【作者】 李晓锋；

【导师】 杜智；

【作者基本信息】 天津医科大学 ， 外科学， 2008， 硕士

【摘要】 目的:1.建立一套高效的无血清体外制备DCs疫苗的方法,为DCs疫苗的临床应用提供实验依据。2.探讨DCs对CIK增殖和杀伤肝癌细胞的影响。方法:1.采集脐血,分离其中的单个核细胞,用无血清DCs培养基(SFM)/含5%胎牛血清的RPMI-1640培养基(SM),并添加rhGM-CSF、rhIL-4联合诱导单个核细胞分化为未成熟DCs,第5天添加肝癌细胞冻融抗原,TNF-α等促成熟因子,第7天收获成熟DCs,以倒置显微镜观察DCs的细胞形态,流式细胞仪测定Des表面标志物的表达;并从细胞形态和表面标志物等方面比较用无血清DCs培养基(SFM)/含5%胎牛血清的RPMI-1640培养基(SM)培养的DCs。2.用细胞因子γ-IFN、IL-2、CD3-Ab、IL-1a诱导脐血单个核细胞为CIK,第7天DCs和CIK共同培养。采用MTT法测定无血清培养基和含5%胎牛血清的培养基产生的DCs刺激CIK的促增殖效应和DC-CIK对肝癌细胞的杀伤效应。结果:1.无论采用无血清树突状细胞培养基(SFM)还是含5%胎牛血清的RPMI-1640培养基,脐血来源的单核细胞在细胞因子rhGM-CSF、rhIL-4、TNF-α的诱导下,并负载7721肝癌细胞冻融抗原后,均能分化成为具有典型形态和表型的成熟DCs,流式结果显示:采用无血清培养基培养的DCs表面标志物表达水平为(%):CD1a 56.6±3.8、CD11c 64.5±4.3、CD14 19.5±1.8、CD4077.8±7.5、CD83 70.8±5.1、CD86 76.5±6.3、HLA-DR 91.5±3.5;采用5%胎牛血清的RPMI-1640培养基培养的SM-DC表面标志物表达水平为(%):CD1a 59.3±5.1、CD11c 59.6±6.2、CD14 20.1±2.2、CD40 75.6±6.4、CD83 65.2±4.4、CD8680.4±6.9、HLA-DR 94.5±2.8;两组DCs的表面标志均无差别,(P＞0.05)。2.混合淋巴细胞反应结果:DCs刺激CIK的刺激指数为:无血清DCs培养基组2.01±0.22,含血清培养基DCs组2.31±0.24;统计分析表明:不同培养基培养的DCs,在促进淋巴细胞增殖作用中无差别,均能有效地刺激CIK增殖,(P＞0.05)。3.杀伤实验结果:DCs激活CIK对肝癌细胞的杀伤率为(%):无血清DCs培养基组SFMDC-CIK 74.5±7.5,含血清培养基组SMDC-CIK 77.9±6.0,单一CIK 54.1±4.9,统计分析表明:①SM-DC和SFM-DC激活的CIK对肝癌细胞的杀伤率无明显差别,(P＞0.05);②SM-DC和SFM-DC激活的CIK对肝癌细胞的杀伤率均高于未被DCs激活的CIK,(P＜0.05)。结论:1.我们初步建立了一套体外无血清制备DCs的方法;无血清树突状细胞培养基在添加细胞因子rhGM-CSF 100ng/ml、rhIL-4、50ng/ml、TNF-α50ng/ml及肝癌抗原后,能够诱导脐血单个核细胞分化为具有典型形态和高度表达DCs表面标志的成熟树突状细胞。2.SM-DC和SFM-DC均能有效地刺激CIK增殖及提高其抗肿瘤作用,DC-CIK治疗肿瘤将具有良好的应用前景。更多还原

【Abstract】 Objectives:1.To establish a highly effective method for preparing the DCs vaccine in serum-free medium in vitro,and provid experimental proof for the clinical antitumor therapy.2.To explore the effect of DCs on the proliferation of CIK and the cytotoxicity of CIK against hepatocellular carcinoma cells.Methods:1.The mononuclear cells separated from cord blood were induced to differentiate into the immature DC by rhGM-CSF and rhIL-4 in serum-free medium (SFM)or 5%fetal calf serum RPMI-1640 medium(SM).TNF-αand the Freeze-thaw lysates of hepatocellular carcinoma cells were was added on the 5th day. On the 7th day,these cells were harvested and identified with light microscope and flow cytometry(FCM).Then,the morphology and surface markers of SM-cultured DCs(SM-DCs)and SFM-derived DCs(SFM-DCs)was compared.2.The mononuclear cells from cord blood were induced to differentiate into CIK by cytokines IFN-γ,IL-2,CD3-Ab,IL-la.DCs were co-cultured with autologous CIK cells on the 7th day.Then,the proliferation and the cytotoxicity of CIK stimulated with DCs was examined by methyl thiazoly tetrazolium(MTT).Results:1.Mononuclear cells separated from the cord blood could be induced into typical and phenotypic DC s with the compatibility of such cellular factors as rhGM-CSF, IL-4,TNF-α,and pulsed antigen.The flow cytometry detection showed:the expression level(%)of cell surface markers of DCs cultured on the SFM-DC:CD1a 56.6±3.8,CD11c 64.5±4.3,CD14 19.5±1.8,CD40 77.8±7.5,CD83 70.8±5.1,CD86 76.5±6.3 HLA-DR 91.5±3.5;The expression level(%)of cell surface markers on the SM-DC:CD1a 59.3±5.1,CD11c 59.6±6.2,CD14 20.1±2.2,CD40 75.6±6.4, CD83 65.2±4.4,CD86 80.4±6.9,HLA-DR 94.5±2.8;The levels of surface markers were not significantly different between SFM-DC and SM-DC,(p＞0.05).2.The results of mixed lymphocyte reaction:The average stimulator index(SI) of CIK actvavted by SFM-DC:2.01±0.22;The average stimulator index(SI)of CIK actvavted by SM-DC:2.31±0.24.The difference of the stimulator index(SI)between SFMDC and SMDC was not significant and they could effectively activate CIK cells to proliferate.3.The results of cytotoxicity assay:The cytotoxicity to hepatocellular carcinoma cells:the cytotoxicity of SFMDC-CIK 74.5±7.5,the cytotoxicity of SMDC-CIK 77.9±6.0,the cytotoxicity of SMDC-CIK 54.1±4.9;The statistical analysis shows:①The difference of the cytotoxicity between SFMDC-CIK and SMDC-CIK was not significantly,(p＞0.05);②SFMDC-CIK and SMDC-CIK possessed higher ability to lyse hepatoma cells than inactivated CIK,(p＜0.05).Conclusion:1.We Preliminarily established a method for preparing the DCs vaccine in serum-free medium in vitro.In serum-free DCs medium with rhGM-CSF 100ng/ml, rhIL-4 50ng/ml,TNF-α50ng/ml,mononuclear ceils from the cord blood could be induced into the Clinical grade DCs with typical modality and Cell surface markers2.Both SFM-DC and SM-DC could Effectively activate CIK cells to have higher proliferative and anti-tumor activity,and DC-CIK will have a good prospect in the treatment of tumors.更多还原