高强度聚焦超声制备肿瘤抗原致敏DC及其体外诱导T细胞产生IFN-γ的研究

发布时间：2018-06-21 05:59

本文选题：树突状细胞 + 高强度聚焦超声　；参考：《泰山医学院》2005年硕士论文

【摘要】：目的：建立体外大量扩增小鼠树突状细胞(dendritic cell, DC)的方法；探讨用高强度聚焦超声(high intensity focused ultrasound, HIFU)辐照肿瘤细胞制备肿瘤抗原致敏DC和制备DC肿瘤疫苗的可行性,为HIFU开辟新的应用领域。方法：①应用1ng/ml IL-4和lOng/ml GM-CSF联合诱导培养小鼠骨髓细胞,光镜及电子显微镜观察DC发育过程中形态学变化；流式细胞术检测DC表面分子CD80、CD86、H-2Kd及I-Ad的表达情况；②固定辐照时间,应用不同功率的HIFU辐照CT26细胞株后,用台盼兰染色、MTT法测定活肿瘤细胞数,同时用台盼兰染色观察肿瘤细胞的形态学变化,分析HIFU剂量与肿瘤细胞存活率的关系,了解剂量-效应关系；③固定辐照时间和超声强度,辐照CT26细胞悬液,制备肿瘤细胞抗原并致敏DC,致敏DC与T细胞功孵育48小时后提取上清,通过ELISA法检测上清中IL-12、IFN-γ的含量,并与冻融组、单纯CT26、对照组进行比较。结果：①用IL-4和GM-CSF联合培养小鼠骨髓细胞3天后,可见细胞形态发生改变,细胞形态不规则。培养第6～8天,细胞表面出现较多毛刺样突起,拉长,为典型的DC特征。流式细胞仪检测DC表面分子有CD80、CD86、H-2Kd及I-Ad表达；②随着HIFU辐照剂量的增加肿瘤细胞的存活率迅速减少,肿瘤细胞的碎片逐渐增多。超声剂量为1000W/cm2×30秒时,无细胞存活,肿瘤细胞完全失去细胞形态,全部被撕裂成碎片;③通过ELISA法检测HIFU、冻融、单纯CT26三组上清中IL-12、IFN-γ的含量显著高于对照组(p均0.05),HIFU、冻融两组上清中IL-12、IFN-γ的含量显著高于单纯CT26组(p均0.05),但H1FU、冻融组之间比较在统计学上无显著性意义(p0.05)。结论：应用IL-4联合GM-CSF培养小鼠骨髓细胞,可大量扩增成熟DC,培养的DC符合其自身的特性;HIFU能灭活肿瘤细胞且能破碎肿瘤细胞,HIFU制备的肿瘤抗原可体外致敏DC,并可使DC成熟分泌大量IL-12,同时能诱导T细胞分泌大量IFN-γ。
[Abstract]:Objective: to establish a method to amplify dendritic cells (DCs) in vitro, and to investigate the feasibility of preparing tumor antigen-sensitized DC and DC tumor vaccine by high intensity focused ultrasound (HIFU) irradiation. To open up a new application field for HIFU. Methods 1ng/ml IL-4 and lOng / ml GM-CSF were used to induce and culture mouse bone marrow cells. The morphological changes of DC were observed by light microscope and electron microscope, and the expression of CD80 CD86 + H-2Kd and I-Ad on DC surface was detected by flow cytometry. CT26 cells were irradiated with HIFU of different power. The number of live tumor cells was determined by Trypan blue colorimetric assay. The morphological changes of tumor cells were observed by trypan blue staining, and the relationship between HIFU dose and survival rate of tumor cells was analyzed. To understand the dose-effect relationship between irradiation time and ultrasound intensity, CT26 cell suspension was irradiated to prepare tumor cell antigen and sensitize DC. the supernatant was extracted after incubation with T cells for 48 hours. The content of IL-12 IFN- 纬 in the supernatant was detected by Elisa. And compared with freeze-thaw group, CT26 and control group. Results the bone marrow cells of mice were cultured with IL-4 and GM-CSF for 3 days. On the 6th day of culture, there were many burrs on the surface of the cells, which were typical DC characteristics. Flow cytometry was used to detect CD80, CD86, H-2Kd and I-Ad expression on DC surface. With the increase of HIFU irradiation dose, the survival rate of tumor cells decreased rapidly, and the fragments of tumor cells increased gradually. When the ultrasound dose was 1000 W / cm 2 脳 30 seconds, no cells survived, the tumor cells completely lost the shape of the cells, all of them were torn into fragments, HIFU3 was detected by Elisa, freeze-thaw, freezing and thawing. The level of IL-12 IFN- 纬 in supernatant of CT26 group was significantly higher than that in control group (P < 0.05), and the content of IL-12 and IFN- 纬 in supernatant of freeze-thaw group was significantly higher than that in CT26 group (P < 0.05). Conclusion: bone marrow cells of mice were cultured with IL-4 combined with GM-CSF. The cultured DC could inactivate tumor cells, and the tumor antigens prepared by HIFU could sensitize DCin vitro, and make DC mature and secrete a large amount of IL-12, and induce T cells to secrete a large amount of IFN- 纬.
【学位授予单位】：泰山医学院
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392

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