调节性T细胞介导的肝内免疫微环境改变对小鼠移植肝内肿瘤生长的影响及其机制研究

发布时间：2018-05-04 08:56

本文选题：小鼠肝移植 + 肝转移瘤模型　；参考：《浙江大学》2012年硕士论文

【摘要】：背景：目前已知调节性T细胞(Tregs)在移植和肿瘤免疫中都发挥了重要的作用。但是,调节性T细胞在肝移植(LT)后对肝内肿瘤的确切影响还不甚清楚。本实验的目的在于研究小鼠肝移植后移植肝内免疫微环境改变对移植肝内肿瘤生长的影响,以期找到预防和控制临床肝移植后肿瘤复发的新方法。方法：本实验采用小鼠肝移植后转移瘤模型,在体内水平研究了移植肝内调节性T细胞介导的肝内免疫微环境改变对移植肝内肿瘤生长的影响；体外肿瘤-淋巴细胞共培养实验被用于检测不同基因背景的淋巴细胞对肿瘤的杀伤能力的差异；一系列免疫组化染色被用于检测和分析移植肝内浸润的淋巴细胞群及两组间的改变；RT-PCR和ELISA被用于检测Tregs和效应性T细胞(Teffs)功能相关的各种调控因子的水平；流式细胞胞内染色用于检测活体Tregs和Teffs的功能状态；淋巴细胞体外混合(Tregs-Teffs)和荷瘤裸鼠过继免疫实验用于验证不同基因背景的Tregs对Teff增殖／凋亡以及肿瘤杀伤能力的影响。结果：异基因小鼠肝移植后,移植肝内的转移瘤生长快于同基因移植组；异基因移植肝中浸润的Teffs功能下调,无法有效杀伤肿瘤细胞；Tregs在异基因移植肝内浸润增多,Tregs的各种免疫负向调节介质表达上调；调节性T细胞通过下调效应T细胞细胞毒性介质及炎性细胞因子的释放,促进效应T细胞凋亡和死亡；体外淋巴细胞共培养实验和荷瘤裸鼠过继免疫实验证实异基因来源的Tregs可以明显促进Teffs凋亡及抑制其增殖,同时可以抑制其肿瘤杀伤能力。结论：异基因小鼠肝移植促进了肿瘤在移植肝中的生长；移植肝中Tregs浸润增多并伴随负向调控功能增强；Tregs通过减弱Teffs的免疫杀伤功能、促进效应T细胞的凋亡等机制为移植肝内的肿瘤提供了免疫庇护；控制和干预调节性T细胞在肝移植后介导的肿瘤免疫逃逸可能成为临床肝移植后预防肿瘤复发的新方法。
[Abstract]:Background: Regulatory T cell Tregs have been known to play an important role in transplantation and tumor immunity. However, the exact effect of regulatory T cells on intrahepatic tumors after liver transplantation is unclear. The purpose of this study was to study the effect of immune microenvironment on tumor growth after liver transplantation in mice, and to find a new method to prevent and control tumor recurrence after liver transplantation. Methods: In this study, the effects of regulatory T cell mediated intrahepatic immune microenvironment changes on the growth of transplanted liver tumors were studied in vivo using a mouse model of metastatic tumor after liver transplantation. In vitro tumor-lymphocyte co-culture was used to detect the cytotoxicity of lymphocytes from different gene backgrounds to tumor. A series of immunohistochemical staining was used to detect and analyze the infiltrating lymphocyte groups in the transplanted liver and the changes between the two groups. RT-PCR and ELISA were used to detect the levels of various regulatory factors related to the function of Tregs and effectual T cell Teffs. Flow cytometry was used to detect the functional status of Tregs and Teffs in vivo, and Tregs-Teffs in vitro and adoptive immunoassay in nude mice bearing tumor were used to test the effects of Tregs with different gene backgrounds on Teff proliferation / apoptosis and tumor killing ability. Results: After allogeneic liver transplantation, the growth of metastatic tumor in allogeneic mice was faster than that in allogeneic transplantation group, and the function of infiltrating Teffs in allogeneic transplanted liver was down-regulated. The expression of Tregs was up-regulated by various immunoregulatory mediators which could not effectively kill tumor cells in allogeneic liver transplantation, and regulatory T cells down-regulated the toxic mediators of T cells and the release of inflammatory cytokines. The co-culture of lymphocytes and adoptive immunoassay in nude mice showed that allogeneic Tregs could significantly promote the apoptosis and inhibit the proliferation of Teffs and inhibit its tumor killing ability. Conclusion: Allogeneic liver transplantation promoted the growth of tumor in the transplanted liver, and the infiltration of Tregs in the transplanted liver was increased, and the negative regulatory function was enhanced by weakening the immune and killing function of Teffs. The mechanism of promoting the apoptosis of effector T cells provides immune shelter for tumor in liver transplantation, and controlling and intervening the immune escape of tumor mediated by regulatory T cells after liver transplantation may become a new method to prevent tumor recurrence after clinical liver transplantation.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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