正常小鼠外周成熟T细胞回归胸腺及其功能表达的探究

发布时间：2018-05-26 16:07

本文选题：胸腺 + T淋巴细胞　；参考：《山东大学》2017年硕士论文

【摘要】：在对胸腺的功能研究中,一直把胸腺当作一个免疫"特赦"器官看待,抗原和大分子不能进入胸腺从而避免了胸腺内发生免疫反应。根据中枢耐受理论,如果在胸腺内植入外界抗原,那么该抗原将会被发育中的T细胞识别并对阳性选择和阴性选择产生影响进而形成耐受。根据我们先前的研究发现,当把肿瘤细胞注射到胸腺后,并没有诱导形成肿瘤细胞的耐受,肿瘤细胞最终会被清除。并且流式分析显示,在注射人肺癌细胞A549后,胸腺内CD4+、CD8+T细胞的数量增加。那么这些增多的淋巴细胞会不会是来自于外周呢?在给免疫缺陷小鼠移植T细胞的实验中发现,异体的成熟T细胞可以进入胸腺,但正常小鼠的外周成熟T细胞是否也具有这种现象还不确定。因此针对以上问题,我们选用正常小鼠作为实验对象,首先在其脾脏中注射CFDA SE荧光染料,通过流式检测胸腺内是否含有表达CFDA SE荧光信号的细胞,检测外周T细胞能迁回胸腺;其次我们在外周免疫过人肺癌细胞A549抗原的小鼠和未免疫小鼠的胸腺中分别注射等量的A549细胞,通过石蜡切片和流式细胞术分析对比胸腺内发生的免疫反应、抗原细胞在胸腺内的状态以及T细胞亚群含量的变化,并对发生于胸腺的应答反应做进一步探究。通过以上实验,我们得到以下结果:1.胸腺内可以检测到CFDA SE阳性细胞,并且这些细胞绝大多数呈CD3阳性;2.石蜡切片结果显示,免疫小鼠的胸腺内发生的免疫反应较未免疫小鼠更加剧烈,胸腺在相对较短的时间内即可恢复正常;3.流式分析结果显示,与未免疫小鼠相比,A549细胞在免疫小鼠胸腺内的存活时间缩短;4.胸腺微创注射后第5天,外周免疫小鼠和未免疫小鼠与未进行胸腺微创注射的小鼠相比,胸腺中CD4+和CD8+T细胞的数量都明显增加,但免疫小鼠的胸腺中CD8+/CD4+的比值较未免疫小鼠显著升高,而未免疫小鼠与未进行胸腺微创注射的小鼠之间无明显差异。通过以上结果,我们得出以下结论:1.正常小鼠的胸腺内存在来自外周的T细胞。这种回迁现象不仅发生于免疫缺陷小鼠,也发生于正常小鼠。2.用抗原外周免疫后,当胸腺内再次出现相同抗原时,胸腺内会发生更加及时、剧烈的免疫反应。根据以上结论,我们推测外周免疫后一部分记忆T细胞进入胸腺,当再次遇到相应抗原时,记忆T细胞迅速增殖并对抗原产生记忆性杀伤。外周成熟T细胞回归胸腺可能是机体保护胸腺免于抗原感染的一种正常机制,防止机体形成对外来抗原的耐受。
[Abstract]:In the study of thymus function, the thymus has been regarded as an immune "amnesty" organ. Antigens and macromolecules can not enter the thymus, thus avoiding the immune reaction in the thymus. According to the central tolerance theory, if an external antigen is implanted in the thymus, the antigen will be recognized by the developing T cells and will affect the positive and negative selection, thus forming tolerance. According to our previous studies, when the tumor cells were injected into the thymus, the tumor cells were not induced to develop tolerance, and the tumor cells were eventually eliminated. Flow analysis showed that the number of CD4 CD8 T cells in thymus increased after injection of human lung cancer cell line A549. So are these increased lymphocytes coming from the periphery? It was found that allogeneic mature T cells could enter the thymus in immunodeficient mice, but it was uncertain whether the peripheral mature T cells in normal mice had the same phenomenon. To solve the above problems, we selected normal mice as the experimental object. First, we injected CFDA SE fluorescent dye into the spleen, and detected whether there were any cells expressing CFDA SE fluorescence signal in the thymus by flow cytometry. Then we injected the same amount of A549 cells into the thymus of mice immunized with human lung cancer cell A549 antigen and non-immunized mice respectively. The immune reaction in thymus, the state of antigen cells in thymus and the content of T cell subsets were analyzed and compared by paraffin section and flow cytometry, and the response reaction occurred in thymus was further explored. Through the above experiment, we get the following result: 1. CFDA SE positive cells can be detected in thymus, and most of these cells are CD3 positive. The results of paraffin section showed that the immune reaction in thymus of immunized mice was more intense than that of unimmunized mice, and the thymus could return to normal in a relatively short time. The results of flow cytometry showed that the survival time of A549 cells in thymus of immunized mice was shorter than that of unimmunized mice. On the 5th day after microinvasive thymus injection, the number of CD4 and CD8 T cells in thymus of peripheral immunized mice and unimmunized mice were significantly increased compared with those of mice without thymus microinvasive injection. However, the ratio of CD8 / CD4 in thymus of immunized mice was significantly higher than that of unimmunized mice, but there was no significant difference between the unimmunized mice and the mice without thymus microinvasive injection. From the above results, we draw the following conclusion: 1. In normal mice thymus there are T cells from the periphery. This migration occurs not only in immunodeficient mice, but also in normal mice. After peripheral immunization with antigens, when the same antigen appears again in the thymus, there will be a more timely and intense immune response in the thymus. According to the above conclusions, we speculate that a part of memory T cells enter the thymus after peripheral immunization. When the corresponding antigens are encountered again, the memory T cells proliferate rapidly and produce memory killing to the antigens. The return of peripheral mature T cells to thymus may be a normal mechanism to protect thymus from antigen infection and prevent the formation of tolerance to foreign antigens.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R392

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