炎症微环境在炎症性结肠癌发生发展过程中的关键作用及机制解析
发布时间：2018-08-21 20:07
【摘要】：[背景]慢性炎症是肿瘤发生发展的主要驱动力。溃疡性结肠炎是慢性炎症性肠病的一种主要形式。与正常人群相比,溃疡性结肠炎病人结肠癌发生率明显增高,并且与其炎症病变的范围、持续时间以及病变程度密切相关。免疫细胞的浸润,炎性因子,趋化因子及其受体,基质金属蛋白酶等构成的炎症微环境对炎症性结肠癌的发生发展起到了关键作用。我们前期的实验结果表明,肿瘤相关中性粒细胞(TANs)经CXCR2-CXCL2趋化轴介导,向结肠炎症部位聚集,促进炎症性肠癌的发生和发展。同时,我们的研究还发现,在建立的小鼠炎症性肠癌模型中,结肠部位有大量的成纤维细胞浸润。据相关报道显示,肿瘤相关成纤维细胞作为肿瘤基质的主要成分对肿瘤的发生和发展起到非常重要的作用,而其在结肠癌中的作用机制还不十分清楚。[目的]1.建立小鼠炎症性结肠癌(CAC)模型,后期给予中性粒细胞表面抗原Ly6G抗体干预,通过阻断中性粒细胞向病变结肠组织聚集,拮抗炎症性肠癌的发展,进一步验证肿瘤相关中性粒细胞(TANs)在CAC发生和发展过程中的关键作用。2.探讨肿瘤相关成纤维细胞(CAFs)在结肠癌发生发展中的作用,阐明FGF1/3-FGFR4信号通路在结直肠癌形成过程中的作用机制。[方法]1.建立AOM/DSS动物模型,在模型建立第56天,随机分为两组,一组连续7天尾静脉注射抗中性粒细胞表面抗原抗体Ly6G即治疗组,另一组连续7天尾静脉注射对照IgG抗体(对照组)。第0、56、67天行乙醚麻醉,脱臼安乐处死小鼠,取结直肠组织保存备用。qRT-PCR法检测治疗前后CXCL2、CXCR2、MMP-9以及中性粒细胞弹性蛋白酶NE的mRNA水平的变化；用HE染色法检测治疗前后结肠组织结构变化；用免疫荧光染色方法检测治疗前后中性粒细胞浸润的变化、中性粒细胞弹性蛋白酶NE蛋白水平的变化；用免疫组织化学染色方法检测治疗前后CXCL2、CXCR2、CD31、 PCNA、MMP-9蛋白水平的变化。2.用免疫组织化学染色方法检测CAC模型0、14、28、35、56天时成纤维细胞,巨噬细胞,T淋巴细胞浸润的动态变化,血管形成指标MMP-7蛋白水平的变化；qRT-PCR法检测各阶段细胞因子和趋化因子mRNA水平的变化,Western-blot检测各个阶段增殖相关指标p-Erk, Erk蛋白水平的变化。3.体外实验选取结肠癌病人的正常组织、癌旁组织、癌组织体外分离培养成纤维细胞,qRT-PCR法检测三种细胞表达的生长因子FGF等mRNA水平的变化；将其上清与结肠癌细胞共培养,Western-Blot检测Mek/Erk、MMP-7的变化：使用受体FGFR4抑制剂和FGF重组蛋白处理结肠癌细胞,Western Blot检测上述相关信号分子的表达变化；CCK8及小管形成实验检测肿瘤相关成纤维细胞CAFs对血管内皮细胞生长的影响。[结果]1.第67天处死小鼠后,与对照组相比较,抗体治疗组肉眼可见肿瘤减少(p0.01)；HE染色镜下可见,注射Ly6G抗体后,结肠肿瘤的组织形态及结构有所恢复。2.与对照组相比,治疗组结肠组织中性粒细胞的浸润明显减少(p0.01)、CXCL2, CXCL5和CXCR2的表达明显降低(p0.05),而且MMP-9表达显著降低(p0.01),新生血管明显减少(p0.01)；NE表达也明显减少(p0.01),细胞增殖活性显著下降(p0.01)。这些结果提示使用抗中性粒细胞表面抗原抗体可以逆转结肠癌的发展。3.在小鼠CAC模型中,成纤维细胞,巨噬细胞和T淋巴细胞的浸润明显增强,炎症因子IL-1α、IL-1β、IL-6、TNF-α在结肠癌形成过程中表达增加(p0.05),此外,CXCL-2及PDGF-α的表达也明显增加(p0.01)。血管形成指标MMP-7 (p0.01)、增殖相关信号分子p-Erk、Erk表达也显著增加。4.体外提取成纤维细胞纯度达90%以上,肿瘤相关成纤维细胞CAFs高表达FGF1和FGF3(p0.05)。将成纤维细胞的上清与结肠癌细胞共培养,与NFs, PFs相比,CAFs可促进结肠癌细胞的增殖,并且与CAFs细胞上清共培养的结肠癌细胞FGFR4自身磷酸化水平(p-Tyrosine) 、p-Mek/Erk、MMP-7的表达上调,而总FGFR4的表达没有明显变化。将成纤维细胞上清与血管内皮细胞共培养,CCK8和小管形成实验结果显示,与CAFs细胞上清共培养,可促进血管内皮细胞的增殖和迁移。5.在含有CAFs上清培养的结肠癌细胞中加入FGFR4抑制剂后,p-Tyrosine, p-Mek、p-Erk、MMP-7水平明显减少。6.在结肠癌细胞中加入重组蛋白FGF-1和FGF-3,Western Blot实验结果显示p-Tyrosine, p-MeK、p-ErK、MMP-7表达显著上调。[结论]综上所述,我们的实验结果揭示：1.注射抗中性粒细胞表面抗原抗体Ly6G可以逆转结肠癌的发展,进一步验证了肿瘤相关中性粒细胞促进炎症性肠癌的发生及发展。2.CAFs通过FGF1/3-FGFR4信号通路,上调结肠癌细胞Mek/Erk增殖信号分子的表达,上调MMP-7的表达,进而促进肿瘤细胞增殖和血管新生,从而导致结肠癌的发生和发展。
[Abstract]:[BACKGROUND] Chronic inflammation is the main driving force for the occurrence and development of tumors. Ulcerative colitis is a major form of chronic inflammatory bowel disease. Compared with normal people, the incidence of colon cancer in patients with ulcerative colitis is significantly higher, and is closely related to the extent, duration and degree of inflammation. Inflammatory microenvironment composed of inflammatory factors, chemokines, chemokines and their receptors, matrix metalloproteinases and so on plays a key role in the development of inflammatory colorectal cancer. Our previous experimental results showed that tumor-associated neutrophils (TANs) aggregated to inflammatory sites of the colon via the CXCR2-CXCL2 chemotactic axis and promoted the development of inflammatory colorectal cancer. At the same time, our study also found that a large number of fibroblasts infiltrated the colon in the inflammatory bowel cancer model of mice. According to related reports, tumor-related fibroblasts as the main component of tumor matrix play a very important role in the occurrence and development of tumors, and their role in colon cancer. [Objective] 1. To establish an inflammatory colon cancer (CAC) model in mice and interfere with neutrophil surface antigen Ly6G antibody in the later stage. By blocking the aggregation of neutrophils into the diseased colon tissues, we can antagonize the development of inflammatory colon cancer and further verify the occurrence and development of tumor-associated neutrophils (TANs) in CAC. Objective: To investigate the role of tumor-associated fibroblasts (CAFs) in the development of colorectal cancer and elucidate the mechanism of FGF1/3-FGFR4 signaling pathway in the development of colorectal cancer. Ly6G was injected into the tail vein of the treatment group and the control group for 7 consecutive days. The mice were sacrificed by ether anesthesia on the 0th, 56th and 67th day. The colorectal tissues were taken for preservation. The mRNA levels of CXCL2, CXCR2, MMP-9 and neutrophil elastase NE were detected by qRT-PCR before and after treatment. The changes of colon tissue structure before and after treatment were detected by staining method; the changes of neutrophil infiltration and neutrophil elastase NE protein level were detected by immunofluorescence staining method; the changes of CXCL2, CXCR2, CD31, PCNA and MMP-9 protein levels were detected by immunohistochemistry staining method before and after treatment. The dynamic changes of infiltration of fibroblasts, macrophages and T lymphocytes and the levels of MMP-7 protein were detected by histochemical staining at 0,14,28,35 and 56 days of CAC model. The levels of cytokines and chemokines mRNA were detected by qRT-PCR and the proliferation-related indexes p-Erk and Erk were detected by Western-blot. Changes in protein levels. 3. In vitro, fibroblasts were isolated and cultured from normal tissues, adjacent tissues and cancer tissues of patients with colon cancer, and the mRNA levels of growth factor FGF and other growth factors were detected by qRT-PCR. The supernatants were co-cultured with colon cancer cells, and the changes of Mek/Erk and MMP-7 were detected by Western-Blot. After the colon cancer cells were treated with somatic fibroblast growth factor receptor 4 inhibitor and recombinant fibroblast growth factor recombinant protein, the expression of the above-mentioned signal molecules was detected by Western Blot, and the effect of CAFs on the growth of vascular endothelial cells was detected by CCK8 and tubule formation assay. [Results] 1. After the mice were sacrificed on the 67th day, the antibody treatment group was compared with the control group. Compared with the control group, the infiltration of neutrophils in the colon tissue of the treatment group was significantly reduced (p0.01), the expression of CXCL2, CXCL5 and CXCR2 was significantly decreased (p0.05), and the expression of MMP-9 was significantly decreased (p0.01), and the neovascularization was obvious. These results suggest that the use of anti-neutrophil surface antigen antibodies can reverse the development of colon cancer. 3. In the mouse CAC model, the infiltration of fibroblasts, macrophages and T lymphocytes was significantly increased, and inflammatory factors IL-1a, IL-1beta, IL-6 were increased. In addition, the expression of CXCL-2 and PDGF-alpha was also significantly increased (p0.01). Angiogenesis markers MMP-7 (p0.01), proliferation-related signal molecules p-Erk and Erk were also significantly increased. 4. The purity of fibroblasts extracted in vitro was over 90%, and the expression of fibroblast growth factor 1 and fibroblast growth factor 3 (p0.01) in tumor-related fibroblasts CAFs was high. 05. Compared with NFs and PFs, CAFs could promote the proliferation of colon cancer cells, and the expression of FGFR4, p-Tyrosine, p-Mek/Erk and MMP-7 was up-regulated in co-cultured colon cancer cells with CFAS cell supernatant, while the expression of total fibroblast growth factor R4 was not changed significantly. CCK8 and vascular endothelial cells co-cultured with the supernatant of CAFs cells can promote the proliferation and migration of vascular endothelial cells. 5. The levels of p-Tyrosine, p-Mek, p-Erk, and MMP-7 in colon cancer cells cultured with CAFs supernatant were significantly reduced by adding FGFR4 inhibitor. The results of histone FGF-1 and FGF-3, Western Blot showed that the expression of p-Tyrosine, p-MeK, p-ErK and MMP-7 was significantly up-regulated. [Conclusion] To sum up, our experimental results revealed that: 1. Injection of anti-neutrophil surface antigen antibody Ly6G can reverse the development of colon cancer, further verify that tumor-associated neutrophils promote inflammatory bowel cancer. CAFs up-regulate the expression of Mek/Erk proliferation signal molecule and up-regulate the expression of MMP-7 through the FGF1/3-FGFR4 signaling pathway, thereby promoting the proliferation and angiogenesis of tumor cells, leading to the occurrence and development of colon cancer.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R735.35
[Abstract]:[BACKGROUND] Chronic inflammation is the main driving force for the occurrence and development of tumors. Ulcerative colitis is a major form of chronic inflammatory bowel disease. Compared with normal people, the incidence of colon cancer in patients with ulcerative colitis is significantly higher, and is closely related to the extent, duration and degree of inflammation. Inflammatory microenvironment composed of inflammatory factors, chemokines, chemokines and their receptors, matrix metalloproteinases and so on plays a key role in the development of inflammatory colorectal cancer. Our previous experimental results showed that tumor-associated neutrophils (TANs) aggregated to inflammatory sites of the colon via the CXCR2-CXCL2 chemotactic axis and promoted the development of inflammatory colorectal cancer. At the same time, our study also found that a large number of fibroblasts infiltrated the colon in the inflammatory bowel cancer model of mice. According to related reports, tumor-related fibroblasts as the main component of tumor matrix play a very important role in the occurrence and development of tumors, and their role in colon cancer. [Objective] 1. To establish an inflammatory colon cancer (CAC) model in mice and interfere with neutrophil surface antigen Ly6G antibody in the later stage. By blocking the aggregation of neutrophils into the diseased colon tissues, we can antagonize the development of inflammatory colon cancer and further verify the occurrence and development of tumor-associated neutrophils (TANs) in CAC. Objective: To investigate the role of tumor-associated fibroblasts (CAFs) in the development of colorectal cancer and elucidate the mechanism of FGF1/3-FGFR4 signaling pathway in the development of colorectal cancer. Ly6G was injected into the tail vein of the treatment group and the control group for 7 consecutive days. The mice were sacrificed by ether anesthesia on the 0th, 56th and 67th day. The colorectal tissues were taken for preservation. The mRNA levels of CXCL2, CXCR2, MMP-9 and neutrophil elastase NE were detected by qRT-PCR before and after treatment. The changes of colon tissue structure before and after treatment were detected by staining method; the changes of neutrophil infiltration and neutrophil elastase NE protein level were detected by immunofluorescence staining method; the changes of CXCL2, CXCR2, CD31, PCNA and MMP-9 protein levels were detected by immunohistochemistry staining method before and after treatment. The dynamic changes of infiltration of fibroblasts, macrophages and T lymphocytes and the levels of MMP-7 protein were detected by histochemical staining at 0,14,28,35 and 56 days of CAC model. The levels of cytokines and chemokines mRNA were detected by qRT-PCR and the proliferation-related indexes p-Erk and Erk were detected by Western-blot. Changes in protein levels. 3. In vitro, fibroblasts were isolated and cultured from normal tissues, adjacent tissues and cancer tissues of patients with colon cancer, and the mRNA levels of growth factor FGF and other growth factors were detected by qRT-PCR. The supernatants were co-cultured with colon cancer cells, and the changes of Mek/Erk and MMP-7 were detected by Western-Blot. After the colon cancer cells were treated with somatic fibroblast growth factor receptor 4 inhibitor and recombinant fibroblast growth factor recombinant protein, the expression of the above-mentioned signal molecules was detected by Western Blot, and the effect of CAFs on the growth of vascular endothelial cells was detected by CCK8 and tubule formation assay. [Results] 1. After the mice were sacrificed on the 67th day, the antibody treatment group was compared with the control group. Compared with the control group, the infiltration of neutrophils in the colon tissue of the treatment group was significantly reduced (p0.01), the expression of CXCL2, CXCL5 and CXCR2 was significantly decreased (p0.05), and the expression of MMP-9 was significantly decreased (p0.01), and the neovascularization was obvious. These results suggest that the use of anti-neutrophil surface antigen antibodies can reverse the development of colon cancer. 3. In the mouse CAC model, the infiltration of fibroblasts, macrophages and T lymphocytes was significantly increased, and inflammatory factors IL-1a, IL-1beta, IL-6 were increased. In addition, the expression of CXCL-2 and PDGF-alpha was also significantly increased (p0.01). Angiogenesis markers MMP-7 (p0.01), proliferation-related signal molecules p-Erk and Erk were also significantly increased. 4. The purity of fibroblasts extracted in vitro was over 90%, and the expression of fibroblast growth factor 1 and fibroblast growth factor 3 (p0.01) in tumor-related fibroblasts CAFs was high. 05. Compared with NFs and PFs, CAFs could promote the proliferation of colon cancer cells, and the expression of FGFR4, p-Tyrosine, p-Mek/Erk and MMP-7 was up-regulated in co-cultured colon cancer cells with CFAS cell supernatant, while the expression of total fibroblast growth factor R4 was not changed significantly. CCK8 and vascular endothelial cells co-cultured with the supernatant of CAFs cells can promote the proliferation and migration of vascular endothelial cells. 5. The levels of p-Tyrosine, p-Mek, p-Erk, and MMP-7 in colon cancer cells cultured with CAFs supernatant were significantly reduced by adding FGFR4 inhibitor. The results of histone FGF-1 and FGF-3, Western Blot showed that the expression of p-Tyrosine, p-MeK, p-ErK and MMP-7 was significantly up-regulated. [Conclusion] To sum up, our experimental results revealed that: 1. Injection of anti-neutrophil surface antigen antibody Ly6G can reverse the development of colon cancer, further verify that tumor-associated neutrophils promote inflammatory bowel cancer. CAFs up-regulate the expression of Mek/Erk proliferation signal molecule and up-regulate the expression of MMP-7 through the FGF1/3-FGFR4 signaling pathway, thereby promoting the proliferation and angiogenesis of tumor cells, leading to the occurrence and development of colon cancer.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R735.35
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