抗原负载的DC与CIK共培养后对MGC-803胃癌细胞株杀伤活性的研究
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摘要
目的:胃癌是对人类健康与生命危害最大的恶性肿瘤之一,目前胃癌临床治疗手段主要是根治性切除手术辅以术前、术后化疗及放射治疗。由于临床所见病例多为中晚期患者,手术及放、化疗效果差,长期生存率较低,因此,如何提高胃癌根治术后效果,改善胃癌晚期患者生活质量,预防胃癌复发及转移一直是临床长期探索的问题。
肿瘤过继性细胞免疫治疗(adoptive cellular immunotherapy,ACI)是继手术、化疗和放疗之后的第四种肿瘤治疗模式。迄今,NK细胞、CTL、MΦ、TIL、LAK、CIK和DC细胞的过继治疗已在临床上得到应用,其中CIK和DC细胞为基础的抗肿瘤免疫治疗以其独特的优势,近年来成为国内外众多学者的研究热点。
细胞因子诱导的杀伤细胞(cytokine-induced killer cells,CIK)是由多种细胞因子如IL-2、IFN-γ和CD3单克隆抗体等诱导而成的对多种肿瘤具有杀伤活性的细胞毒性T细胞,其溶瘤活性具有非MHC限制性,是一种非特异性的免疫杀伤细胞。CIK细胞能分泌多种细胞因子(如IL-4、IFN-γ等),且具有比LAK、CD3AK细胞更强的杀伤活性。树突状细胞(dendritic cells,DC)是功能强大的主要的抗原递呈细胞,分布于除脑和睾丸以外身体各部的任何组织,DC借助膜表面不同受体可有效捕获低浓度抗原,并能与这些抗原表面的MHC I类和II类分子结合,刺激初始型CD8+T细胞和CD4+T细胞活化。综合国内外研究表明,将负载肿瘤抗原的DC细胞和具有高效杀伤活性的CIK细胞联合应用治疗恶性肿瘤,将有望起到协同作用。本研究将利用MTT法和流式细胞仪分别完成负载抗原的DC和CIK的联合培养物对MGC-803的杀伤活性和凋亡机制的初步研究,为DC和CIK联合治疗胃癌应用于临床打下科研基础。
材料与方法:
1 PBMC来源的CIK和DC细胞制备:提取人外周血单个核细胞(PBMC) ,在体外以基因重组人白介素-2(interleukine-2,IL-2)、鼠抗人CD3单抗(anti-CD3McAb)、基因重组人白介素-1α(interleukin-1α,IL-1α)、基因重组人干扰素-γ(interferon-γ,INF-γ)等细胞因子刺激非贴壁细胞获得成熟CIK;以基因重组人粒细胞/巨噬细胞集落刺激因子(granulocyte-macrophage CSF,GM-CSF)和基因重组人白介素-4(interleukine-4,IL-4)刺激贴壁细胞获得DC,并以流式细胞仪检测细胞表型。
2 MGC-803胃癌细胞可溶性抗原制备及定量:以液氮反复冻融MGC-803细胞,过滤,制成用以刺激DC细胞的可溶性抗原,以考马斯亮蓝法对蛋白抗原进行定量测定。
3不同效靶比和培养天数下,不同效应细胞组杀伤功能的比较:将效应细胞分作3组:单纯CIK组、未负载MGC-803抗原的DC+CIK组和负载MGC-803抗原的DC+CIK组,分别记做:CIK、DC-CIK和Ag-DC-CIK。效应细胞培养12d后,以MTT法测定CIK、DC-CIK和Ag-DC-CIK对MGC-803杀伤作用,效靶比分别为:2.5:1、5:1、10:1、20:1,并测定不同时间对其作用的影响。
4酶联免疫吸附法检测外分泌细胞因子水平:提取培养6d、12d和21d的Ag-DC-CIK细胞上清液,检测细胞外分泌细胞因子IL-2、TNF-α、INF-γ含量随培养天数的变化。
5 Ag-DC-CIK对MGC-803细胞增殖及凋亡的作用:将培养18d的效应细胞和靶细胞混合培养(E:T=20:1)24h后,收集细胞,加入Hoechst 33342至终浓度为1ug/ml,37℃孵育10min,离心,弃染液。加入PI染液重悬避光染色。流式细胞仪分析蓝色荧光对红色荧光的散点图或地形图。正常细胞为低蓝光/低红光,凋亡细胞为高蓝光/低红光,坏死细胞为低蓝光/高红光。
结果:
1单独培养的CIK细胞在第3d开始增殖,第5-6d进入快速增殖期,CIK细胞和DC(包括负载和未负载MGC-803抗原)共培养后的第12d开始,共培养细胞组的增殖速度开始明显增快,并快于同源CIK细胞(p<0. 05)。
2健康成年PBMC在体外可以分别由IL-2、CD3McAb、IL-1α、INF-γ和GM-CSF、IL-4诱导出经表型鉴定为成熟的CIK和DC细胞。其中有(79.64±3.41)%的细胞表达成熟DC特异性表面标志CD83,(95.17±1.22)%的细胞表达HLA-DR,(94.66±2.42)%的细胞表达CD86,(86.59±1.09)%的细胞表达CD40。与DC共培养的CIK细胞在培养第14d时CD3+CD8+、CD3+CD56+双阳性细胞的百分含量进一步升高到59.39%-60.46%和35.67%-36.90%,经抗原刺激过的DC与CIK共培养14d后,混合细胞中CD3+CD8+、CD3+CD56+细胞最多,达到63.63%-65.42%和51.03%-53.15%,与CIK组和DC-CIK组相比均具有显著性差异(p<0.05)。
3在2.5:1-20:1效靶比范围内,DC-CIK对MGC-803靶细胞的杀伤活性一般均高于同源CIK细胞,经MGC-803细胞冻融物致敏的Ag-DC-CIK共培养细胞对MGC-803靶细胞的杀伤活性有进一步的提高,与单纯CIK细胞组比较差异显著(p<0.05),与DC-CIK共培养细胞组比较也有显著性差异(p<0.05)。
4 IFN-γ在21d的混合细胞上清液中表达较高,TNF-α在各个时期均能检测到,但表达水平变化很明显,在第6d时低表达,21d时较高。促炎因子IL-2变化正好与TNF-α相反,主要在第6d高表达,21d时表达相对较低或不表达。
5 24h组凋亡早期、中期、晚期细胞及正常细胞在FCM散点图中占绝大多数比例,而48h组以坏死细胞为主,凋亡细胞已较24h明显减少。
结论:
1负载MGC-803胃癌细胞抗原的DC可使CIK的增殖速度进一步提高。
2负载MGC-803抗原的DC与CIK共培养后可使具备攻击能力的主要细胞群CD3+CD8+、CD3+CD56+双阳性细胞群比例分别进一步提高。
3经抗原冲击的DC与CIK共培养后能增强其对胃癌细胞MGC-803的杀伤活性。
4 Ag-DC-CIK在体外培养过程中不仅需要持续来源于外界细胞因子的存在,而且该效应细胞组还能够向细胞外分泌IFN-γ、TNF-α、IL-2影响其自身的功能。
5在效靶相互作用初期,Ag-DC-CIK对MGC-803的杀伤作用主要通过诱导肿瘤细胞凋亡,同时发生自身凋亡来实现;在相互作用的中晚期,细胞开始趋向坏死。
Objective: Gastric cancer is one of the most harmful malignant tumors to human health and life and the clinical therapeutic tools of gastric cancer is mainly radical excision assistant with chemo and radiation therapy both before and after operation. Most clinical cases are found to be middle or advanced stage, the curative effects of operation, chemo and radiation are not satisfactory, and thus the long term survival is relatively low. So improving the curing effects and quality of life for patients of gastric cancer and preventing the relapse and metastasis is the issue which clinicians have been exploring. The adoptive cellular immunotherapy (ACI) is the fourth oncotherapy mode after operation, chemo and radiation. Until now the adoptive therapy of NK, CTL, MΦ, TIL, LAK, CIK and DC cells has been well applied clinically, in which the antitumor immunotherapy based on CIK and DC has been warm spots for researchers both in home and abroad recently. The cytokine-induced killer cell(CIK) is a kind of cytotoxic T cell which can be induced by various cytokines such as IL-2, IFN-γand monoclonal antibody CD3. The tumor-lytic activity is non-MHC restricted for which the CIK cells can be termed as non-specific immuno-killer. Moreover, it can excrete kinds of cytokines(IL-4, IFN-γ,et al) and has a killing activity to tumor cells much stronger than LAK and CD3AK. Dendritic cell (DC) is regarded as a powerful antigen-presenting cells, which can exist in all parts of human body except in the brain and didymus. DC can capture low-density antigen effectively by receptors on its cell membrane, combine with MHC I and MHC II molecules on the antigen surfaces and stimulate the activation of na?ve CD8+ and CD4+ T cells. According to the researches in home an abroad, combination of DC pulsed with antigen and the effective killing cells of CIK to treat malignant tumors is a hopeful way of synergistic effect. My initial reseach use the MTT and FCM to detect the toxic activity to MGC-803 and apoptosis mechanisms of co-cultivation of DC pulsed with antigen cocultured with CIK, and do some foundation research for clinical use of CIK and DC in treatment of gastric cancer.
Methods:
1 Preparations of CIK and DC derived from PBMC: Extract PBMC, stimulate the non-adherent cell with interleukine-2, anti-CD3McAb, IL-1αand INF-γto obtain CIK; stimulate the adherent cells with granulocyte-macrophage CSF and IL-4 to obtain DC; test the phenotype of cells with FCM.
2 Preparation and quantitation of MGC-803 soluble antigen: freeze thawing MGC-803 repeatedly with liquid nitrogen, filter and make into soluble antigen to stimulate DC; quantitative assay of antigen with Coomassie brilliant blue.
3 Comparison of killing function between three effective cell groups, with different E:T, in different days: The effector cells are divided into three groups: pure CIK group, non MGC-803 antigen pulsed DC with CIK group and MGC-803 antigen pulsed DC with CIK group. They are labeled as CIK, DC-CIK and Ag-DC-CIK. In Day 12 of co-culture, test the killing function of CIK, DC-CIK and Ag-DC-CIK groups with MTT, under the ratio of effective cells : target cells, 2.5:1, 5:1, 10:1 and 20:1; test the impact of different days.
4 Test the level of excreting cytokines with enzyme linked immunosorbent assay(ELISA): extract the clear supernatant liquid of Ag-DC-CIK in Day 12 and 21, test the contents of IL-2, TNF-αand INF-γ.
5 The proliferation and apoptosis of Ag-DC-CIK to MGC-803: co-cultivate the effector cells with the target cells (E:T=20:1)in Day 18 for 24h, collect cells, add Hoechst 33342 to the final concentration of 1ug/ml, incubate under the temperature of 37℃for 10min, centrifuge and draw off the staining solution; add PI, suspend, and incubate away from light; analyze the scatterplot or tonographic of blue fluorescence over the red; low blue/low red for the normal cells, high blue/low red for the apoptosis cell, low blue/high red for the necrosis cells.
Results:
1 CIK begins to multiply from Day3 and enter the multiplication period in Day5-6. DC-CIK and Ag-DC-CIK begin to proliferate fast from Day12, with the speed faster than CIK (p<0.05).
2 The PBMC from healthy adults can derive into mature CIK and DC with the existing of IL-2, CD3McAb, IL-1α, INF-γ, GM-CSF and IL-4. (79.64±3.41)% of the cells present DC specific surface marker CD83,(95.17±1.22)% of HLA-DR, (94.66±2.42)% of CD86 and (86.59±1.09)% of CD40. DC-CIK in Day14 has a higher percentage of CD3+CD8+ and CD3+CD56+ cells of 59.39%-60.46% and 35.67%-36.90%. Ag-DC-CIK in Day14 has the highest percentage of the two cell populations of 63.63%-65.42% and 51.03%-53.15% and has significant difference (p<0.05) compared with CIK and DC-CIK groups.
3 The killing activity of DC-CIK is generally stronger than CIK in the E:T range of 2.5:1-20:1. When DC is pulsed with MGC-803 antigen and co-cultured with CIK, the lytic activity of effector cells is strengthened and has significant difference (p<0.05) compared with CIK and DC-CIK.
4 IFN-γhas a high concentration in the clear supernatant of co-culturing cells in Day21. TNF-αcan be detected in all periods with an obvious fluctuation, low in Day6 while high in Day21. On the contrary, the proinflammatory factor IL-2 has a high expression in Day6 and low or no expression in Day21.
5 The 24h group shows a dominant percentage of early, middle and late period of apoptosis cells and normal cells in the FCM scatterplot. And 48h group is mainly composed of necrosis cells, with a obviously descending percentage of apoptosis cells than 24h group.
Conclusions:
1 DC pulsed with MGC-803 antigen can further improve the CIK proliferation rate.
2 DC pulsed with MGC-803 antigen can elevate the percentage of CIK attacking cell populations: the CD3+CD8+ and CD3+CD56+ cells.
3 Antigen-pulsed DC or non-antigen-pulsed DC can both strengthen the MGC-803 lytic activity of CIK.
4 Ag-DC-CIK need cytokines to maintain culturing in vitro, and also excrete IFN-γ, TNF-αand IL-2 which influence the function of the cells.
5 During the earlier period of the interaction of effectors and targets, MGC-803-DC-CIK kills MGC-803 mainly through inducing apoptosis of tumor cells and at the same time they themselves enter the process of apoptosis; in the late period of interactions, both of the targets and effectors tend to die of necrosis.
肿瘤过继性细胞免疫治疗(adoptive cellular immunotherapy,ACI)是继手术、化疗和放疗之后的第四种肿瘤治疗模式。迄今,NK细胞、CTL、MΦ、TIL、LAK、CIK和DC细胞的过继治疗已在临床上得到应用,其中CIK和DC细胞为基础的抗肿瘤免疫治疗以其独特的优势,近年来成为国内外众多学者的研究热点。
细胞因子诱导的杀伤细胞(cytokine-induced killer cells,CIK)是由多种细胞因子如IL-2、IFN-γ和CD3单克隆抗体等诱导而成的对多种肿瘤具有杀伤活性的细胞毒性T细胞,其溶瘤活性具有非MHC限制性,是一种非特异性的免疫杀伤细胞。CIK细胞能分泌多种细胞因子(如IL-4、IFN-γ等),且具有比LAK、CD3AK细胞更强的杀伤活性。树突状细胞(dendritic cells,DC)是功能强大的主要的抗原递呈细胞,分布于除脑和睾丸以外身体各部的任何组织,DC借助膜表面不同受体可有效捕获低浓度抗原,并能与这些抗原表面的MHC I类和II类分子结合,刺激初始型CD8+T细胞和CD4+T细胞活化。综合国内外研究表明,将负载肿瘤抗原的DC细胞和具有高效杀伤活性的CIK细胞联合应用治疗恶性肿瘤,将有望起到协同作用。本研究将利用MTT法和流式细胞仪分别完成负载抗原的DC和CIK的联合培养物对MGC-803的杀伤活性和凋亡机制的初步研究,为DC和CIK联合治疗胃癌应用于临床打下科研基础。
材料与方法:
1 PBMC来源的CIK和DC细胞制备:提取人外周血单个核细胞(PBMC) ,在体外以基因重组人白介素-2(interleukine-2,IL-2)、鼠抗人CD3单抗(anti-CD3McAb)、基因重组人白介素-1α(interleukin-1α,IL-1α)、基因重组人干扰素-γ(interferon-γ,INF-γ)等细胞因子刺激非贴壁细胞获得成熟CIK;以基因重组人粒细胞/巨噬细胞集落刺激因子(granulocyte-macrophage CSF,GM-CSF)和基因重组人白介素-4(interleukine-4,IL-4)刺激贴壁细胞获得DC,并以流式细胞仪检测细胞表型。
2 MGC-803胃癌细胞可溶性抗原制备及定量:以液氮反复冻融MGC-803细胞,过滤,制成用以刺激DC细胞的可溶性抗原,以考马斯亮蓝法对蛋白抗原进行定量测定。
3不同效靶比和培养天数下,不同效应细胞组杀伤功能的比较:将效应细胞分作3组:单纯CIK组、未负载MGC-803抗原的DC+CIK组和负载MGC-803抗原的DC+CIK组,分别记做:CIK、DC-CIK和Ag-DC-CIK。效应细胞培养12d后,以MTT法测定CIK、DC-CIK和Ag-DC-CIK对MGC-803杀伤作用,效靶比分别为:2.5:1、5:1、10:1、20:1,并测定不同时间对其作用的影响。
4酶联免疫吸附法检测外分泌细胞因子水平:提取培养6d、12d和21d的Ag-DC-CIK细胞上清液,检测细胞外分泌细胞因子IL-2、TNF-α、INF-γ含量随培养天数的变化。
5 Ag-DC-CIK对MGC-803细胞增殖及凋亡的作用:将培养18d的效应细胞和靶细胞混合培养(E:T=20:1)24h后,收集细胞,加入Hoechst 33342至终浓度为1ug/ml,37℃孵育10min,离心,弃染液。加入PI染液重悬避光染色。流式细胞仪分析蓝色荧光对红色荧光的散点图或地形图。正常细胞为低蓝光/低红光,凋亡细胞为高蓝光/低红光,坏死细胞为低蓝光/高红光。
结果:
1单独培养的CIK细胞在第3d开始增殖,第5-6d进入快速增殖期,CIK细胞和DC(包括负载和未负载MGC-803抗原)共培养后的第12d开始,共培养细胞组的增殖速度开始明显增快,并快于同源CIK细胞(p<0. 05)。
2健康成年PBMC在体外可以分别由IL-2、CD3McAb、IL-1α、INF-γ和GM-CSF、IL-4诱导出经表型鉴定为成熟的CIK和DC细胞。其中有(79.64±3.41)%的细胞表达成熟DC特异性表面标志CD83,(95.17±1.22)%的细胞表达HLA-DR,(94.66±2.42)%的细胞表达CD86,(86.59±1.09)%的细胞表达CD40。与DC共培养的CIK细胞在培养第14d时CD3+CD8+、CD3+CD56+双阳性细胞的百分含量进一步升高到59.39%-60.46%和35.67%-36.90%,经抗原刺激过的DC与CIK共培养14d后,混合细胞中CD3+CD8+、CD3+CD56+细胞最多,达到63.63%-65.42%和51.03%-53.15%,与CIK组和DC-CIK组相比均具有显著性差异(p<0.05)。
3在2.5:1-20:1效靶比范围内,DC-CIK对MGC-803靶细胞的杀伤活性一般均高于同源CIK细胞,经MGC-803细胞冻融物致敏的Ag-DC-CIK共培养细胞对MGC-803靶细胞的杀伤活性有进一步的提高,与单纯CIK细胞组比较差异显著(p<0.05),与DC-CIK共培养细胞组比较也有显著性差异(p<0.05)。
4 IFN-γ在21d的混合细胞上清液中表达较高,TNF-α在各个时期均能检测到,但表达水平变化很明显,在第6d时低表达,21d时较高。促炎因子IL-2变化正好与TNF-α相反,主要在第6d高表达,21d时表达相对较低或不表达。
5 24h组凋亡早期、中期、晚期细胞及正常细胞在FCM散点图中占绝大多数比例,而48h组以坏死细胞为主,凋亡细胞已较24h明显减少。
结论:
1负载MGC-803胃癌细胞抗原的DC可使CIK的增殖速度进一步提高。
2负载MGC-803抗原的DC与CIK共培养后可使具备攻击能力的主要细胞群CD3+CD8+、CD3+CD56+双阳性细胞群比例分别进一步提高。
3经抗原冲击的DC与CIK共培养后能增强其对胃癌细胞MGC-803的杀伤活性。
4 Ag-DC-CIK在体外培养过程中不仅需要持续来源于外界细胞因子的存在,而且该效应细胞组还能够向细胞外分泌IFN-γ、TNF-α、IL-2影响其自身的功能。
5在效靶相互作用初期,Ag-DC-CIK对MGC-803的杀伤作用主要通过诱导肿瘤细胞凋亡,同时发生自身凋亡来实现;在相互作用的中晚期,细胞开始趋向坏死。
Objective: Gastric cancer is one of the most harmful malignant tumors to human health and life and the clinical therapeutic tools of gastric cancer is mainly radical excision assistant with chemo and radiation therapy both before and after operation. Most clinical cases are found to be middle or advanced stage, the curative effects of operation, chemo and radiation are not satisfactory, and thus the long term survival is relatively low. So improving the curing effects and quality of life for patients of gastric cancer and preventing the relapse and metastasis is the issue which clinicians have been exploring. The adoptive cellular immunotherapy (ACI) is the fourth oncotherapy mode after operation, chemo and radiation. Until now the adoptive therapy of NK, CTL, MΦ, TIL, LAK, CIK and DC cells has been well applied clinically, in which the antitumor immunotherapy based on CIK and DC has been warm spots for researchers both in home and abroad recently. The cytokine-induced killer cell(CIK) is a kind of cytotoxic T cell which can be induced by various cytokines such as IL-2, IFN-γand monoclonal antibody CD3. The tumor-lytic activity is non-MHC restricted for which the CIK cells can be termed as non-specific immuno-killer. Moreover, it can excrete kinds of cytokines(IL-4, IFN-γ,et al) and has a killing activity to tumor cells much stronger than LAK and CD3AK. Dendritic cell (DC) is regarded as a powerful antigen-presenting cells, which can exist in all parts of human body except in the brain and didymus. DC can capture low-density antigen effectively by receptors on its cell membrane, combine with MHC I and MHC II molecules on the antigen surfaces and stimulate the activation of na?ve CD8+ and CD4+ T cells. According to the researches in home an abroad, combination of DC pulsed with antigen and the effective killing cells of CIK to treat malignant tumors is a hopeful way of synergistic effect. My initial reseach use the MTT and FCM to detect the toxic activity to MGC-803 and apoptosis mechanisms of co-cultivation of DC pulsed with antigen cocultured with CIK, and do some foundation research for clinical use of CIK and DC in treatment of gastric cancer.
Methods:
1 Preparations of CIK and DC derived from PBMC: Extract PBMC, stimulate the non-adherent cell with interleukine-2, anti-CD3McAb, IL-1αand INF-γto obtain CIK; stimulate the adherent cells with granulocyte-macrophage CSF and IL-4 to obtain DC; test the phenotype of cells with FCM.
2 Preparation and quantitation of MGC-803 soluble antigen: freeze thawing MGC-803 repeatedly with liquid nitrogen, filter and make into soluble antigen to stimulate DC; quantitative assay of antigen with Coomassie brilliant blue.
3 Comparison of killing function between three effective cell groups, with different E:T, in different days: The effector cells are divided into three groups: pure CIK group, non MGC-803 antigen pulsed DC with CIK group and MGC-803 antigen pulsed DC with CIK group. They are labeled as CIK, DC-CIK and Ag-DC-CIK. In Day 12 of co-culture, test the killing function of CIK, DC-CIK and Ag-DC-CIK groups with MTT, under the ratio of effective cells : target cells, 2.5:1, 5:1, 10:1 and 20:1; test the impact of different days.
4 Test the level of excreting cytokines with enzyme linked immunosorbent assay(ELISA): extract the clear supernatant liquid of Ag-DC-CIK in Day 12 and 21, test the contents of IL-2, TNF-αand INF-γ.
5 The proliferation and apoptosis of Ag-DC-CIK to MGC-803: co-cultivate the effector cells with the target cells (E:T=20:1)in Day 18 for 24h, collect cells, add Hoechst 33342 to the final concentration of 1ug/ml, incubate under the temperature of 37℃for 10min, centrifuge and draw off the staining solution; add PI, suspend, and incubate away from light; analyze the scatterplot or tonographic of blue fluorescence over the red; low blue/low red for the normal cells, high blue/low red for the apoptosis cell, low blue/high red for the necrosis cells.
Results:
1 CIK begins to multiply from Day3 and enter the multiplication period in Day5-6. DC-CIK and Ag-DC-CIK begin to proliferate fast from Day12, with the speed faster than CIK (p<0.05).
2 The PBMC from healthy adults can derive into mature CIK and DC with the existing of IL-2, CD3McAb, IL-1α, INF-γ, GM-CSF and IL-4. (79.64±3.41)% of the cells present DC specific surface marker CD83,(95.17±1.22)% of HLA-DR, (94.66±2.42)% of CD86 and (86.59±1.09)% of CD40. DC-CIK in Day14 has a higher percentage of CD3+CD8+ and CD3+CD56+ cells of 59.39%-60.46% and 35.67%-36.90%. Ag-DC-CIK in Day14 has the highest percentage of the two cell populations of 63.63%-65.42% and 51.03%-53.15% and has significant difference (p<0.05) compared with CIK and DC-CIK groups.
3 The killing activity of DC-CIK is generally stronger than CIK in the E:T range of 2.5:1-20:1. When DC is pulsed with MGC-803 antigen and co-cultured with CIK, the lytic activity of effector cells is strengthened and has significant difference (p<0.05) compared with CIK and DC-CIK.
4 IFN-γhas a high concentration in the clear supernatant of co-culturing cells in Day21. TNF-αcan be detected in all periods with an obvious fluctuation, low in Day6 while high in Day21. On the contrary, the proinflammatory factor IL-2 has a high expression in Day6 and low or no expression in Day21.
5 The 24h group shows a dominant percentage of early, middle and late period of apoptosis cells and normal cells in the FCM scatterplot. And 48h group is mainly composed of necrosis cells, with a obviously descending percentage of apoptosis cells than 24h group.
Conclusions:
1 DC pulsed with MGC-803 antigen can further improve the CIK proliferation rate.
2 DC pulsed with MGC-803 antigen can elevate the percentage of CIK attacking cell populations: the CD3+CD8+ and CD3+CD56+ cells.
3 Antigen-pulsed DC or non-antigen-pulsed DC can both strengthen the MGC-803 lytic activity of CIK.
4 Ag-DC-CIK need cytokines to maintain culturing in vitro, and also excrete IFN-γ, TNF-αand IL-2 which influence the function of the cells.
5 During the earlier period of the interaction of effectors and targets, MGC-803-DC-CIK kills MGC-803 mainly through inducing apoptosis of tumor cells and at the same time they themselves enter the process of apoptosis; in the late period of interactions, both of the targets and effectors tend to die of necrosis.
引文
1 Mehta-Damani A,Markowicz S,Engleman EG,et al.Generation of antigen specific CD8+ CTLs from naive precursors [J]. Immunol,1994, 153(3):996-1003
2 陈峻青,王舒宝,齐春莲等.胃癌外科治疗中的若干问题[J].中华外科杂志,1991,24:223
3 刘琳,王哲海.胃癌肝转移的治疗现状和进展[J].实用癌症杂志.2007,22(5):535-537
4 Schmidt R,Murray C,Daley J,et al.A subset of natural killer cells in peripheral blood displays a mature T cell phenotype[J]. Exp Med,1986;164;351-356
5 Schmidt-Wolf IG,Lefterova P,Mehta BA,et al.Phenotypic charact-erization and identification of effector cells involved in tumor cell recognition of cytokine-inducedkiller cells.Exp Hematol,1993,21(13):1673-1679
6 黄文荣等.细胞因子诱导的杀伤细胞(CIK)的研究现状[J].肿瘤防治研究.2000,27(2):157
7 任欢,徐红薇,宋英晖等.CIK 细胞的体外增殖及杀瘤活性的实验研究.中国免疫杂志,1998 ,6: 406-408
8 杜清友等.新型免疫活性细胞 CIK 体外对肝癌细胞的杀伤.世界华人消化杂志,2000,8 (8) : 863-866
9 Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature,1998;392:245-52
10 张利旺,张红梅,贾军等.以AFP为靶点的肝癌树突状细胞免 疫 治 疗 的 实 验 研 究 [J]. 现 代 肿 瘤 医学,2005,(06):736-739
11 Siders WM, Vergilis KL, Johnson C,et al. Induction of specific antitumor immunity in the mouse with the electrofusion product of tumor cells and dendritic cells[J]. Mol Ther. 2003, (4):498-505
12 Trefzer U, Walden P,et al. Hybrid-cell vaccines for cancer immune therapy[J].Mol Biotechnol,2003,25(1):63-69
13 Ojima T, Iwahashi M, Nakamura M,et al. The boosting effect of co-transduction with cytokine genes on cancer vaccine therapy using genetically modified dendritic cells expressing tumor-associated antigen[J].Int J Oncol,2006, 28(4):947-953
14 Marten A, Flieger D, Renoth S,et al. Therapeutic vaccination against metastatic renal cell carcinoma by autologous dendritic cells: preclinical results and outcomeof a first clinical phase I/II trial[J].Cancer Immunol Immunothe, 2002,51(11-12): 637-644
15 Wierecky J, Muller MR, Wirths S,et al. Immunologic and clinical responses after vaccination with peptide-pulsed dendritic cells in metastatic renal cancer patients.[J] Cancer Res.2006,66(11):5910-5918
16 Small EJ, Vogelzang NJ, Sosman A,et al. Safety and biological activity of repeated doses of recombinant human Flt3 ligand in patients with bone scan-negative hormone-refractory prostate cancer[J]. J Clin Cancer Res.2004,10(4):1219-1225
17 Mu LJ, Kyte JA, Kvalheim G,et al. Immunotherapy with allotumour mRNA-transfected dendritic cells in androgen- resistant prostate cancer patients[J]. Br J Cancer. 2005, 93(7): 749-756
18 O’Rourke MG, Johnson M, Lanagan C,et al. Durable complete clinical responses in a phase I/II trial using an autologous melanoma cell/dendritic cell vaccine[J]. Cancer Immunol Immunother,2003,52(6):387-395
19 Shi YJ,Cen XN,et al.Large-capacity expanded cytokine- induced killer cells and its cytotoxic activity[J].Sheng Wu Yi Xue Gong Cheng Xue Za Zhi,2001,18(1):94-96
20 李翠萍,陈宝安,陈津等.脐血来源的 CIK 细胞大容量制备方法的建立[J].临床肿瘤学杂志.2005,10(3):291-292
21 Schmidt-Wolf,Finke S,Huhn D,et al.Phase I clinical study applying autologous immunological effector cellstransfected with the interleukin-2 gene in patients with metastatic renal cancer,colorectal cancer and lymphoma. Br J Cancer, 1999, 81(6):1009-1016
22 Chen FX,Liu JQ,Zhang NZ,et al.Clinical observation on adoptive immunotherapy with autologous cytokine- induced killer cells for advanced malignant tumor[J].Ai Zheng, 2002, 21(7):797-801
23 Marten A,Schottker B,Ziske C,et al.Increase of the immuno- stimulatory effect of dendritic cells by pulsing with CA19-9 protein.J.Immunotherapy,2000,23:464-472
24 Zhu HH,Xu KL,Pan XY,et al.Specific anti-leukemic cell effect mediated by dendritic cells pulsed with chronic myelogenous leukemia lysate antigen in vitro[J].Zhongguo Shi Yan Xue Ye Xue Za Zhi,2003,11(11):278-281
25 Linn YC,Hui KM.Cytokine-induced killer cells:NK-like T cells with cytotolytic specificity against leukemia[J].Leuk Lymphoma,2003,44(9):1457-1462
26 Ge W,Li CH,Zhang W,et al.Coculture of dendritic cell with cytokine-induced killer results in a significant increase in cytotoxic activity of CIK to tumor cells in vitro and in vivo [J]. Zhonghua Xue Ye Xue Za Zhi,2004,25(5):277-280
27 雷静,钱旻,吴芝莉等.体外 CIK 细胞的细胞因子表达谱研究的初步探讨[J].中国免疫学杂志,2005,21(8):589-593
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2 陈峻青,王舒宝,齐春莲等.胃癌外科治疗中的若干问题[J].中华外科杂志,1991,24:223
3 刘琳,王哲海.胃癌肝转移的治疗现状和进展[J].实用癌症杂志.2007,22(5):535-537
4 Schmidt R,Murray C,Daley J,et al.A subset of natural killer cells in peripheral blood displays a mature T cell phenotype[J]. Exp Med,1986;164;351-356
5 Schmidt-Wolf IG,Lefterova P,Mehta BA,et al.Phenotypic charact-erization and identification of effector cells involved in tumor cell recognition of cytokine-inducedkiller cells.Exp Hematol,1993,21(13):1673-1679
6 黄文荣等.细胞因子诱导的杀伤细胞(CIK)的研究现状[J].肿瘤防治研究.2000,27(2):157
7 任欢,徐红薇,宋英晖等.CIK 细胞的体外增殖及杀瘤活性的实验研究.中国免疫杂志,1998 ,6: 406-408
8 杜清友等.新型免疫活性细胞 CIK 体外对肝癌细胞的杀伤.世界华人消化杂志,2000,8 (8) : 863-866
9 Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature,1998;392:245-52
10 张利旺,张红梅,贾军等.以AFP为靶点的肝癌树突状细胞免 疫 治 疗 的 实 验 研 究 [J]. 现 代 肿 瘤 医学,2005,(06):736-739
11 Siders WM, Vergilis KL, Johnson C,et al. Induction of specific antitumor immunity in the mouse with the electrofusion product of tumor cells and dendritic cells[J]. Mol Ther. 2003, (4):498-505
12 Trefzer U, Walden P,et al. Hybrid-cell vaccines for cancer immune therapy[J].Mol Biotechnol,2003,25(1):63-69
13 Ojima T, Iwahashi M, Nakamura M,et al. The boosting effect of co-transduction with cytokine genes on cancer vaccine therapy using genetically modified dendritic cells expressing tumor-associated antigen[J].Int J Oncol,2006, 28(4):947-953
14 Marten A, Flieger D, Renoth S,et al. Therapeutic vaccination against metastatic renal cell carcinoma by autologous dendritic cells: preclinical results and outcomeof a first clinical phase I/II trial[J].Cancer Immunol Immunothe, 2002,51(11-12): 637-644
15 Wierecky J, Muller MR, Wirths S,et al. Immunologic and clinical responses after vaccination with peptide-pulsed dendritic cells in metastatic renal cancer patients.[J] Cancer Res.2006,66(11):5910-5918
16 Small EJ, Vogelzang NJ, Sosman A,et al. Safety and biological activity of repeated doses of recombinant human Flt3 ligand in patients with bone scan-negative hormone-refractory prostate cancer[J]. J Clin Cancer Res.2004,10(4):1219-1225
17 Mu LJ, Kyte JA, Kvalheim G,et al. Immunotherapy with allotumour mRNA-transfected dendritic cells in androgen- resistant prostate cancer patients[J]. Br J Cancer. 2005, 93(7): 749-756
18 O’Rourke MG, Johnson M, Lanagan C,et al. Durable complete clinical responses in a phase I/II trial using an autologous melanoma cell/dendritic cell vaccine[J]. Cancer Immunol Immunother,2003,52(6):387-395
19 Shi YJ,Cen XN,et al.Large-capacity expanded cytokine- induced killer cells and its cytotoxic activity[J].Sheng Wu Yi Xue Gong Cheng Xue Za Zhi,2001,18(1):94-96
20 李翠萍,陈宝安,陈津等.脐血来源的 CIK 细胞大容量制备方法的建立[J].临床肿瘤学杂志.2005,10(3):291-292
21 Schmidt-Wolf,Finke S,Huhn D,et al.Phase I clinical study applying autologous immunological effector cellstransfected with the interleukin-2 gene in patients with metastatic renal cancer,colorectal cancer and lymphoma. Br J Cancer, 1999, 81(6):1009-1016
22 Chen FX,Liu JQ,Zhang NZ,et al.Clinical observation on adoptive immunotherapy with autologous cytokine- induced killer cells for advanced malignant tumor[J].Ai Zheng, 2002, 21(7):797-801
23 Marten A,Schottker B,Ziske C,et al.Increase of the immuno- stimulatory effect of dendritic cells by pulsing with CA19-9 protein.J.Immunotherapy,2000,23:464-472
24 Zhu HH,Xu KL,Pan XY,et al.Specific anti-leukemic cell effect mediated by dendritic cells pulsed with chronic myelogenous leukemia lysate antigen in vitro[J].Zhongguo Shi Yan Xue Ye Xue Za Zhi,2003,11(11):278-281
25 Linn YC,Hui KM.Cytokine-induced killer cells:NK-like T cells with cytotolytic specificity against leukemia[J].Leuk Lymphoma,2003,44(9):1457-1462
26 Ge W,Li CH,Zhang W,et al.Coculture of dendritic cell with cytokine-induced killer results in a significant increase in cytotoxic activity of CIK to tumor cells in vitro and in vivo [J]. Zhonghua Xue Ye Xue Za Zhi,2004,25(5):277-280
27 雷静,钱旻,吴芝莉等.体外 CIK 细胞的细胞因子表达谱研究的初步探讨[J].中国免疫学杂志,2005,21(8):589-593
28 Shiver J W, Lishan S , Henkart P A. Cytotoxicity with target DNA breakdown by rat basophilic leukemia cells expressing both cytolysin and granzyme A[J ] . Cell,1992,71(2):315-322
29 Hoyle C,Bangs C D,Negrin R S,et al. Expansion of Philadelphia chromosome2negative CD3+56+ cytotoxic cells f rom chronic myeloid leukemia patient s : in vitro and in vivo efficacy in severe combined immunodeficiency disease mice[J ] . Blood , 1998 ,92 (9) : 3318-3327
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