似文献
15.
M Murase M Kano T Tsukahara A Takahashi T Torigoe S Kawaguchi S Kimura T Wada Y Uchihashi T Kondo T Yamashita N Sato 《British journal of cancer》2009,101(8):1425-1432
Background:
Several human cancers have been found to contain cancer stem-like cells (CSCs) having cancer-initiating ability. However, only a few reports have shown the existence of CSCs in bone and soft tissue sarcomas. In this study, we identified and characterised side population (SP) cells that showed drug-resistant features in human bone sarcoma cell lines.Methods:
In seven osteosarcoma cell lines (OS2000, KIKU, NY, Huo9, HOS, U2OS and Saos2) and in one bone malignant fibrous histiocytoma (MFH) cell line (MFH2003), the frequency of SP cells was analysed. Tumourigenicity of SP cells was assessed in vitro and in vivo. Gene profiles of SP cells and other populations (main population; MP) of cells were characterised using cDNA microarrays.Results:
SP cells were found in NY (0.31%) and MFH2003 (5.28%). SP cells of MFH2003 formed spherical colonies and re-populated into SP and MP cells. In an NOD/SCID mice xenograft model, 1 × 103 sorted SP cell-induced tumourigenesis. cDNA microarray analysis showed that 23 genes were upregulated in SP cells.Conclusions:
We showed that SP cells existed in bone sarcoma cell lines. SP cells of MFH2003 had cancer-initiating ability in vitro and in vivo. The gene profiles of SP cells could serve as candidate markers for CSCs in bone sarcomas. 相似文献16.
Xingbing Wang Xin Chen Jun Liu Zimin Sun Shiang Huang 《中德临床肿瘤学杂志》2008,7(3):164-169
Objective To investigate the effects of soluble factors secreted by acute myeloid leukemia (AML) cells on the phenotypical and functional properties of DCs derived from normal mononuclear cells. Methods Mononuclear cells were cultured with interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF), in the presence or absence of 24 h culture supernatants from fresh primary AML cells, to generate immature DCs. The maturation of DCs was induced by cytokines IL-1beta, IL-6, tumor necrosis factor-alpha (TNF-alpha), and prostaglandin-2 (PGE-2). The phenotypic alterations of DCs and DCs-primed CD4 T cells were evaluated using flow cytometry. Precursor frequency (PF) was calculated to monitor the allostimulatory effects of DCs on CD4 and CD8 T cells. ResultsAML cell supernatant-treated DCs showed significantly lower expression of co-stimulatory molecules CD80 and CD86, and reduced response to cytokines IL-1beta, IL-6, TNF-alpha,and PGE-2. The allostimulatory effects of AML cell supernatant-treated DCs on CD4 and CD8 T cells were significantly lower than those of normal mature DCs [PF (1.8±0.5)% vs. (5.2±1.6)% for CD4 T cells, (2.1±0.6)% vs. (6.5±2.0)%for CD8 T cells, P<0.01]. These AML supematant-induced DCs could also induce allogeneic CD4 T cells to differentiate into CD4 CD25high T cells, which had immunophenotyping characteristics of regulatory T cells, i.e. they expressed Foxp3 but not active maker CD69. Conclusion This study demonstrates that soluble factors secreted by AML cells can inhibit development and functions of DCs. In addition, AML supematant-induced DCs can induce the generation of CD4 CD25high T cells from CD4 T cells, which may be a mechanism of increased prevalence of CD4 CD25high regulatory T cells and immune dysfunction in AML patients. 相似文献
17.
Carcinoma cells in PIN are situated above a layer of basal epithelial cells, which shield the tumor cells from stimulation by factors from the prostate stroma. During progression to invasive carcinoma, the basal cell layer becomes disrupted and tumor cells adhere to the basement membrane. The close proximity of basal epithelial cells to tumor cells in the early stages of prostate oncogenesis raises the possibility that basal epithelial cells participate in tumor cell invasion. Here, we investigated the migration-promoting activity of secreted factors from basal epithelial cells on BPH-1 cells, which we used as an in vitro model of preinvasive prostate cancer cells. We showed that the conditioned medium of basal epithelial cells (PEC-CM) contains adhesion proteins and chemotactic factors that stimulate adhesion, planar polarization, migration, and phosphorylation of Akt and that LY294002 and Wortmannin partially inhibit PEC-CM-triggered migration. We identified laminin-5 as a major migration-stimulating protein for BPH-1 cells in PEC-CM. Laminin-5 induced migration is completely inhibited by LY294002 or Wortmannin. In addition, antibody-depletion of laminin-5 from PEC-CM significantly diminishes the migration of BPH-1 cells. These results demonstrated, that laminin-5 is secreted by basal prostate epithelial cells in vivo and in vitro and stimulates migration of BPH-1 cells through a PI3-kinase dependent mechanism. Altogether, the possibility that basal epithelial cells assist in the invasion of in situ carcinoma cells is supported by the results from our in vitro system. 相似文献
18.
Scientists have tried for decades to understand cancer development in the context of therapeutic strategies. The realization that cancers may rely on "cancer stem cells" that share the self-renewal feature of normal stem cells has changed the perspective with regard to new approaches for treating the disease. In this review, we propose that one of the differences between normal stem cells and cancer stem cells is their degree of dependence on the stem cell niche, a specialized microenvironment in which stem cells reside. The stem cell niche in adult somatic tissues plays an essential role in maintaining stem cells or preventing tumorigenesis by providing primarily inhibitory signals for both proliferation and differentiation. However, the niche also provides transient signals for stem cell division to support ongoing tissue regeneration. The balance between proliferation-inhibiting and proliferation-promoting signals is the key to homeostatic regulation of stem cell maintenance versus tissue regeneration. Loss of the niche can lead to loss of stem cells, indicating the reliance of stem cells on niche signals. Therefore, cancer stem cells may arise from an intrinsic mutation, leading to self-sufficient cell proliferation, and/or may also involve deregulation or alteration of the niche by dominant proliferation-promoting signals. Furthermore, the molecular machinery used by normal stem cells for homing to or mobilizing from the niche may be "hijacked" by cancer stem cells for invasion and metastasis. We hope this examination of the interaction between stem cells and their niche will enhance understanding of the process of cancer development, invasiveness, and metastasis and reveal possible targets for cancer treatment. 相似文献
19.
Follicular lymphoma B cells induce the conversion of conventional CD4+ T cells to T-regulatory cells
Ai WZ Hou JZ Zeiser R Czerwinski D Negrin RS Levy R 《International journal of cancer. Journal international du cancer》2009,124(1):239-244
There has been accumulating evidence that CD4(+)CD25(+) FoxP3 expressing regulatory T cells (Treg) are highly concentrated in tumors, thereby fostering an immune-privileged microenvironment. Some studies have shown that T-cell receptor (TCR) stimulation can convert conventional T cells into Treg. Follicular lymphoma (FL) B cells can enhance this Treg conversion. We investigated whether FL tumor B cells, as opposed to normal B cells, are unique in their ability to convert effector T cells into Treg. We found that tumor B cells alone, without artificial TCR stimulation, could induce conventional T cells to express FoxP3 and to acquire regulatory function. In contrast to their malignant counterpart, normal B cells did not induce Treg conversion. Treg conversion was independent of the T cell background, as T cells isolated from FL or normal peripheral blood were equally susceptible to being converted by tumor B cells. Our study provides evidence for a tumor-specific mechanism by which FL tumor cells promote immune escape through the induction of Treg. 相似文献
20.
Mesothelial cells stimulate the anchorage-independent growth of human ovarian tumour cells 总被引:1,自引:0,他引:1
A P Wilson 《British journal of cancer》1989,59(6):876-882
Results are presented which show that mesothelial cells (MC) from ovarian cancer patients can both stimulate and inhibit the clonogenic growth of ovarian tumour cells (TC) in a dose-dependent fashion. TC lines from both non-ovarian and ovarian tumours were variable in their response to MC. Colony formation was rarely induced when the TC population was non-clonogenic and a bladder cell line showed inhibition of colony formation in the presence of MC. Primary tumour cultures from ovarian cancer patients also showed a variable response to MC. Fibroblasts from malignant, benign and non-neoplastic sources were significantly less effective in stimulating the clonogenic growth of responsive cell lines. Conditioned medium was a poor substitute for the presence of intact viable cells, and distance between feeder cell and TC was an important factor in determining the magnitude of response. A significant relationship between the feeder effect of MC and their proliferation in soft agar was observed when epidermal growth factor was used in the medium. The relevance of the findings in the context of the pattern of spread of ovarian cancer is discussed. 相似文献