本文選題：胃癌　切入點：腫瘤相關(guān)成纖維細胞　出處：《浙江大學》2016年博士論文　論文類型：學位論文

【摘要】：背景：胃癌是全球最常見的惡性腫瘤之一,其發(fā)病率位居第四,腫瘤相關(guān)死亡率位居第二。腫瘤血管形成促進腫瘤生長和轉(zhuǎn)移,VEGF/VEGFR信號通路在腫瘤血管生成中起主導作用。因此抗腫瘤血管生成治療多以VEGF/VEGFR為靶點。Apatinib作為VEGFR抑制劑,晚期胃癌患者接受apatinib治療使生存獲益,但是缺乏能夠預測apatinib獲益人群的生物標志物。腫瘤相關(guān)成纖維細胞(cancer-associated fibroblasts, CAFs)分泌多種細胞因子促進腫瘤血管生成。Galectin-1在腫瘤細胞和CAFs中均可表達。近年來,多項研究報道galectin-1通過多種途徑促進腫瘤血管生成。然而,galectin-1在CAFs中表達與胃癌apatinib敏感性的關(guān)系,目前尚無報道。第一部分：研究背景：腫瘤進展是腫瘤細胞和CAFs相互促進的結(jié)果,腫瘤細胞分泌的細胞因子如TGF-p等,誘導正常成纖維細胞(normal fibroblasts, NFs)向CAFs轉(zhuǎn)化,而CAFs活化后促進腫瘤進展。Galectin-1在腫瘤細胞和CAFs均可表達。目前galectin-1/TGF-β信號軸在腫瘤細胞與CAFs相互促進過程中所起的中介作用尚不清楚,因此我們將對galectin-1/TGF-β在腫瘤細胞與CAFs間所起的聯(lián)系進行探索。方法：分離原代胃癌CAFs和NFs,并應(yīng)用免疫熒光和western blotting進行鑒定。應(yīng)用real-time PCR和western blotting比較CAFs和NFs中g(shù)alectin-1的表達水平。利用galectin-1過表達的慢病毒載體感染NFs上調(diào)galectin-1表達(NFs-Gall), Small interfering RNA (siRNA)轉(zhuǎn)染CAFs沉默galectin-1表達(CAFs-siGal1)。通過腫瘤細胞上清液誘導NFs向CAFs轉(zhuǎn)化的模型,探索galectin-1/TGF-β信號軸在腫瘤細胞與CAFs相互作用中的聯(lián)系。免疫組織化學法檢測胃癌組織中g(shù)alectin-1、TGF-β的表達情況,明確galectin-1與TGF-β的關(guān)聯(lián)。Kaplan-Meier曲線描述胃癌患者的生存期,并采用Log-rank檢驗對臨床資料和數(shù)據(jù)進行統(tǒng)計分析。Cox regression風險回歸模型對胃癌預后的各臨床參數(shù)進行多因素分析。結(jié)果：Galectin-1在CAFs中高表達,并且促進NFs向CAFs轉(zhuǎn)化。腫瘤細胞分泌TGF-β誘導NFs表達galectin-1, galectin-1正向調(diào)節(jié)TGF-β誘發(fā)的NFs向CAFs轉(zhuǎn)化。Galectin-1表達與TGF-β表達呈正相關(guān)(r= 0.765, P0.001)。Galectin-1表達水平與腫瘤分化程度、淋巴轉(zhuǎn)移、浸潤深度和腫瘤TNM分期有關(guān)。Galectin-1高表達的胃癌患者總生存期短,并且是獨立的預后因素。結(jié)論：Galectin-1促進TGF-p所誘導的NFs向CAFs轉(zhuǎn)化的過程,并且其表達水平與胃癌患者的預后呈負相關(guān)。第二部分研究背景：第一部分實驗結(jié)果表明CAFs中g(shù)alectin-1表達升高,并且galectin-1表達水平與胃癌患者預后呈負相關(guān)。我們將通過一系列的體外體內(nèi)功能學實驗探索galectin-1促進腫瘤進展的機制。方法：通過不同Galectin-1表達水平的成纖維細胞條件培養(yǎng)基(CMs)培養(yǎng)胃癌細胞,采用流式細胞儀(flow cytometry, FC)檢測腫瘤細胞凋亡,transwell實驗檢測腫瘤細胞侵襲和遷移。Western blotting等實驗方法對作用機制進行探討。建立裸鼠皮下荷瘤模型,明確成纖維細胞galectin-1表達水平對腫瘤生長的影響。結(jié)果：CAFs明顯抑制腫瘤細胞凋亡,促進侵襲和遷移(P0.05)。沉默CAFs中g(shù)alectin-1表達,抗腫瘤細胞凋亡及促侵襲的作用被逆轉(zhuǎn)。CAFs促進腫瘤細胞中Bcl-2和MMP9表達,抑制Bax表達。裸鼠荷瘤實驗結(jié)果顯示,galectin-1高表達的成纖維細胞明顯促進腫瘤生長。結(jié)論：Galectin-1高表達的CAFs通過調(diào)節(jié)凋亡相關(guān)蛋白和MMP9的表達水平,分別影響腫瘤細胞凋亡、侵襲和轉(zhuǎn)移的過程。第三部分研究背景：Apatinib靶向VEGFR,使晚期胃癌患者的生存顯著獲益,但是缺乏能夠預測apatinib療效的生物標志物。Galectin-1在腫瘤血管生成中起重要作用,因此,我們將明確galectin-1表達水平與胃癌apatinib敏感性的關(guān)系。方法：通過CCK-8及體外HUVEC小管形成實驗,探索CAFs中g(shù)alectin-1的表達水平與apatinib敏感性的關(guān)系。NFs-Gal1、NFs-Ctr分別與腫瘤細胞共同接種,建立裸鼠皮下荷瘤模型,從體內(nèi)水平評價apatinib的療效。結(jié)果：Galectin-1促進CAFs表達bFGF,使apatinib抑制HUVEC增殖及小管形成的作用受限。上調(diào)NFs中g(shù)alectin-1表達能夠抑制apatinib抗腫瘤生長及抗血管生成的作用。結(jié)論：Galectin-1表達導致apatinib耐藥的發(fā)生。
[Abstract]:Background: gastric cancer is one of the most common malignant tumor in the world, the incidence rate was fourth, tumor related mortality was second. Tumor angiogenesis and promote tumor growth and metastasis, VEGF/VEGFR signaling pathway plays a dominant role in tumor angiogenesis. Therefore, anti angiogenesis therapy targeting VEGF/VEGFR.Apatinib as a VEGFR inhibitor to survive benefit from apatinib therapy in patients with advanced gastric cancer, but the lack of biomarkers to predict apatinib benefit groups. Tumor associated fibroblasts (cancer-associated, fibroblasts, CAFs) secreting a variety of cytokines.Galectin-1 can promote tumor angiogenesis in tumor cells and CAFs expression. In recent years, several studies reported that galectin-1 promote tumor angiogenesis through a variety of ways. However, the expression of galectin-1 and gastric carcinoma apatinib sensitivity in CAFs, the current is still No reports. The first part: BACKGROUND: tumor cells and CAFs tumor progression is the result of mutual promotion, tumor cells secrete cytokines such as TGF-p, normal fibrolasts (normal fibroblasts, NFs) and transformed to CAFs, after the activation of CAFs promotes the expression of.Galectin-1 in tumor cells and tumor progression. The CAFs can be galectin-1/TGF- beta signaling axis in tumor cells and CAFs mutual promotion has a mediating effect in the process is not clear, so we will galectin-1/TGF- beta plays in tumor cells and to explore the relations between CAFs. Methods: isolated primary gastric cancer CAFs and NFs, and the application of immunofluorescence and Western blotting were used to identify the expression level of galectin-1 application. Real-time PCR and Western blotting CAFs and NFs. Using the galectin-1 Lentivirus Expression Vector NFs infection up regulates the expression of galectin-1 (NFs-Gall, Small) Interfering RNA (siRNA) transfection and expression of CAFs silencing (CAFs-siGal1) induced by NFs. Galectin-1 to CAFs conversion by the supernatant of tumor cells in tumor cell model, explore the interaction between CAFs and galectin-1/TGF- in the beta signaling axis. Immunohistochemistry was used to detect the gastric cancer tissues galectin-1, the expression of TGF- beta,.Kaplan-Meier curve galectin-1 clear association with the TGF- beta description of the survival of patients with gastric cancer, and Log-rank test was used for statistical analysis of clinical parameters of.Cox regression on the prognosis of gastric cancer risk regression model of clinical data and the data were analyzed by multivariate analysis. Results: the expression of Galectin-1 in CAFs, and promote the transformation of NFs to CAFs. The tumor cells secrete TGF- beta induced NFs expression galectin-1, transformation of galectin-1 positively regulates TGF- beta induced NFs to CAFs the expression of.Galectin-1 was positively correlated with the expression of TGF- beta (r= 0.765, P0 .001).Galectin-1 expression and tumor differentiation, lymph node metastasis, invasion depth and overall survival of patients with gastric cancer tumor TNM stage.Galectin-1 expression is short, and is an independent prognostic factor. Conclusion: Galectin-1 promotes the process of transformation induced by TGF-p NFs to CAFs, and negatively correlated with the expression level and prognosis of gastric cancer with the research background. The second part: the first part of the experiment results show that the increase of expression of galectin-1 in CAFs, and the expression level of galectin-1 was negatively correlated with prognosis in patients with gastric cancer. We will go through the function of a series of studies in vitro and in vivo experiments to explore the mechanism of galectin-1 promoting tumor progression. Methods: the different expression levels of Galectin-1 fibroblast conditioned medium (CMs) cultured gastric cancer cells by flow cytometry (flow cytometry FC) to detect the apoptosis of tumor cells, Transwell assay, tumor Cell invasion and migration of.Western blotting and other experimental methods to explore the mechanism. To establish the model of nude mice bearing tumor, clear fibroblasts galectin-1 expression level of tumor growth. Results: CAFs can significantly inhibit the apoptosis of tumor cells, promote the invasion and migration (P0.05). The expression of galectin-1 CAFs in silence, and promote the apoptosis of tumor cells the invasion effect was reversed by.CAFs promotes the expression of Bcl-2 and MMP9 in tumor cells, inhibit the expression of Bax in tumor bearing nude mice. The experimental results show that the high expression of galectin-1 fibroblasts significantly promote tumor growth. Conclusion: high expression of Galectin-1 CAFs by regulating the expression level of apoptosis related protein and MMP9, respectively. The apoptosis of tumor cells and process the invasion and metastasis. The third part is the research background: Apatinib targeting VEGFR, the survival of a significant benefit for patients with advanced gastric cancer, but the lack of able to predict APA Biomarkers.Galectin-1 effect of tinib play an important role in tumor angiogenesis, therefore, we will clear the relationship between the expression level of galectin-1 and apatinib in gastric carcinoma. Methods: the formation of sensitivity experiments by CCK-8 and in vitro HUVEC tubules,.NFs-Gal1 to explore the relationship between CAFs galectin-1 expression level and the sensitivity of apatinib, NFs-Ctr were co inoculated with tumor cells the establishment of subcutaneous xenograft model in nude mice, and evaluate the effect of apatinib from the body level. Results: Galectin-1 promotes the expression of CAFs bFGF, the role of the limited apatinib inhibits proliferation and tubule formation of HUVEC. Up regulation of NFs expression of galectin-1 in apatinib can inhibit the anti-tumor growth and anti angiogenesis effect. Conclusion: the expression of Galectin-1 apatinib lead to the occurrence of drug resistance.

【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R735.2

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