【作者】 刘苗；

【导师】 金润铭；

【作者基本信息】 华中科技大学 ， 儿科学， 2010， 博士

【摘要】 第一部分：细胞因子诱导的杀伤细胞体外生物活性的研究目的：探讨细胞因子诱导的杀伤细胞(DC-CIK)的体外生物活性。方法：从白血病患儿外周血分离淋巴细胞,经过IFN-γ、CD3McAb、IL-2诱导并培养,获得大量的DC-CIK。光镜下观察其细胞形态学及动态增殖情况,流式细胞仪检测DC-CIK细胞免疫表型,MTT法研究DC-CIK细胞对多种白血病细胞株的杀伤活性,ELISA方法测定DC-CIK细胞释放细胞因子IL-12、IFN-γ、TNF-α的表达水平。结果：诱导后的DC-CIK细胞形态规则,DC-CIK细胞起初增殖缓慢,第4-8天快速增殖,于第9-10天达到增殖高峰,扩增约100倍。DC-CIK细胞中主要效应细胞是CD3+CD8+双阳性细胞和CD3+CD56+双阳性细胞。MTT结果示DC-CIK对肿瘤细胞B95杀伤作用最强,对Jhhan细胞和M07e细胞杀伤作用较弱。ELISA方法检测,发现诱导培养的DC-CIK细胞上清液中细胞因子IL-12、IFN-γ、TNF-α的分泌水平较高。结论：细胞因子诱导培养的杀伤细胞扩增能力和杀伤能力均较强。Th1细胞因子如IL-12、IFN-γ、TNF-α分泌较高,提示DC-CIK可能通过Th1途径发挥杀伤作用。第二部分：LFA-1/ICAM-1介导的细胞因子诱导的杀伤细胞体外杀瘤机制的研究目的：探讨LFA-1/ICAM-1介导的细胞因子诱导杀伤细胞的体外杀瘤机制。方法：从白血病患儿外周血分离淋巴细胞,经过IFN-γ、CD3McAb、IL-2诱导并培养,获得大量的DC-CIK。在予以10μg/ml、20μg/ml等不同浓度小鼠抗人LFA-1单克隆抗体处理后,采用MTT法研究DC-CIK细胞对多种白血病细胞株的杀伤活性,RT-PCR与Western-Blot方法检测GATA-3和T-bet基因表达水平的变化。ELISA方法测定DC-CIK细胞释放细胞因子IL-12、IFN-γ、TNF-α的表达水平。结果：诱导后的DC-CIK细胞形态规则,在予以10μg/ml、20μg/ml等不同浓度的LFA-1单克隆抗体处理后,MTT结果表明与阴性对照组相比,20μg/ml LFA-1单克隆抗体封闭的实验组DC-CIK细胞对B95细胞杀伤作用下降最为明显,差异有统计学意义(P<0.05),而对Jhhan细胞和M07e细胞杀伤作用均下降,但差异没有统计学意义(P>0.05; P>0.05)。RT-PCR与Western-Blot结果表明与阴性对照组相比,20μg/mlLFA-1单克隆抗体封闭的B95细胞实验组GATA-3基因mRNA水平和蛋白水平表达增加最为明显,差异有统计学意义(P<0.05；P<0.05)。20μg/mlLFA-1单克隆抗体封闭的B95细胞实验组T-bet基因mRNA水平和蛋白水平表达降低最为明显,差异有统计学意义(P<0.05;P<0.05)。ELISA结果表明与阴性对照组相比,20μg/mlLFA-1单克隆抗体封闭的B95细胞实验组DC-CIK细胞上清液中细胞因子IL-12、IFN-γ、TNF-α分泌水平下降最为明显,差异有统计学意义(P<0.05；P<0.05；P<0.05)。结论：GATA-3和T-bet基因参与了LFA-1/ICAM-1介导的DC-CIK杀瘤途径,并且通过分泌Thl型细胞因子IL-12、IFN-γ、TNF-α等发挥杀瘤作用。第三部分：T-bet介导的细胞因子诱导的杀伤细胞体外杀瘤机制的研究目的：研究T-bet介导的细胞因子诱导的杀伤细胞体外杀瘤的作用机制。方法：从白血病患儿外周血分离淋巴细胞,经过IFN-γ、CD3McAb、IL-2诱导,经树突细胞共培养后,获得大量的DC-CIK。在予以1μg/ml、5μg/ml、10μg/ml等不同浓度小鼠抗人T-bet单克隆抗体处理后,采用MTT法研究DC-CIK细胞对多种白血病细胞株的杀伤活性,流式细胞仪检测CD4+CD25+Treg细胞比例,RT-PCR与Western-Blot方法分别检测Foxp3和GATA-3基因表达水平的变化,ELISA方法测定DC-CIK细胞释放细胞因子IL-12、IFN-γ、TNF-a的表达水平。结果：诱导后的DC-CIK细胞形态规则,在予以1μg/ml、51μg/ml、10μg/ml等不同浓度小鼠抗人T-bet单克隆抗体处理后,MTT结果表明与阴性对照组相比,10μg/mlT-bet单克隆抗体封闭的实验组DC-CIK细胞对B95细胞杀伤作用下降最为明显,差异有统计学意义(P<0.05),而对Jhhan细胞和M07e细胞杀伤作用均下降,但差异没有统计学意义(P>0.05；P>0.05)。流式结果表明与阴性对照组相比,10μg/mlT-bet单克隆抗体封闭的B95细胞实验组CD4+CD25+Treg细胞表达上调最为明显,差异有统计学意义(P<0.05),而Jhhan细胞实验组和M07e细胞实验组CD4+CD25+Treg细胞表达也上调,但差异没有统计学意义(P>0.05;P>0.05)。RT-PCR与Western-Blot结果表明与阴性对照组相比,10μg/mlT-bet单克隆抗体封闭的B95细胞实验组Foxp3基因mRNA水平和蛋白水平表达增加最为明显,差异有统计学意义(P<0.05;P<0.05)。10μg/m1T-bet单克隆抗体封闭的B95细胞实验组GATA-3基因mRNA水平和蛋白水平表达增加最为明显,差异有统计学意义(P<0.05；P<0.05)。ELISA结果表明与阴性对照组相比,10μg/m1T-bet单克隆抗体封闭的B95细胞实验组DC-CIK细胞上清液中细胞因子IL-12、IFN-γ、TNF-a分泌水平下降最为明显,差异有统计学意义(P<0.05；P<0.05；P<0.05)。结论：Foxp3和GATA-3基因参与了T-bet介导的DC-CIK细胞杀瘤途径,其杀瘤机制主要表现为Thl途径活化,而Th2和Treg途径受抑。更多还原

【Abstract】 Part 1 The biological characteristics of cytokine-induced killer cells in vitroObjective:To investigate the biological activity of cytokine-induced killer (CIK) cells in vitro.Methods:Lymphocytes isolated from peripheral blood of leukemia children were induced with interferon-gamma (IFN-γ), anti-CD3 monoclonal antibody (CD3McAb) and interleukin-2 (IL-2) and co-cultured with dendrite cells (DCs) to generate DC-CIK cells. The morphology and immunophenotype of these cells were determined by electron microscopy and flow cytometry, respectively. Cytotoxicity of DC-CIK cells against leukemia cell lines was measured by the MTT assay. Interleukin-12 (IL-12), interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α) levels released by DC-CIK cells were quantified by enzyme-linked immunosorbent assays (ELISA).Results:Induced DC-CIK cells were regular, round and transparent with variable cell volume and cellular aggregation. At first, its amplification was very slow, and it increased quickly at the 4th-8th day, it reached its peak amplification at the 9th-10th day, at approximately 100-fold.The main effectors cells in this population were CD3+CD8+ cells and CD3+CD56+cells. DC-CIK cells were cytotoxic to B95 cells, Jhhan cells and M07e cells, with the highest cytotoxicity towards B95 cells. The expression levels of IL-12, IFN-γ, and TNF-αin supernatant were very high. Conclusion:DC-CIK cells induced with cytokines displayed powerful amplification and strongly killing activities in vitro. It suggested that DC-CIK cells induced with cytokines may play killing activities through Thl pathway in vitro, as a result of high secretion of Thl cytokines, such as IL-12, IFN-y, and TNF-α. Part 2 The mechanism of LFA-1/ICAM-1 mediated anti-neoplastic effects of cytokine-induced killer cellsObjective:To investigate the molecular mechanism underlying LFA-1/ICAM-1 mediated anti-neoplastic effects of cytokine induced killer (CIK) cells.Methods:Lymphocytes isolated from peripheral blood of leukemia children were induced with interferon-gamma (IFN-γ), anti-CD3 monoclonal antibody (CD3McAb) and interleukin-2 (IL-2) and co-cultured with dendrite cells (DCs) to generate DC-CIK cells. When treated with LFA-1 monoclonal antibody, cytotoxicity of DC-CIK cells against leukemia cell lines was measured by the MTT assay, while RT-PCR and Western-blot were used to determine mRNA and protein expressions of GATA-3 and T-bet in DC-CIK cells, respectively. IL-12, IFN-γand TNF-αlevels released by DC-CIK cells were quantified by ELISA.Results:Induced DC-CIK cells were regular, round and transparent with variable cell volume and cellular aggregation. When treated with mouse anti-human LFA-1 monoclonal antibody, the cytotoxicity decreased mostly towards B95 cells under 20μg/ml LFA-1 monoclonal antibody in comparison with the control group (P<0.05), while it also resulted in a decrease of the cytotoxicity towards Jhhan cells and M07e cells, however there was no statistically significant differences (P> 0.05;P> 0.05). It led to a highest elevation of GATA-3 mRNA and protein levels (P<0.05; P<0.05)and a highest decrease of T-bet mRNA and protein levels (P<0.05; P<0.05)under 20μg/ml LFA-1 monoclonal antibody in B95 cells group in comparison with the control group. The expression levels of IL-12, IFN-γ, and TNF-αin supernatant were lowest under 20μg/ml LFA-1 monoclonal antibody in B95 cells group in comparison with the control group (P<0.05;P<0.05;P<0.05).Conclusion:GATA-3 and T-bet were implicated in the LFA-1/ ICAM-1 mediated anti-neoplastic effects of DC-CIK cells via activation of the Thl pathway, with high secretion of Th1 cytokines, such as IL-12, IFN-γ, and TNF-α. Part 3 The mechanism of T-bet mediated anti-neoplastic effects of cytokine-induced killer cellsObjective:To investigate the molecular mechanism underlying T-bet mediated anti-neoplastic effects of cytokine induced killer (CIK) cells.Methods:Lymphocytes isolated from peripheral blood of leukemia children were induced with interferon-gamma (IFN-γ), anti-CD3 monoclonal antibody (CD3McAb) and interleukin-2 (IL-2) and co-cultured with dendrite cells (DCs) to generate DC-CIK cells. When treated with T-bet monoclonal antibody, cytotoxicity of DC-CIK cells against leukemia cell lines was measured by the MTT assay. FCM was used to detect CD4+CD25+Treg cells, while RT-PCR and Western-blot were used to determine mRNA and protein expressions of Foxp3 and GATA-3 in DC-CIK cells,respectively. IL-12, IFN-γand TNF-a levels released by DC-CIK cells were quantified by ELISA.Results:Induced DC-CIK cells were regular, round and transparent with variable cell volume and cellular aggregation. When treated with mouse anti-human T-bet monoclonal antibody, the cytotoxicity decreased mostly towards B95 cells under 10μg/ml T-bet monoclonal antibody in comparison with the control group (P<0.05), while it also resulted in a decrease of the cytotoxicity towards Jhhan cells and M07e cells, however there was no statistically significant differences (P> 0.05;P> 0.05). Treatment with 10μg/ml T-bet monoclonal antibody resulted in a highest increase in the proportion of CD4+CD25+Treg cells in B95 cells group in comparison with the control group (P<0.05), and it also resulted in an increase in Jhhan cells group and M07e cells group, however there was no statistically significant differences (P> 0.05;P> 0.05). It led to a highest elevation of Foxp3 and GATA-3 mRNA and protein levels(P<0.05;P<0.05;P<0.05;P<0.05)under 10μg/ml T-bet monoclonal antibody in B95 cells group in comparison with the control group. The expression levels of IL-12, IFN-γ, and TNF-αin supernatant were lowest under 10μg/ml T-bet monoclonal antibody in B95 cells group in comparison with the control group (P<0.05;P<0.05;P<0.05).Conclusion:Foxp3 and GATA-3 were implicated in the T-bet mediated anti-neoplastic effects of DC-CIK cells via activation of the Thl pathway and suppression of the Th2 and Treg pathways.更多还原