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1.
取外科手术切除的新鲜胃腺癌组织标本22例,分离单个胃癌细胞,反复冻融后经0.22μm微孔滤膜过滤,获冻融液作为粗提可溶性抗原.用该抗原刺激患者自身外周血单个核细胞,与抗CD3单克隆抗体和重组白细胞介素2一起共同培养,体外诱导细胞毒T淋巴细胞(CTL),并用流式细胞仪测定其培养前后的表型变化.分别用自身胃癌细胞、人胃腺癌细胞株(BGC-823)和人胃粘液腺癌细胞株(MGC-803)作为靶细胞,用MTT比色法测定CTL的体外杀伤活性.结果表明,此种方法诱导的CTL对自体癌细胞杀伤活性为89.53%±3.1%,明显高于对胃癌细胞株的杀伤活性(P<0.01);对同一组织分型的胃腺癌细胞株BGC-823的杀伤活性为63.05%±5.6%,高于对MGC-803的杀伤活性40.09%±  相似文献   

2.
自体宫颈癌-树突细胞疫苗激活的CTL杀伤效应   总被引:17,自引:0,他引:17  
Zhou CJ  Ma W  Zhou JD  Zhao YX  Xie HQ 《癌症》2006,25(2):143-147
背景与目的:树突细胞(dendriticcells,DC)是目前已知的功能最强的抗原递呈细胞(antigen-presentingcell,APC),它可以在体内、外向T淋巴细胞递呈抗原,并诱发细胞毒T淋巴细胞(cytotoxicTlymphocyte,CTL)反应。本研究旨在探讨负载自体宫颈癌抗原的DC体外激发的CTL对自体宫颈癌细胞的杀伤效应。方法:先冻融宫颈癌细胞制备抗原,然后以GM-CSF、IL-4诱导自体外周血单个核细胞(peripheralbloodmononuclearcell,PBMC)获得DC并负载抗原,刺激自体T淋巴细胞制备宫颈癌抗原特异性CTL,观察CTL对宫颈癌细胞的杀伤活性。结果:负载自体宫颈癌抗原DC诱导的特异性CTL对自体宫颈癌细胞的体外杀伤率高达79.32%~89.27%,显著高于淋巴因子激活的杀伤细胞(lymphokine-activatedkillingcells,LAK)的杀伤率(t≥2.89,P<0.05);且对宫颈癌HeLa细胞株具有一定杀伤效应(40.35%~58.09%),但低于自体癌细胞组(t≥2.97,P<0.05);特异性CTL对HepG2、MCF7、A549、MGC803细胞无明显杀伤效应。结论:自体宫颈癌-树突细胞疫苗体外诱导的CTL具有高效而特异的抗自体宫颈癌细胞免疫活性,可望成为宫颈癌生物治疗的一个有力手段。  相似文献   

3.
目的 探讨原发性肝癌患者外周血树突状细胞 (DC)经自体肝癌细胞抗原致敏后诱导的体外抗肿瘤作用。方法 对肝癌患者外周血采用密度梯度离心法分离 ,获得DC前体细胞 ,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DC。制备自体肝癌细胞抗原 ,体外脉冲DC ,检测DC诱导自体T细胞增殖能力及细胞毒性T细胞 (CTL )在体外对自体肝癌细胞的杀伤活性 ,并检测肿瘤抗原致敏DC分泌的IL 12水平。结果 经自体肝癌细胞抗原致敏的DC能分泌IL 12和诱导较强的自体T细胞增殖 ,且能诱导特异性CTL ,该CTL对自体肝癌细胞具有很强的杀伤活性 ,杀伤率显著高于DC、未经肝癌细胞抗原致敏的DC激活的CTL及T淋巴细胞的杀伤率 ,而对CT 2 6细胞、BEL 740 2细胞无明显的杀伤作用。结论 肝癌患者外周血DC经自体肝癌细胞抗原致敏后能诱导高效而特异的抗肝癌免疫 ,其机制可能与增强T细胞应答和诱导机体产生肿瘤特异CTL从而发挥特异性的抗肿瘤作用有关。  相似文献   

4.
目的 研究人外周血单核细胞来源的树突状细胞(DC)转染含甲胎蛋白(AFP,137-145)片段的重组腺相关病毒后所诱导的特异性T细胞对肝癌细胞株HepG2和SMMC-7721的体外杀伤作用.方法 抽取健康志愿者外周血,分离单核细胞,体外培养,使用含AFP片断的重组腺相关病毒转染未成熟DC,诱导特异性T细胞.检测体外培养的DC和细胞毒性T细胞(CTL)活性,应用四甲基偶氮唑蓝(MTT)法检测CTL对HepG2和SMMC-7721细胞的杀伤作用.结果 转染或未转染的体外培养的成熟DC高表达CD40、CD86和IL12,成熟DC诱导的CTL高表达IFNγ;修饰成熟后的DC后体外能诱导特异性CTL,该CTL在休外对肝癌细胞株HepG2和SMMC-7721均有杀伤作用.结论 重组腺相关病毒转染DC,不明显改变DC表型和刺激淋巴细胞增殖和分化功能,可诱导自体CTL增殖,含AFP(137~145)片断的腺相关病毒转染DC诱导自体CTL对肝癌细胞株HepG2和SMMC-7721细胞有明显杀伤作用,DC疫苗可以作为肝癌患者免疫治疗的有效补充.  相似文献   

5.
目的 探讨原发性肝癌患者外周血树突状细胞(DC)体外经自体肝癌细胞抗原致敏后诱导的抗肿瘤作用。方法肝癌患者外周血经梯度密度离心法分离,获得DC前体细胞,用重组人粒细胞-巨噬细胞集落刺激因子(rhGM—CSP)和重组人白细胞介素-4(rhIL-4)联合培养,诱导扩增DC。制备自体肝癌细胞抗原,体外脉冲DC,检测DC诱导自体T细胞增殖能力及细胞毒性T细胞(CTL)在体外对自体肝癌细胞的杀伤活性,并检测肿瘤抗原致敏DC分泌的IL-12水平。结果经自体肝癌细胞抗原致敏的DC能分泌IL-12和诱导较强的自体T细胞增殖,且能诱导特异性CTL,该CTL对自体肝癌细胞具有很强的杀伤活性,杀伤率明显高于DC、未经肝癌细胞抗原致敏的DC激活的CTL及T淋巴细胞的杀伤率,而对3LLLEWIS肺癌细胞、H22肝癌细胞则无明显的.杀伤作用。结论肝癌患者外周血DC经自体肝癌细胞抗原致敏后能诱导高效而特异的抗肝癌免疫,其机制可能与增强T细胞应答和诱导机体产生肿瘤特异CTL而发挥特异性的抗肿瘤作用有关。  相似文献   

6.
目的:探讨HLA限制性EB病毒(Epstein-Barr virus,EBV)表位肽EBV-潜伏膜蛋白2A (EBV-latent membrane protein 2A,EBV-LMP2A)诱导的细胞毒性T淋巴细胞(CTL)对EBV阳性胃癌细胞的体外杀伤作用.方法:选用南京大学医学院附属肿瘤医院肿瘤中心HLA-A2阳性胃癌患者外周血单个核细胞(PBMC)、人胃腺癌细胞株(AGS)和人EBV阳性胃腺癌细胞株(AGS-EBV),通过改良的体外细胞培养技术用HLA-A2限制性的EBV-LMP2A抗原肽从人PBMC中诱导扩增出特异性CTL,采用四聚体技术和流式细胞术检测抗原肽诱导产生的特异性CTL的含量,通过FITC-PI标记流式术检测EBV-CTL对EBV阳性和EBV阴性的胃癌细胞的体外杀伤作用.结果:经改良的细胞培养技术和抗原肽双重刺激后EBV-LMP2A抗原肽可以诱导产生出高比例的抗原特异性CTL[EBV-LMP2A-356特异性T细胞占CD8+T细胞的(47.1±5.2)%];EBV-CTL对EBV阳性胃癌细胞的体外杀伤作用较EBV阴性的胃癌细胞明显增强[(45.1±9.3)%s(19.4±2.5)%,P<0.05].结论:通过改良的细胞培养技术使用EBV-LMP2A抗原肽能诱导产生高比例的EBV特异性CTL,其对EBV阳性胃癌细胞有较强的特异性杀伤作用.  相似文献   

7.
目的:比较热休克凋亡和冻融裂解人胃癌细胞株抗原负载树突状细胞(dendritic cell,DC)体外刺激细胞毒T细胞(cytotoxic Tlymphocyte,CTL)的活性。方法:分别制备热休克诱导凋亡胃癌细胞株和肿瘤细胞冻融裂解物,同时分离12例胃癌患者外周血单核细胞体外诱导DC进行负载,并以两种不同方式负载的DC进行淋巴细胞增殖反应及CTL杀伤实验,比较其刺激淋巴细胞增殖及激活的淋巴细胞杀伤靶细胞能力的差异。结果:负载热休克胃癌细胞的DC与负载反复冻融肿瘤细胞裂解物的DC都显示对靶细胞CTL活性,但前者的CTL活性显著高于后者(P<0.05)。结论:用热休克肿瘤细胞负载DC是一个更为有效的负载方式,为临床应用提供了一种可选择的负载方式。  相似文献   

8.
程琪  朱长焜  叶枫  陈怀增  谢幸 《实用癌症杂志》2005,20(2):113-115,119
目的探讨卵巢癌冻融抗原对T淋巴细胞的增殖作用及其诱导的细胞毒性T淋巴细胞(CTL)在体外杀伤卵巢癌细胞的抗原特异性细胞毒性效应。方法采用免疫磁珠分离法(magnetic activated cell sorting,MACS)分离纯化正常人外周血CD3^ 细胞,体外以SKOV3卵巢癌细胞中提取的可溶性肿瘤抗原加以刺激。溴标法检测肿瘤抗原对T细胞增殖的影响;MTT法测定肿瘤抗原诱导的CTL体外杀伤卵巢癌细胞的能力;利用绒毛膜癌细胞抗原对比观察肿瘤抗原诱导CTL杀伤肿瘤细胞的抗原特异性。结果卵巢癌细胞抗原有很强的促进T淋巴细胞增殖的作用,且存在时间依赖性,在24h时促进作用最强。SKOV3抗原诱导的CTL在体外对SKOV3细胞的杀伤率为68.38%,显著高于它对JAR绒毛膜癌细胞的杀伤率(18.31%);而JAR细胞抗原诱导的CTL对JAR和SKOV3细胞的杀伤率分别为61.02%和14.79%,有显著的抗原特异性(P=0.0001)。结论卵巢癌细胞冻融抗原诱导的CTL在体外具有很强的增殖能力和抗原特异性杀伤卵巢癌细胞的作用。  相似文献   

9.
 目的探讨负载自体抗原DC诱导的CTLs对自体乳腺癌细胞的杀伤作用。方法以负载自体癌细胞抗原的DCs体外诱导CTLs,用ELISA法检测IFN-γ和IL-12的表达水平,用LDH法检测CTL对自体癌细胞的杀伤作用。结果负载自体抗原DC组IFN-γ和IL-12的浓度高于未致敏DC组、抗原组及单核细胞对照组(P<0.05),且负载抗原DC组所刺激的CTLs的杀伤作用也强于未致敏DC组、抗原组及单核细胞组(P<0.01)及对照的MCF-7组和HT-29组(P<0.01)。结论肿瘤裂解物致敏DC可持续刺激特异性CTLs从而可在体外杀伤乳腺癌患者的自体肿瘤细胞,说明肿瘤抗原致敏的DC疫苗对于治疗残留的和(或)对化疗耐药乳腺癌可能是适合的,因而有可能成为标准的补救措施。  相似文献   

10.
目的:研究重组人热休克蛋白70(rhHSP70)联合肝癌组织冻融抗原修饰的树突状细胞(dendritic cell,DC)诱导对肝癌细胞的免疫杀伤效应.方法:外周血单个核细胞经粒-巨噬细胞集落刺激因子(GM-CSF)、白细胞介素4 (IL-4)诱导生成DC,负载冻融抗原的同时加入rhHSP70,不同分组致敏的DC激活淋巴细胞生成肿瘤抗原特异性细胞毒性T淋巴细胞(cytotoxic T cells,CTL),四甲基偶氮唑蓝(MTT)法及3H-TdR法检测DC刺激淋巴细胞增殖能力, MTT法检测CTL对肝癌细胞的体外杀伤活性,酶联免疫吸附试验(ELISA)测定细胞因子的分泌,流式细胞术(FCM)检测DC表型变化.结果:冻融抗原致敏的DC可明显促进淋巴细胞增殖,能有效呈递肝癌冻融抗原,诱导产生抗原特异性CTL,联合rhHSP70能进一步增强CTL对肝癌细胞的杀伤作用.结论:肝癌冻融抗原联合rhHSP70修饰的DC诱导CTL对肝癌细胞能产生高效杀伤作用.  相似文献   

11.
Cytotoxic T lymphocytes (CTL) against autologous malignant brain tumor were generated in peripheral blood lymphoid cells (PBL) prepared from a patient with a malignant brain tumor by stimulation of the cultured PBL for 7 days with attenuated Crossreactive malignant melanoma (MM2) cells pretreated with mitomycin C. The Crossreactive MM2 cells were effective for antigen stimulation for CTL induction in place of autologous glioblastoma cells, which are difficult to expand in culture. The optimal ratio between nylon wool-passed T lymphocytes and nylon wool-adherent accessory cells to induce CTL in the patient's PBL was found to be 25 to 1. In vitro -activated CTLs induced by MM2 were cytotoxic not only to MM2, but also to the autologous tumor cells in an HLA class I-restricted manner, and their surface phenotype was found to be CD3+ and CD8+. CTL therapy using cross-reactive allogeneic tumor cells as the stimulator could be clinically valuable to treat malignant brain tumors.  相似文献   

12.
目的 探讨转化生长因子-β1(TGF-β1)/丝裂原活化的蛋白激酶(MAPK)通路在调节胃癌细胞MMP-2和MMP-9表达中的作用。方法 以MKN45和BGC823胃癌细胞系为研究对象。明胶酶谱法检测TGF-β1对各细胞MMP-2和MMP-9表达的影响,Western蛋白印迹检测在TGF-β1作用下各细胞中MAPK(P38、JNK、ERK)的活化情况。用相应MAPK通路的特异抑制剂对TGF-β1调节各细胞MMP-2和MMP-9的表达进行阻断研究。结果 TGF-β1可上调MKN45和BGC823细胞MMP-2和MMP-9的表达;TGF-β1可诱导BGC823细胞P38、MKN45和BGC823细胞ERK及JNK的活化;ERK特异抑制剂PD98059可明显抑制TGF-β1诱导MKN45和BGC823细胞MMP-2和MMP-9的表达,但P38特异抑制剂SB203580和JNK特异抑制剂SP600125对TGF-β1诱导这两种细胞MMP-2和MMP-9的表达无明显影响。结论 TGF-β1可通过活化ERK信号通路上调MKN45和BGC823胃癌细胞 MMP-2和MMP-9的表达。  相似文献   

13.
TIL were cultured from human renal-cell carcinoma (RCC) in 1,000 U/ml rIL2 and restimulated with autologous tumor in efforts to establish which conditions would best expand the number of lymphocytes cytotoxic for autologous tumor. Greater cell yields resulted from multiple restimulations of TIL with autologous tumor. In most instances, these TIL lysed autologous tumor better than TIL grown in rIL2 alone. Enhanced proliferation was seen also after restimulation of TIL with allogeneic RCC as well as with tumor cells of non-renal origin. Although in some instances lysis of autologous tumor appeared to be specific, restimulation with autologous tumor did not consistently result in the generation of specific cytolytic T cells. Attempts to culture more specific cytolytic T cells by using 50 U/ml rIL2 were successful in expanding TIL with enhanced lytic activity; however, this activity was not specific for autologous tumor. The phenotype of the tumor-restimulated TIL generally did not change. In most of the TIL cultures, CD3+CD4+ cells were predominant with low numbers of CD3+Leu19+ cells and minimal numbers of CD3-Leu19+ cells. Thus, the cytotoxic response to tumor was mediated by T cells and not NK cells. Overall, our data indicate that restimulation of TIL with autologous tumor may be beneficial for growing larger numbers of cells which have increased lytic activity and for prolonging the presence of lytic activity among the expanded TIL.  相似文献   

14.
T Ebihara  S Koyama 《Gan no rinsho》1990,36(5):604-610
The functional and phenotypic characteristics of carcinomatous pleural or peritoneal lymphoid cells cultivated with either rIL 2 or TCGF have been investigated. The cultivation of the lymphoid cells with cytokines was initiated by a mixture of coexisting, viable carcinoma cells for 14 days. Results have indicated that cytokine-activated lymphoid cells from malignant pleural and peritoneal effusions showed considerable cytolytic activity against K562 and Daudi cells. The cell population responsible for LAK and/or CTL effector cells of TCGF-activated lymphoid cells were CD8+ CD11- cells. Further, in rIL 2-expanded cultures from pleural and peritoneal lymphoid cells, the CD4+ Leu 8- population was found to contain effector cells of cytotoxic activity against the tumor cells. It further was seen that the TCGF-activated CD8+ CD11- T cells possessed a more potent killing activity, in comparison to the rIL 2-activated CD4+ Leu8- T cells. However, rIL 2-activated lymphoid cells from ascites in liver cirrhosis (used for a control) showed a higher tumoricidal activity.  相似文献   

15.
目的:观察不同浓度菝葜皂苷元对体外培养胃癌BGC-823细胞增殖、凋亡的影响.方法:BGC-823细胞经不同浓度菝葜皂苷元处理后,采用MTT法检测细胞的生长活性,流式细胞仪检测细胞凋亡.结果:随菝葜皂苷元浓度的增加,胃癌BGC-823细胞增殖抑制率、凋亡率亦升高.结论:菝葜皂苷元对胃癌BGC-823细胞的增殖有抑制作用,并可诱导细胞凋亡.  相似文献   

16.
As part of a phase-II clinical trial of post-operative active specific immunization (ASI) with virus-modified autologous tumour cells (AuTu) in colorectal carcinoma patients, we have analyzed in vitro anti-AuTu immune responses with lymphocytes isolated from the peripheral blood (PBL) of 5 treated patients. The PBL of 3 "responder patients", those who developed a positive DTH reaction to AuTu, when stimulated in standard in vitro autologous lymphocyte tumour-cell cultures (ALTC), showed cytotoxic anti-AuTu reactivity only in association with natural-killer-cell(NK)-like activity. We removed nonspecific cytotoxic cells (CD56-positive) from PBL of colon carcinoma or melanoma patients and positively selected T cells with strong CD8 staining (CD8hi) using FACS. Following in vitro stimulation, specific cytotoxic T cells (CTL) directed against either autologous EBV-transformed B cells (AuEBV-B) or autologous melanoma cells were identified in the CD8hi T-cell population. However, even using this novel technique, no specific CTL against autologous colon carcinoma cell lines were detected in PBL from ASI-treated patients (2 DTH responders and 2 DTH non-responders). If AuTu-specific CTL precursors existed in these blood samples, their frequency must have been very low (less than 1 in 8 x 10(4) CD8 positive T cells). Sorted CD4 T cells from these patients, in the presence of autologous antigen-presenting cells, showed no specific anti-tumour proliferative response, and in one instance we observed inhibition of proliferation in the presence of tumour cells.  相似文献   

17.
 目的 研究血管性血友病因子(von Willebrand factor,vWF)对胃癌细胞BGC 823增殖和黏附的影响,并初步探讨相关机制。 方法 体外培养人胃癌细胞株BGC-823,以vWF抗体(vWF Ab) 处理;噻唑蓝比色(MTT)法检测细胞增殖活性和细胞黏附能力 的变化;用倒置显微镜观察细胞形态学变化。 结果 vWF Ab能够抑制 BGC-823细胞的增殖,抑制作用呈剂量及时间依赖性;细胞形态学观察vWF Ab作用对细胞生长具有明显的细 胞毒作用;MTT黏附实验结果显示vWF Ab处理后细胞的黏附率较对照组明显下降(P<0.05),Ⅳ型胶原高浓度组较低 浓度组细胞黏附率增大(P<0.01)。 结论 vWF在体外能够影响胃癌细胞BGC-823的增殖,并诱导细胞形态改变,促进细胞与细胞外基质的黏附。  相似文献   

18.
目的:探讨环氧化酶2(COX-2)抑制剂塞来昔布对人胃癌BGC823细胞株的放射增敏作用及其机制。方法:MTT实验测定塞来昔布对胃癌BGC823细胞株的抑制率,计算半数抑制浓度(IC50)。克隆形成实验检测塞来昔布联合不同剂量X线照射后细胞的存活率,计算放射敏感性相关参数,评价增敏效果。流式细胞仪(FCM)分析细胞周期分布情况。结果:MTT实验显示塞来昔布对BGC823细胞作用24h、48h、72h的IC50分别是162.6μmol/L、95.4μmol/L、62.1μmol/L;克隆形成实验测得联合组的D0、Dq及SF2均明显低于单纯照射组(1.705VS 2.185,1.032VS 2.327,0.495 VS 0.745),SER为1.3。流式细胞仪检测联合组G0/G1期细胞增多,S期细胞减少。结论:塞来昔布能增强胃癌BGC823细胞的放射敏感性,其机制可能与减少肿瘤细胞亚致死性损伤修复和细胞周期再分布有关。  相似文献   

19.
目的 观察西妥昔单抗(C225)联合放疗对人胃癌细胞株MGC803及BGC823增殖的影响,并探讨其机制。方法 (1)采用MTT法观察C225联合放疗对细胞生长增殖的影响,评价两者联合效应;(2)采用FCM检测C225联合放疗对细胞周期分布及细胞凋亡的影响;(3)通过Western blot法检测C225联合放疗对EGFR细胞信号蛋白及其下游信号蛋白AKT、MAPK磷酸化的影响。结果 (1)当C225联合三种不同剂量放疗时,均可显著抑制细胞的增殖,两者具有协同作用。(2)单用C225可使细胞阻滞于G0/G1期,并减少S期细胞比例。在C225作用后,G0/G1期及S期MGC803细胞比例分别为67.3%及24.8%,BGC823细胞则分别为71.9%及18.8%。单用放疗可使细胞阻滞于G2/M期。在放疗作用于MGC803细胞及BGC823细胞后,G2/M期比例分别为18.2%及19.5%。当C225与放疗联合作用时,G2/M期MGC803细胞及BGC823细胞比例进一步升高,分别为25.7%及26.4%,而S期细胞比例进一步下降,分别为18.6%及13.5%。(3)在C225处理后MGC803细胞及BGC823细胞的凋亡率分别为4.9%及9.9%,放疗处理后MGC803细胞及BGC823细胞的凋亡率分别为11.7%及19.8%,与对照组相比差异均具有统计学意义。当C225与放疗联合作用时,MGC803细胞及BGC823细胞的凋亡率分别为20.1%及47.3%,与单用放疗组相比差异均具有统计学意义。(4)C225与放疗联合应用时,可使细胞EGFR信号蛋白表达下降,并能使其下游信号蛋白AKT、MAPK磷酸化活性降低。结论 (1)西妥昔单抗可增强放疗对MGC803和BGC823细胞株的生长抑制作用;(2)其增敏作用的机制可能与抑制EGFR信号转导通路、诱导细胞周期阻滞和细胞凋亡增加等有关。  相似文献   

20.
Tumor-infiltrating lymphocytes (TIL) isolated from 11 gastric carcinoma were studied. TIL could grow for a long-term in medium containing recombinant interleukin-2(rIL-2). The mean expansion fold achieved in 6 long-term cultures of 11 specimens was 15.1 RIL-2 expanded gastric TIL exhibited significant cytotoxicity against K562, BGC823, MCF-7 and more effective antitumor cytotoxicity against fresh autologous tumor targets and human gastric cancer cell line. Peak cytotoxicity was shown in the third or fourth week after cultures. Cryopreservation of gastric TIL didn’t influence their expansion capacity and antitumor activity. Phenotypic analysis was demonstrated in this study. The results of present study indicate that TIL from human gastric carcinoma could be expanded and reach high levels of antitumor effector function in long-term cultures with rIL-2. Their function may be of clinical importance.  相似文献   

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