【摘要】：[背景]慢性炎癥是腫瘤發(fā)生發(fā)展的主要驅(qū)動(dòng)力。潰瘍性結(jié)腸炎是慢性炎癥性腸病的一種主要形式。與正常人群相比,潰瘍性結(jié)腸炎病人結(jié)腸癌發(fā)生率明顯增高,并且與其炎癥病變的范圍、持續(xù)時(shí)間以及病變程度密切相關(guān)。免疫細(xì)胞的浸潤,炎性因子,趨化因子及其受體,基質(zhì)金屬蛋白酶等構(gòu)成的炎癥微環(huán)境對(duì)炎癥性結(jié)腸癌的發(fā)生發(fā)展起到了關(guān)鍵作用。我們前期的實(shí)驗(yàn)結(jié)果表明,腫瘤相關(guān)中性粒細(xì)胞(TANs)經(jīng)CXCR2-CXCL2趨化軸介導(dǎo),向結(jié)腸炎癥部位聚集,促進(jìn)炎癥性腸癌的發(fā)生和發(fā)展。同時(shí),我們的研究還發(fā)現(xiàn),在建立的小鼠炎癥性腸癌模型中,結(jié)腸部位有大量的成纖維細(xì)胞浸潤。據(jù)相關(guān)報(bào)道顯示,腫瘤相關(guān)成纖維細(xì)胞作為腫瘤基質(zhì)的主要成分對(duì)腫瘤的發(fā)生和發(fā)展起到非常重要的作用,而其在結(jié)腸癌中的作用機(jī)制還不十分清楚。[目的]1.建立小鼠炎癥性結(jié)腸癌(CAC)模型,后期給予中性粒細(xì)胞表面抗原Ly6G抗體干預(yù),通過阻斷中性粒細(xì)胞向病變結(jié)腸組織聚集,拮抗炎癥性腸癌的發(fā)展,進(jìn)一步驗(yàn)證腫瘤相關(guān)中性粒細(xì)胞(TANs)在CAC發(fā)生和發(fā)展過程中的關(guān)鍵作用。2.探討腫瘤相關(guān)成纖維細(xì)胞(CAFs)在結(jié)腸癌發(fā)生發(fā)展中的作用,闡明FGF1/3-FGFR4信號(hào)通路在結(jié)直腸癌形成過程中的作用機(jī)制。[方法]1.建立AOM/DSS動(dòng)物模型,在模型建立第56天,隨機(jī)分為兩組,一組連續(xù)7天尾靜脈注射抗中性粒細(xì)胞表面抗原抗體Ly6G即治療組,另一組連續(xù)7天尾靜脈注射對(duì)照IgG抗體(對(duì)照組)。第0、56、67天行乙醚麻醉,脫臼安樂處死小鼠,取結(jié)直腸組織保存?zhèn)溆�。qRT-PCR法檢測治療前后CXCL2、CXCR2、MMP-9以及中性粒細(xì)胞彈性蛋白酶NE的mRNA水平的變化；用HE染色法檢測治療前后結(jié)腸組織結(jié)構(gòu)變化；用免疫熒光染色方法檢測治療前后中性粒細(xì)胞浸潤的變化、中性粒細(xì)胞彈性蛋白酶NE蛋白水平的變化；用免疫組織化學(xué)染色方法檢測治療前后CXCL2、CXCR2、CD31、 PCNA、MMP-9蛋白水平的變化。2.用免疫組織化學(xué)染色方法檢測CAC模型0、14、28、35、56天時(shí)成纖維細(xì)胞,巨噬細(xì)胞,T淋巴細(xì)胞浸潤的動(dòng)態(tài)變化,血管形成指標(biāo)MMP-7蛋白水平的變化；qRT-PCR法檢測各階段細(xì)胞因子和趨化因子mRNA水平的變化,Western-blot檢測各個(gè)階段增殖相關(guān)指標(biāo)p-Erk, Erk蛋白水平的變化。3.體外實(shí)驗(yàn)選取結(jié)腸癌病人的正常組織、癌旁組織、癌組織體外分離培養(yǎng)成纖維細(xì)胞,qRT-PCR法檢測三種細(xì)胞表達(dá)的生長因子FGF等mRNA水平的變化；將其上清與結(jié)腸癌細(xì)胞共培養(yǎng),Western-Blot檢測Mek/Erk、MMP-7的變化：使用受體FGFR4抑制劑和FGF重組蛋白處理結(jié)腸癌細(xì)胞,Western Blot檢測上述相關(guān)信號(hào)分子的表達(dá)變化；CCK8及小管形成實(shí)驗(yàn)檢測腫瘤相關(guān)成纖維細(xì)胞CAFs對(duì)血管內(nèi)皮細(xì)胞生長的影響。[結(jié)果]1.第67天處死小鼠后,與對(duì)照組相比較,抗體治療組肉眼可見腫瘤減少(p0.01)；HE染色鏡下可見,注射Ly6G抗體后,結(jié)腸腫瘤的組織形態(tài)及結(jié)構(gòu)有所恢復(fù)。2.與對(duì)照組相比,治療組結(jié)腸組織中性粒細(xì)胞的浸潤明顯減少(p0.01)、CXCL2, CXCL5和CXCR2的表達(dá)明顯降低(p0.05),而且MMP-9表達(dá)顯著降低(p0.01),新生血管明顯減少(p0.01)；NE表達(dá)也明顯減少(p0.01),細(xì)胞增殖活性顯著下降(p0.01)。這些結(jié)果提示使用抗中性粒細(xì)胞表面抗原抗體可以逆轉(zhuǎn)結(jié)腸癌的發(fā)展。3.在小鼠CAC模型中,成纖維細(xì)胞,巨噬細(xì)胞和T淋巴細(xì)胞的浸潤明顯增強(qiáng),炎癥因子IL-1α、IL-1β、IL-6、TNF-α在結(jié)腸癌形成過程中表達(dá)增加(p0.05),此外,CXCL-2及PDGF-α的表達(dá)也明顯增加(p0.01)。血管形成指標(biāo)MMP-7 (p0.01)、增殖相關(guān)信號(hào)分子p-Erk、Erk表達(dá)也顯著增加。4.體外提取成纖維細(xì)胞純度達(dá)90%以上,腫瘤相關(guān)成纖維細(xì)胞CAFs高表達(dá)FGF1和FGF3(p0.05)。將成纖維細(xì)胞的上清與結(jié)腸癌細(xì)胞共培養(yǎng),與NFs, PFs相比,CAFs可促進(jìn)結(jié)腸癌細(xì)胞的增殖,并且與CAFs細(xì)胞上清共培養(yǎng)的結(jié)腸癌細(xì)胞FGFR4自身磷酸化水平(p-Tyrosine) 、p-Mek/Erk、MMP-7的表達(dá)上調(diào),而總FGFR4的表達(dá)沒有明顯變化。將成纖維細(xì)胞上清與血管內(nèi)皮細(xì)胞共培養(yǎng),CCK8和小管形成實(shí)驗(yàn)結(jié)果顯示,與CAFs細(xì)胞上清共培養(yǎng),可促進(jìn)血管內(nèi)皮細(xì)胞的增殖和遷移。5.在含有CAFs上清培養(yǎng)的結(jié)腸癌細(xì)胞中加入FGFR4抑制劑后,p-Tyrosine, p-Mek、p-Erk、MMP-7水平明顯減少。6.在結(jié)腸癌細(xì)胞中加入重組蛋白FGF-1和FGF-3,Western Blot實(shí)驗(yàn)結(jié)果顯示p-Tyrosine, p-MeK、p-ErK、MMP-7表達(dá)顯著上調(diào)。[結(jié)論]綜上所述,我們的實(shí)驗(yàn)結(jié)果揭示：1.注射抗中性粒細(xì)胞表面抗原抗體Ly6G可以逆轉(zhuǎn)結(jié)腸癌的發(fā)展,進(jìn)一步驗(yàn)證了腫瘤相關(guān)中性粒細(xì)胞促進(jìn)炎癥性腸癌的發(fā)生及發(fā)展。2.CAFs通過FGF1/3-FGFR4信號(hào)通路,上調(diào)結(jié)腸癌細(xì)胞Mek/Erk增殖信號(hào)分子的表達(dá),上調(diào)MMP-7的表達(dá),進(jìn)而促進(jìn)腫瘤細(xì)胞增殖和血管新生,從而導(dǎo)致結(jié)腸癌的發(fā)生和發(fā)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.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R735.35

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