论文标题:CIK细胞治疗恶性实体瘤的临床前研究
Pre-clinical Study on the Treatment of Malignant Solid Tumors with Cytokine-Induced Killer Cells
论文作者 岳欣
论文导师 郝希山；王家仓；任秀宝,论文学位 博士,论文专业 肿瘤学
论文单位 天津医科大学,点击次数 213,论文页数 126页File Size11718k
2005-06-01论文网 http://www.lw23.com/lunwen_63988532/ 脑肿瘤;动物模型;血管生成;病理学;血管生成因子;灌注;神经上皮肿瘤;脑脓肿;转移瘤;体层摄影术;X线计算机;磁共振成像
Brain neoplasms; Animal model; Angiogenesis factor; Perfusion; Pathology; Tumors of neuroepithelial tissue; Metastasis; Brain abscess; Tomography, X-ray computed; Magnetic resonance imaging
第一部分 CIK细胞的制备及基本特征 目的:CIK细胞是一种新型的应用于过继免疫治疗的免疫活性细胞,本研究旨在建立包含多细胞因子的培养体系体外扩增外周血单个核细胞制备CIK细胞,进而通过检测CIK细胞的表型特点、多种细胞因子的分泌和对于胃癌细胞和结肠癌细胞的杀伤活性以探讨其应用于抗肿瘤治疗的可能。 材料和方法: 1.CIK细胞的体外制备:采集人外周血单个核细胞,按特定的次序加入CD3McAb、IL-2、IFN-γ、IL-1α等多种细胞因子,每3日更换新鲜培养液并补充IL-2和IFN-γ,经过12～14天的体外培养收获CIK细胞,并检测其增殖概况和细胞活率。 2.CIK细胞的表型特征和细胞因子的分泌:以流式细胞技术对CIK细胞进行表型特征分析,用ELISA方法检测CIK细胞分泌的IL-2、IFN-γ和IL-10等细胞因子。 3.CIK细胞对肿瘤的细胞毒活性:以胃癌细胞系(823细胞)和结肠癌细胞系(HCT-8细胞)为靶细胞,用LDH法检测CIK细胞对肿瘤细胞的细胞毒活性。 结果: 1.经过体外培养,细胞总数扩增为初始细胞数目的49.8倍,活细胞率比例在95%以上。 2.经过多种细胞因子的诱导,CIK细胞的CD3、CD3/CD8、CD3/CD4、
Part Ⅰ: CIK cells preparation and biological charactersPurpose: CIK cells are new immunological competent cells that are employed in adoptive cellular immunotherapy. In this study, we established a multi-cytokine culture system to prepare CIK cells by amplifying peripheral blood mononuclear cells in vitro. We evaluated the phenotype, multiple cytokines secretion of CIK cells and anti-tumor activities of CIK cells against the gastric carcinoma cell line and the colon cancer cell line. Based on these experiment results, we shed lights on the possibility of the application of CIK cells in clinical treatment against cancer.Material and Method:1. Preparation of CIK cells in vitro:Peripheral blood mononuclear cells (PBMC) from donor were incubated in vitro with various cytokines, such as CD3 monoclonal antibody (CD3McAb), interleukin-2 (IL-2), interferon-gamma (IFN-Y) and interleukin-1α (IL-1α), in a certain sequence. Cells were incubated in a culture system that is renewed every 3 days with fresh complete medium and various types of cytokines such as IL-2 and IFN-γ. After 12 to 14 days, CIK cells were harvested and tested for proliferation profiles and survival rates.2. The phenotype and cytokine secretion of CIK cells:Flow cytometry analysis was used to analyze the phenotype of CIK cells. ELISA was used to test cytokines, such as IL-2、 IFN-γ and IL-10, that are secreted fromCIK cells.3. Anti-tumor cytotoxicity of CIK cells:The gastric carcinoma cell line (823 cells) and the colon cancer cell line (HCT-8 cells) were used as target cells. The LDH method was employed to measure the cytotoxicity of CIK cells against cancer cells.Results:1. Cells were amplified to a total population of 49.8 times of initial cellular population after in vitro culture and incubation. The survival rate of CIK cells is above 95%.2. After the induction of various cytokins, the phenotypes of CIK cells, such as CD3, CD3/CD8> CD3/CD4> CD3/CD56, have more than that of peripheral blood mononuclear cells. Cytokines secreted from CIK cells , such as IL-2> IFN-y, are more than that from peripheral blood mononuclear cells. In the contrast, cytokine of IL-10 from CIK cells is less than that from peripheral blood mononuclear cells.3. Results from in vitro cytotoxicity experiment show that CIK cells have potent killing activity to 823 cells and HCT-8 cells.Conclusion: The multi-cytokine culture system is capable of amplifying peripheral blood mononuclear cells into CIK cells in vitro. The CD3+/CD56+ cell, which is the major effect cell of CIK cells, responds with a consequence of prompt proliferation and productive secretion of cytokines. These downstream events have potent killing activity to cancer cells that can be applied towards adoptive immunotherapy against malignant solid tumors.Part II: Impacts of cancer cells on CIK cells and Joint abilities of CIK cells and 5FU to kill cancer cells in vitroPurpose: To discuss the impact of malignant cancer cells on the preparation, proliferation inhibition and induced apoptosis of CIK cells. To discuss the possibility of combinational application of chemotherapy and CIK cells in clinical treatment against cancer.Material and Method:1. Supernatant from 823 cells and HCT- cells culture system and the chemotherapy drug (5FU) were added separately into the culture system of CIK cells. Experiment data, such as CIK cells proliferation rate, percentage of the major effect cell (CD3+/CD56+ cells) and killing activities was taken individually to evaluate the impact of each experiment condition on CIK cells.2. CIK cells and cancer cells are co-cultured in medium. Flow cytometry analysis was used to measure the influence of cancer cells to CIK cells in respect of the proliferation inhibition and induced apoptosis.3. ELISA was used to measure the content of immunoinhibitory cytokines in the culture supernatant from CIK cells. Ultracentrifugation was used to separate exosome from culture system. Those experiment data were analyzed to evaluate their inhibitory effect on CIK cells.4. The LDH assay and the MTT a
|
