调节性T细胞通过TGFβ-IL6-STAT3信号通路促进脑胶质瘤细胞的干性

发布时间：2018-03-28 19:13

本文选题：脑胶质瘤　切入点：肿瘤细胞干性　出处：《郑州大学》2017年硕士论文

【摘要】：目的脑胶质瘤(Glioma)是颅内常见的恶性肿瘤之一,发病率呈逐年上升趋势,且因易复发,病人死亡率高。脑胶质瘤干细胞(Glioma stem cells,GSCs)是一群决定脑胶质瘤发生、发展、复发及耐药的关键细胞,因此寻找影响肿瘤细胞干性的机制具有重要的理论意义和实用价值。由各种基质细胞和免疫细胞及其分泌的细胞因子所组成的肿瘤微环境是GSCs存活和干性维持的基本条件。最新研究表明,肿瘤相关巨噬细胞(Tumor-associated macrophages,TAMs)和骨髓来源的抑制性细胞(Myeloid-derived suppressor cells,MDSCs)能够通过分泌细胞因子促进肿瘤细胞的干性,然而调节性T细胞(Regulatory T cells,Tregs)作为在肿瘤微环境部位发挥免疫抑制功能的一群重要免疫细胞,其与脑胶质瘤干细胞之间的相互作用机制研究仍不清楚。本研究目的是探讨Tregs与GSCs之间相互作用及详细机制。方法收集2013年6月至2016年6月就诊于郑州大学第一附属医院的脑胶质瘤患者癌组织与配对癌旁组织86对,采用荧光定量法检测CD133等基因表达水平;免疫组化法验证CD133在肿瘤组织中的高表达;收取脑胶质瘤患者的外周血,密度梯度离心法获得外周血单个核细胞(Peripheral Blood Mononuclear Cells,PBMCs),磁珠分选法获得纯化的Treg细胞;Treg细胞分别与脑胶质瘤细胞系U251、U87在Transwell板中共孵育;流式细胞术、荧光定量法和低粘附板成球法和检测孵育后肿瘤细胞干性的变化;多因子法筛选细胞因子TGF-β、IL-6;荧光定量和免疫荧光鉴定TGF-β的来源;Western-Blot技术检测信号通路上p-NF-κB、p-STAT3、CD133、SOX2等蛋白的表达水平,慢病毒转染法构建IL-6稳定敲低的细胞系。结果1.CD133在脑胶质瘤组织中表达明显高于癌旁组织,并且CD133的表达与病人的生存和预后呈负相关,CD133高表达的病人预后更差;2.Tregs能够通过分泌TGF-β促进脑胶质瘤细胞U251和U87干性基因和成球能力上调;3.TGF-β作用于肿瘤细胞后,通过激活ΝF-κB信号通路促进肿瘤细胞自分泌IL-6;4.当阻断肿瘤细胞分泌IL-6时,TGF-β促进肿瘤细胞的干性的能力也明显下调。并且IL-6的表达与病人的生存和预后呈负相关,IL-6低表达的病人具有一个较好的预后;4.肿瘤细胞自分泌的IL-6通过激活STAT3信号通路从而促进肿瘤细胞的干性。结论在脑胶质瘤肿瘤微环境中,Treg通过分泌TGF-β促进肿瘤细胞自分泌ΙL-6,从而促进肿瘤细胞干性,TGF-β和IL-6均可作为脑胶质瘤治疗的潜在靶点,为脑胶质瘤的临床治疗提供了坚实的理论依据。
[Abstract]:Objective Glioma (Glioma) is one of the most common malignant tumors in the brain. The incidence of Glioma is increasing year by year, and the mortality of patients is high because of its easy recurrence. Glioma stem cells are a group of gliomas that determine the occurrence and development of gliomas. Key cells for relapse and drug resistance, Therefore, it is of great theoretical significance and practical value to explore the mechanism of affecting the dryness of tumor cells. The tumor microenvironment composed of various stromal cells, immune cells and cytokines secreted by them is the survival and dry maintenance of GSCs. Basic conditions. Recent research shows that. Tumor-associated macrophages (Tumor-associated macrophages) and bone marrow-derived inhibitory cells (Myeloid-derived suppressor cells / MDSCs) can promote the dryness of tumor cells by secreting cytokines. However, regulatory T cells T cells Tregs are a group of important immune cells that play an immunosuppressive role in the tumor microenvironment. The purpose of this study was to explore the interaction between Tregs and GSCs and its detailed mechanism. Methods the first visit to Zhengzhou University from June 2013 to June 2016 was conducted. 86 pairs of brain glioma tissues and matched paracarcinomatous tissues in affiliated hospitals, Fluorescence quantitative method was used to detect the expression of CD133 and other genes, immunohistochemical method was used to verify the high expression of CD133 in tumor tissue, and peripheral blood was collected from glioma patients. Peripheral Blood Mononuclear cells of peripheral blood were obtained by density gradient centrifugation, and purified Treg cells were incubated with glioma cell line U251 U87 on Transwell plate, respectively. Fluorescence quantitative assay and low adhesion plate granulation assay were used to detect the changes of tumor cell dryness after incubation. The cytokines TGF- 尾 and IL-6 were screened by multifactor method. Western-Blot technique was used to detect the expression level of p-NF- 魏 Bmp-STAT3, CD133SOX2 and other proteins in the signaling pathway, and the origin of TGF- 尾 was identified by Western-Blot technique. IL-6 stable knockout cell lines were constructed by lentivirus transfection. Results the expression of 1.CD133 in gliomas was significantly higher than that in paracancerous tissues. Moreover, the expression of CD133 was negatively correlated with the survival and prognosis of the patients. The prognosis of patients with high expression of CD133 was even worse. 2. TGF- 尾 could promote the upregulation of the dry genes and the ability of granulation of glioma cells by secreting TGF- 尾. 3. TGF- 尾 acted on the tumor cells. The ability of TGF- 尾 to promote the dryness of tumor cells was also down-regulated when tumor cells were blocked from secreting IL-6. And the expression of IL-6 was negatively correlated with the survival and prognosis of patients. The autocrine IL-6 of tumor cells promotes the dryness of tumor cells by activating STAT3 signaling pathway. Conclusion in the microenvironment of glioma tumors, Treg promotes tumor cells by secreting TGF- 尾. Autocrine I L-6, thus promoting tumor cell dryness, TGF- 尾 and IL-6, may be potential targets for the treatment of glioma. It provides a solid theoretical basis for the clinical treatment of glioma.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.41

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