结直肠癌细胞EGFR信号通路调节巨噬细胞极性的机制研究

发布时间：2018-07-07 17:32

本文选题：TAM + 肿瘤微环境　；参考：《北京协和医学院》2016年博士论文

【摘要】：机体内肿瘤细胞不是孤立存在的,肿瘤细胞与肿瘤相关成纤维细胞、内皮细胞、免疫细胞、巨噬细胞以及细胞间的因子、基质金属蛋白酶(matrix metalloproteinases, MMPs)组分、细胞外基质(Extracellular matrix, ECM)等共同构成了肿瘤微环境。肿瘤微环境不仅对肿瘤的发生发展及侵袭转移至关重要,而且也会影响肿瘤的放化疗效果。同时放化疗也可以影响肿瘤微环境。肿瘤相关巨噬细胞(tumor-associated macrophages, TAM)是肿瘤微环境中的主要细胞成分,TAM通过分泌生长因子和基质金属蛋白酶、提高血管的生成能力等促进肿瘤生长。因此,靶向TAM治疗可以加强肿瘤的治疗效果。本研究主要探讨靶向表皮生长因子受体EGFR(epidermal growth factor receptor)治疗除了直接抑制肿瘤细胞生长之外,如何改变肿瘤微环境,进而影响到肿瘤相关巨噬细胞的极性。首先构建AOM/DSS炎症相关结肠癌动物模型,利用EGFR单抗cetuximab进行治疗,可以明显抑制结直肠肿瘤的生长。Western blot结果表明,在小鼠肿瘤组织中EGFR和p-EGFR(Y1068)蛋白表达水平升高,同时精氨酸酶-1(arginase-1, Arg1)和可诱导的一氧化氮合酶(inducible nitric oxide synthase, iNOS)蛋白表达水平也升高。当利用cetuximab治疗后,组织中EGFR、p-EGFR(Y1068)和Argl蛋白表达水平与非治疗组相比显著下降,而iNOS蛋白表达水平没有明显变化。此外,流式检测结果也表明,cetuximab治疗组中总巨噬细胞F4/80+/CD11b+和M2巨噬细胞F4/80+/CD206+数量与未治疗组相比,显著降低。这说明cetuximab不仅可以抑制肿瘤细胞生长,而且也降低了总巨噬细胞和M2巨噬细胞的数量。由于在巨噬细胞中未检测到EGFR的表达,因此,cetuximab可能是通过改变肿瘤细胞外泌蛋白进而调控巨噬细胞极性。在细胞模型中利用HCT116和SW480细胞培养上清可以诱导Ana-1细胞向M2型巨噬细胞极化,其M2型相关分子标志物Arg1、CD206、IL-4等表达升高。敲降HCT1 16和SW480细胞的EGFR后,上述两种细胞培养上清诱导Ana-1向M2型极化能力减弱,其M2型相关分子标志物表达下降,而M1型相关分子标志物表达升高。此外,裸鼠成瘤实验结果也表明M2型巨噬细胞可以促进肿瘤细胞的生长,而EGFR敲除可以抑制巨噬细胞向M2型极化。为了探究敲降EGFR前后结肠癌细胞培养上清中诱导巨噬细胞极化的具体成分,我们利用细胞因子芯片检测了敲降EGFR前后结肠癌细胞培养上清中分泌的细胞因子情况,发现IGF-1细胞因子在EGFR敲降后的细胞培养上清中显著下降,ELISA结果也证实了EGFR表达与IGF-1分泌具有相关性。利用IGF-1可以诱导Ana-1细胞Argl表达水平升高。此外,通过IGF1R的特异性抑制剂AG1024抑制Anna-1细胞的IGF1R信号通路后,肿瘤细胞上清诱导巨噬细胞向M2极化能力减弱。这些结果提示肿瘤细胞中EGFR信号通路的改变,影响细胞因子IGF-1的分泌,同时外泌的IGF-1在诱导巨噬细胞极化过程中起着关键作用。综上所述,靶向EGFR治疗可以抑制巨噬细胞向M2型极化,进一步抑制肿瘤生长。细胞因子IGF-1在结肠癌细胞培养上清诱导巨噬细胞极化过程中起着重要作用。我们的结果揭示肿瘤细胞培养上清诱导巨噬细胞极化的可能机制,同时也提示抑制巨噬细胞极化或许能成为一种新的有前途的肿瘤治疗方式。
[Abstract]:Tumor cells are not isolated in isolation. Tumor cells and tumor related fibroblasts, endothelial cells, immune cells, macrophages and intercellular factors, matrix metalloproteinases (matrix metalloproteinases, MMPs) components, extracellular matrix (Extracellular matrix, ECM) together constitute the tumor microenvironment. Tumor microring It is not only essential for tumor development and invasion and metastasis, but also affects the effect of radiotherapy and chemotherapy. At the same time, chemoradiotherapy can also affect the microenvironment of tumor. Tumor-associated macrophages (TAM) is the main cell component in the tumor microenvironment. TAM is used to secrete growth factors and matrix metal eggs. White enzyme, enhancing angiogenesis, and so on promoting tumor growth. Therefore, targeted TAM therapy can enhance the therapeutic effect of tumor. This study mainly discusses how the target epidermal growth factor receptor EGFR (epidermal growth factor receptor) can change the tumor microenvironment, and then affect the tumor, in addition to the direct inhibition of tumor cell growth. The polarity of related macrophages. First, the AOM/DSS inflammation related colon cancer animal model was constructed and the EGFR monoclonal antibody cetuximab was used to inhibit the growth of the colorectal tumor. The.Western blot results showed that the expression of EGFR and p-EGFR (Y1068) protein in the tumor tissues of mice increased, and the arginase -1 (arginase-1, Arg1) was at the same time. The expression level of the inducible nitric oxide synthase (inducible nitric oxide synthase, iNOS) protein also increased. When cetuximab was used, the expression level of EGFR, p-EGFR (Y1068) and Argl proteins in the tissues decreased significantly compared with those in the non treatment group, but the expression level of iNOS protein was not significantly changed. Furthermore, the flow test results also showed that cetuximab was the same. The number of F4/80+/CD11b+ and M2 macrophages F4/80+/CD206+ in the total macrophages in the treatment group was significantly lower than that in the untreated group. This indicates that cetuximab not only inhibits the growth of tumor cells, but also reduces the number of total macrophages and M2 macrophages. Since the expression of EGFR is not detected in the macrophages, cetuximab may be possible. HCT116 and SW480 cell culture supernatant can induce the polarization of Ana-1 cells to M2 type macrophages in the cell model, and the expression of M2 related molecular markers Arg1, CD206, IL-4 and so on. After knocking down HCT1 16 and SW480 cells, the above two cells are cultured. The supernatant induced the decrease of Ana-1 type polarization to M2 type, and the expression of M2 type related molecular markers decreased, while the expression of M1 related molecular markers increased. In addition, the results of tumor formation in nude mice showed that M2 macrophages could promote the growth of tumor cells, and EGFR knockout could inhibit the polarization of macrophage to M2 type. In order to explore the knockdown EGFR In the culture supernatant of colon cancer cells, the specific components of the macrophage polarization were induced. We detected the cytokines secreted in the culture supernatant of colon cancer cells before and after the knock down EGFR by the cytokine chip, and found that the IGF-1 cytokine decreased significantly in the cell culture supernatant after the EGFR knock down, and the ELISA results also confirmed the expression of EGFR and IGF- 1 secretion is related. The use of IGF-1 can induce the increase of Argl expression in Ana-1 cells. In addition, after the IGF1R specific inhibitor AG1024 inhibits the IGF1R signaling pathway of Anna-1 cells, the cell supernatant induces the decrease of macrophage's polarization to M2. These results suggest that the EGFR signaling pathway changes in the tumor cells and affects the cells. The secretion of factor IGF-1 and the simultaneous secretion of IGF-1 play a key role in inducing the polarization of macrophages. To sum up, targeted EGFR therapy can inhibit the M2 type polarization of macrophages and further inhibit the growth of tumor. Cytokine IGF-1 plays an important role in the process of inducing macrophage polarization in colon cancer cell culture supernatant. The results reveal the possible mechanism of macrophage polarization induced by the culture supernatant of tumor cells, and also suggest that inhibiting the polarization of macrophages may be a promising new method for cancer treatment.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.34

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