Galectin-1介导NFs向CAFs转化与胃癌apatinib敏感性的关系的研究

发布时间：2018-03-18 20:42

  本文选题：胃癌　切入点：肿瘤相关成纤维细胞　出处：《浙江大学》2016年博士论文　论文类型：学位论文

【摘要】：背景：胃癌是全球最常见的恶性肿瘤之一,其发病率位居第四,肿瘤相关死亡率位居第二。肿瘤血管形成促进肿瘤生长和转移,VEGF/VEGFR信号通路在肿瘤血管生成中起主导作用。因此抗肿瘤血管生成治疗多以VEGF/VEGFR为靶点。Apatinib作为VEGFR抑制剂,晚期胃癌患者接受apatinib治疗使生存获益,但是缺乏能够预测apatinib获益人群的生物标志物。肿瘤相关成纤维细胞(cancer-associated fibroblasts, CAFs)分泌多种细胞因子促进肿瘤血管生成。Galectin-1在肿瘤细胞和CAFs中均可表达。近年来,多项研究报道galectin-1通过多种途径促进肿瘤血管生成。然而,galectin-1在CAFs中表达与胃癌apatinib敏感性的关系,目前尚无报道。第一部分：研究背景：肿瘤进展是肿瘤细胞和CAFs相互促进的结果,肿瘤细胞分泌的细胞因子如TGF-p等,诱导正常成纤维细胞(normal fibroblasts, NFs)向CAFs转化,而CAFs活化后促进肿瘤进展。Galectin-1在肿瘤细胞和CAFs均可表达。目前galectin-1/TGF-β信号轴在肿瘤细胞与CAFs相互促进过程中所起的中介作用尚不清楚,因此我们将对galectin-1/TGF-β在肿瘤细胞与CAFs间所起的联系进行探索。方法：分离原代胃癌CAFs和NFs,并应用免疫荧光和western blotting进行鉴定。应用real-time PCR和western blotting比较CAFs和NFs中galectin-1的表达水平。利用galectin-1过表达的慢病毒载体感染NFs上调galectin-1表达(NFs-Gall), Small interfering RNA (siRNA)转染CAFs沉默galectin-1表达(CAFs-siGal1)。通过肿瘤细胞上清液诱导NFs向CAFs转化的模型,探索galectin-1/TGF-β信号轴在肿瘤细胞与CAFs相互作用中的联系。免疫组织化学法检测胃癌组织中galectin-1、TGF-β的表达情况,明确galectin-1与TGF-β的关联。Kaplan-Meier曲线描述胃癌患者的生存期,并采用Log-rank检验对临床资料和数据进行统计分析。Cox regression风险回归模型对胃癌预后的各临床参数进行多因素分析。结果：Galectin-1在CAFs中高表达,并且促进NFs向CAFs转化。肿瘤细胞分泌TGF-β诱导NFs表达galectin-1, galectin-1正向调节TGF-β诱发的NFs向CAFs转化。Galectin-1表达与TGF-β表达呈正相关(r= 0.765, P0.001)。Galectin-1表达水平与肿瘤分化程度、淋巴转移、浸润深度和肿瘤TNM分期有关。Galectin-1高表达的胃癌患者总生存期短,并且是独立的预后因素。结论：Galectin-1促进TGF-p所诱导的NFs向CAFs转化的过程,并且其表达水平与胃癌患者的预后呈负相关。第二部分研究背景：第一部分实验结果表明CAFs中galectin-1表达升高,并且galectin-1表达水平与胃癌患者预后呈负相关。我们将通过一系列的体外体内功能学实验探索galectin-1促进肿瘤进展的机制。方法：通过不同Galectin-1表达水平的成纤维细胞条件培养基(CMs)培养胃癌细胞,采用流式细胞仪(flow cytometry, FC)检测肿瘤细胞凋亡,transwell实验检测肿瘤细胞侵袭和迁移。Western blotting等实验方法对作用机制进行探讨。建立裸鼠皮下荷瘤模型,明确成纤维细胞galectin-1表达水平对肿瘤生长的影响。结果：CAFs明显抑制肿瘤细胞凋亡,促进侵袭和迁移(P0.05)。沉默CAFs中galectin-1表达,抗肿瘤细胞凋亡及促侵袭的作用被逆转。CAFs促进肿瘤细胞中Bcl-2和MMP9表达,抑制Bax表达。裸鼠荷瘤实验结果显示,galectin-1高表达的成纤维细胞明显促进肿瘤生长。结论：Galectin-1高表达的CAFs通过调节凋亡相关蛋白和MMP9的表达水平,分别影响肿瘤细胞凋亡、侵袭和转移的过程。第三部分研究背景：Apatinib靶向VEGFR,使晚期胃癌患者的生存显著获益,但是缺乏能够预测apatinib疗效的生物标志物。Galectin-1在肿瘤血管生成中起重要作用,因此,我们将明确galectin-1表达水平与胃癌apatinib敏感性的关系。方法：通过CCK-8及体外HUVEC小管形成实验,探索CAFs中galectin-1的表达水平与apatinib敏感性的关系。NFs-Gal1、NFs-Ctr分别与肿瘤细胞共同接种,建立裸鼠皮下荷瘤模型,从体内水平评价apatinib的疗效。结果：Galectin-1促进CAFs表达bFGF,使apatinib抑制HUVEC增殖及小管形成的作用受限。上调NFs中galectin-1表达能够抑制apatinib抗肿瘤生长及抗血管生成的作用。结论：Galectin-1表达导致apatinib耐药的发生。
[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.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.2

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