A549细胞受照后诱导的树突状细胞的抗肿瘤免疫研究

发布时间：2018-05-10 21:16

本文选题：肺癌 + 树突状细胞　；参考：《泰山医学院》2011年硕士论文

【摘要】：目的肺癌作为全球范围内对人们健康威胁最大的肿瘤之一,辐射及化学药物诱导细胞死亡(包括细胞凋亡和坏死)是目前已十分明确的一种治疗癌症的重要手段。近年来,随着免疫学的发展,已有研究表明,以高温、射线等手段处理肿瘤细胞引起其死亡的同时,肿瘤细胞表面表达可以引起机体免疫攻击的蛋白分子,启动免疫系统从而引起抗肿瘤免疫反应。通过研究以辐射诱导的方法制备能被DC更好利用的肿瘤抗原,观察并评价射线是否具有增强DC诱导的特异性CTL的抗肿瘤作用,以明确射线诱导细胞死亡与免疫应答之间的相互作用过程及发生机制,为探索辐射联合生物治疗恶性肿瘤的新模式提供实验依据。方法 1、体外利用PBMC联合应用rhIL-4和rhGM-CSF培养树突状细胞DC,并用流式细胞仪检测细胞表型。 2、采用Annexin-V-FITC/PI法检测体外不同剂量X线照射后A549细胞凋亡率。 3、不同浓度的抗原冲击未成熟DC,加入TNF-α促使imDC成熟,成熟后DC诱导T细胞产生特异性杀伤作用杀伤肿瘤细胞。 4、不同方法体外杀伤肿瘤细胞。 5、采用ELISA酶联免疫吸附实验检测各实验组培养上清液中IL-12、IL-2、IFN-γ分泌水平。结果 1、联合应用rhIL-4、rhGM-CSF与TNF-α。使用健康人外周血可诱导培养出成熟DC,细胞具有树突状细胞的典型形态,经流式检测证实获得的细胞为成熟DC。 2、A549细胞在一定照射剂量范围内,凋亡率随照射剂量的增加而升高。 3、采用不同浓度的抗原冲击未成熟DC,其成熟后诱导T细胞产生特异性杀伤肿瘤细胞的能力有所不同。DC与抗原浓度比为1:10-1时DC诱导的CTL作用最强(0.2446±0.0490, P0.01)。 4、辐射联合组与对照组、单纯照射组、无抗原组、冻融组相比可以更好的杀伤肿瘤细胞(p0.01)。 5、联合组培养上清液中IL-12、IL-2、IFN-γ明显高于对照组(P0.01)。结论辐射诱导肿瘤细胞凋亡联合DC介导的免疫应答以清除肿瘤细胞的方法,能有效的激活免疫效应细胞发挥作用,诱导免疫调节分子表达等一系列反应,并创造适宜的抗肿瘤微环境,从而达到更确切的杀伤肿瘤的效果,成为治疗肺癌的一条新途径。
[Abstract]:Purpose Lung cancer is one of the most dangerous tumors in the world. Radiation and chemotherapy-induced cell death (including apoptosis and necrosis) is an important method to treat cancer. In recent years, with the development of immunology, it has been shown that when tumor cells die due to high temperature and radiation, the tumor cells express protein molecules that can cause immune attack. Activate the immune system to trigger an anti-tumor immune response. By studying the method of radiation-induced preparation of tumor antigens which can be used better by DC, we observed and evaluated whether the radiation enhanced the anti-tumor effect of specific CTL induced by DC. In order to clarify the interaction process and mechanism between radiation-induced cell death and immune response, it provides experimental basis for exploring a new mode of radiation combined with biological therapy for malignant tumors. Method 1. Dendritic cells were cultured by PBMC combined with rhIL-4 and rhGM-CSF in vitro, and the phenotypes were detected by flow cytometry. 2. Annexin-V-FITC/PI assay was used to detect the apoptosis rate of A549 cells after different doses of X-ray irradiation in vitro. (3) imDC maturation was induced by different concentrations of antigens, and T cells were induced to kill tumor cells by DC. 4. Different methods of killing tumor cells in vitro. 5. ELISA enzyme linked immunosorbent assay (Elisa) was used to detect the level of IL-12 and IL-2 IFN- 纬 in the supernatant of each experimental group. Result 1. RhIL-4 rhGM-CSF combined with TNF- 伪. Mature DCs can be induced by using healthy human peripheral blood. The cells have typical morphology of dendritic cells, and the cells obtained by flow cytometry are mature DCs. 2 the apoptosis rate of A549 cells increased with the increase of irradiation dose. 3. When different concentration of antigen was used to shock immature DCs, the ability of inducing T cells to produce specific cytotoxicity to tumor cells was different after maturation. When the ratio of DC to antigen concentration was 1: 10-1, DC could induce the strongest CTL effect (0.2446 卤0.0490, P 0.01). 4, radiation combined group and control group, irradiation group, no antigen group, freeze-thaw group can kill tumor cells better than p0.01. 5. The levels of IL-12 and IL-2 IFN- 纬 in the culture supernatant of the combined group were significantly higher than those in the control group (P 0.01). Conclusion Radiation induced apoptosis of tumor cells combined with immune response mediated by DC to clear tumor cells can effectively activate immune effector cells and induce a series of responses such as immunomodulatory molecule expression. It is a new way to treat lung cancer by creating appropriate antitumor microenvironment so as to achieve more accurate effect of killing tumor.
【学位授予单位】：泰山医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

【相似文献】