γδT细胞体外扩增，i-IELs分离和免疫荧光技术的建立

发布时间：2019-04-03 13:47

【摘要】：目的：免疫实验技术是从事免疫研究工作的基石,是实验进度得以维持的保证。因此我把建立免疫实验技术做为自己硕士研究生期间的重要工作。在三年的学习工作中,我主要建立了三套免疫实验技术,它们分别为：小鼠γδT细胞的体外扩增技术；小鼠小肠上皮淋巴细胞(i-IELs)的分离技术以及免疫荧光技术在肿瘤微环境研究中的应用。 1.小鼠γδT细胞的体外扩增技术主要解决的是小鼠外周淋巴系统中γδT细胞稀少,无法获得足够的细胞用以完成小鼠淋巴细胞回输重构实验的问题。使用的方法是利用抗γδTCR和CD28的抗体包板,在IL-2的协同下对γδT细胞进行刺激,从而实现体外增殖。 2.小鼠小肠上皮淋巴细胞(i-IELs)的分离技术主要解决的是无法有效研究肠道上皮中γδT细胞的问题。方法依赖在缺氧环境下,小肠上皮容易脱离且EDTA具有螯合钙、镁离子的特性,通过物理方法完成小鼠小肠上皮淋巴细胞(i-IELs)的分离。分离得到的细胞可以按照实验要求进行后续实验。 3.免疫荧光技术主要解决的是研究Vγ1γδT细胞和Vγ4γδT细胞在皮下黑色素瘤中共定位的问题。问题的主要难点在于：皮下黑色素瘤的摘取,组织的预处理,冰冻切片的最适温度以及荧光抗体的选择。通过解决这一技术问题,不仅满足了研究γδT细胞亚群在皮下黑色素瘤中定位的需要,还满足了研究CD4阳性T细胞,CD8阳性T细胞以及树突状细胞在其他类型肿瘤中的浸润情况的需求。
[Abstract]:Objective: immune experimental technology is the cornerstone of immune research and the guarantee to maintain the progress of the experiment. Therefore, I take the establishment of immunization experiment technology as my master's degree during the important work. During my three years of study, I have mainly set up three sets of immune test techniques, which are: amplification of mouse 纬未 T cells in vitro; Isolation of mouse small intestinal epithelial lymphocytes (i-IELs) and application of immunofluorescence in the study of tumor microenvironment. 1. In vitro amplification of mouse 纬未 T cells mainly solves the problem of the scarcity of 纬未 T cells in the peripheral lymphoid system of mice and the inability to obtain enough cells to complete the retransmission and remodeling experiment of mouse lymphocytes. The anti-纬未 TCR and CD28 antibodies were used to stimulate 纬未 T cells in combination with IL-2, so that the proliferation of 纬未 T cells could be realized in vitro. 2. The separation of mouse intestinal epithelial lymphocytes (i-IELs) mainly solves the problem that 纬未 T cells in intestinal epithelium cannot be studied effectively. Methods the intestinal epithelial cells (i-IELs) of mice were isolated by physical method depending on the characteristics of EDTA chelating calcium and magnesium ions. The isolated cells can be followed up according to the experimental requirements. 3. Immunofluorescence technique mainly solves the problem of co-localization of V 纬 1 纬未 T cells and V 纬 4 纬未 T cells in subcutaneous melanoma. The main difficulties are: extraction of subcutaneous melanoma, pretreatment of tissue, optimum temperature of frozen section and selection of fluorescent antibody. By solving this technical problem, not only the need to study the localization of 纬未 T cell subsets in subcutaneous melanoma, but also the study of CD4 positive T cells. Demand for infiltration of CD8-positive T cells and dendritic cells in other types of tumors.
【学位授予单位】：南开大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

【共引文献】