肿瘤FOXP3促胰腺导管腺癌招募Treg及机制研究

发布时间：2018-05-04 02:14

本文选题：胰腺导管腺癌 + c-FOXP3　；参考：《天津医科大学》2016年博士论文

【摘要】：背景FOXP3是主要表达于CD4+CD25+调节性T细胞的标志性转录因子[1]。它的主要功能是促进T细胞向调节性T细胞的分化,促进机体的免疫抑制功能[2]。然而,近期多项研究显示,除外T细胞,FOXP3也被发现表达于多种肿瘤细胞,如乳腺癌细胞、肾癌细胞与黑色素瘤细胞等等[3-5]。研究肿瘤源性FOXP3(cancer-FOXP3,c-FOXP3)的功能,可能为胰腺导管腺癌的靶向治疗提供新的靶点。我们在前期实验与文献阅读中发现,相较于正常组织和细胞系,胰腺导管腺癌组织及细胞系高表达c-FOXP3[6]。因此,我们设计了该课题以进一步明确c-FOXP3在胰腺导管腺癌组织及细胞中的表达水平、功能效应、以及背后的调控机制;旨从临床患者水平、细胞功能水平、分子生物学水平及动物实验水平深入探索胰腺导管腺癌中c-FOXP3的功能与意义。方法1.应用胰腺导管腺癌组织的石蜡标本切片进行FOXP3的免疫组化染色,分析c-FOXP3蛋白在胰腺导管腺癌组织中的表达水平;同时收集整理患者的临床病例资料并行预后随访,分析胰腺导管腺癌中c-FOXP3的表达水平与各项临床病理指标之间的关系。2.分析胰腺导管腺癌组织标本中c-FOXP3表达水平与Treg细胞在肿瘤局部富集程度的相关性;并分析c-FOXP3表达于Treg细胞聚集对胰腺导管腺癌患者临床病理指标及预后的共同影响。3.应用Western blot实验技术检测4个胰腺癌细胞系及正常胰腺导管细胞系的c-FOXP3的表达水平,并构建c-FOXP3过表达与降表达稳定转染细胞系;用Western blot验证构建的稳系所表达c-FOXP3的水平。通过细胞凋亡、细胞周期、Edu增殖掺入实验与免疫缺陷动物模型等多种方法检测c-FOXP3对肿瘤细胞的直接作用;通过免疫正常的动物模型检测c-FOXP3对肿瘤免疫微环境的作用。4.分离人源外周血单个核细胞(Peripheral Mononuclear Cells,PBMCs)与调节性T细胞(Regulatory T Cells,Treg cells)。利用共培养系统检测c-FOXP3对Treg细胞的促增殖作用;利用Transwell系统在体外模拟Treg细胞向肿瘤微环境的募集过程,检测Treg细胞的趋化水平。构建裸鼠胰腺癌原位成瘤模型,同时由鼠尾静脉注射人外周血单个核细胞(PBMCs),体内验证c-FOXP3促Treg细胞向胰腺癌微环境的趋化作用,5.应用RT-PCR的方法筛选c-FOXP3促进肿瘤细胞所分泌的趋化因子谱;应用Western blot、ELISA及免疫组化染色等实验验证RT-PCR结果;应用CHIP及双荧光素酶实验检测c-FOXP3调控趋化因子表达分泌的分子机制。6.通过趋化因子阻断试验确定c-FOXP3促进Treg细胞向肿瘤微环境趋化过程的中介因子。构建C57/BL黑鼠皮下成瘤动物模型,通过阻断趋化因子进一步确定c-FOXP3引起Treg细胞向肿瘤微环境募集的通路及该通路的生物学功能,确定胰腺导管腺癌靶向治疗目标,从而实现临床转化。结果1.c-FOXP3表达及Treg细胞聚集在胰腺导管腺癌组织中的意义。通过对120例胰腺导管腺癌组织标本进行免疫组织化学染色发现,c-FOXP3在胰腺导管腺癌中多数表达阳性,而对应的癌旁正常胰腺组织不表达或表达程度极弱;分析该120例患者的病例资料,我们发现c-FOXP3高表达的占63.3%(76例),c-FOXP3低表达的占36.6%(44例);进一步研究c-FOXP3蛋白表达水平与临床病理指标之间的关系,我们发现高表达c-FOXP3的胰腺导管腺癌患者的总生存期(中位数:24 vs 15个月)与无复发生存期(中位数:15 vs 9个月)均明显低于短于c-FOXP3低表达组的患者(p0.05*)。同时,FOXP3阳性的Treg细胞在胰腺癌组织中也有不同程度的表达。我们深入研究了胰腺导管腺癌肿瘤细胞中c-FOXP3的表达与FOXP3阳性Treg细胞在肿瘤局部微环境中聚集程度的相关性,发现两者呈明显的正相关(r=0.537,p0.001**)。统计分析后我们发现,c-FOXP3高表达同时伴随Treg细胞在肿瘤中高度聚集的患者的总生存期及无复发生存期明显短于c-FOXP3高表达Treg低浸润的患者,同时,c-FOXP3高表达同时Treg细胞高度浸润的患者的肿瘤大小也明显大于c-FOXP3高表达Treg低浸润的患者;这些结果提示,Treg细胞浸润程度在c-FOXP3高表达对患者生存期的影响中发挥了重要的生物学功能,引起胰腺导管腺癌的进展。2.c-FOXP3对胰腺导管腺癌细胞系的直接与间接影响。利用Western blot验证了c-FOXP3在4个胰腺癌细胞系(PANC-1、MIA Pa Ca-2、As PC-1及Bx PC-3)均有不同程度的表达,而在正常胰腺细胞系HPDE6C7中表达极弱;并且成功构建了2个过表达c-FOXP3的胰腺癌细胞系(PANC-1及As PC-1)和2个降表达c-FOXP3的细胞系(MIA Pa Ca-2和Bx PC-3),构建的稳系经Western blot验证了c-FOXP3表达的变化。通过细胞凋亡、细胞周期、Edu增殖掺入与免疫缺陷动物模型证明c-FOXP3对肿瘤细胞无明显的直接影响。3.免疫系统完整的小鼠体内皮下成瘤实验证实,降表达c-FOXP3的胰腺癌细胞成瘤大小明显小于其对照组细胞成瘤,并且其瘤块中Treg浸润也明显少于对照组;同时,CD25抗体特异清除免疫系统完整小鼠Treg细胞后,降表达c-FOXP3的胰腺癌细胞成瘤与对照组成瘤大小无统计学差别,证明了Treg细胞参与c-FOXP3对肿瘤细胞生物学功能的间接影响。4.利用体外共培养肿瘤细胞与Treg细胞实验,通过Edu增殖掺入检测发现c-FOXP3不能促进Treg细胞自身增殖。利用体外Transwell趋化实验,通过流式细胞术检测发现,胰腺导管腺癌细胞过表达c-FOXP3后对Treg细胞的趋化能力增强;而降表达c-FOXP3后对Treg的趋化能力下降。裸鼠原位成瘤实验证实,过表达c-FOXP3的人源胰腺癌细胞成瘤对经由鼠尾注射的人外周血单个核细胞中Treg细胞的趋化能力明显强于未过表达c-FOXP3的对照组成瘤。5.实时定量PCR筛选出趋化因子CCL5表达水平随c-FOXP3表达变化而变化的程度最为显著,Western blot证实,胰腺癌细胞内CCL5表达水平受c-FOXP3的调控;ELISA实验证实,胰腺癌细胞向外释放CCL5的水平也受c-FOXP3调控;同时免疫组化染色发现,c-FOXP3与CCL5的表达胰腺癌组织中呈现共定位,并有明显的正相关性(r=0.681,P0.001**)。5.利用Panc-1、Pan02和293T细胞系,我们行Ch IP实验发现人源与鼠源转录因子FOXP3均可分别直接结合于相应种属CCL5的启动子区;同时双荧光素酶实验证实,过表达c-FOXP3后CCL5启动子转录活性增强,而突变上述FOXP3与CCL5结合位点后,其转录活性的增强即被反转,从而证实c-FOXP3可以直接调控CCL5的表达。6.体外Transwell趋化模型阻断实验发现,通过对CCL5进行中和阻断可明显减弱过表达c-FOXP3的胰腺癌细胞对Treg细胞的趋化作用;体内胰腺皮下成瘤实验发现,CCL5阻断不仅减弱了Treg细胞向肿瘤局部的浸润,同时抑制了肿瘤的生长,并且该现象在高表达c-FOXP3的肿瘤中更为明显。结论1.c-FOXP3蛋白在胰腺导管腺癌组织及细胞系中呈现阳性表达;c-FOXP3表达水平与Treg细胞聚集程度正相关;c-FOXP3表达水平较高且Treg细胞比例高的患者预后较差。2.c-FOXP3直接结合至CCL5的启动子区,并促进其转录翻译过程,上调CCL5在胞内的表达及其向胞外的分泌。3.以CCL5为介导,c-FOXP3促进了胰腺导管腺癌细胞对Treg细胞的趋化能力。高表达的c-FOXP3与Treg细胞在肿瘤微环境中的高度浸润共同作用,促进胰腺导管腺癌肿瘤的生长。4.对高表达c-FOXP3的胰腺导管腺癌肿瘤进行CCL5的中和阻断,实现了对Treg浸润和肿瘤生长的抑制作用。
[Abstract]:Background FOXP3 is a marker transcription factor [1]., which is mainly expressed in CD4+CD25+ regulatory T cells. Its main function is to promote the differentiation of T cells to regulatory T cells and promote the immune inhibitory function of the body. However, a number of recent studies have revealed that FOXP3 is also found to be expressed in a variety of tumor cells, such as breast cancer cells, and kidney cancer, with the exception of T cells. Cells and melanoma cells and so on [3-5]. studies the function of the tumor derived FOXP3 (cancer-FOXP3, c-FOXP3), which may provide new targets for the targeting therapy of pancreatic ductal adenocarcinoma. We found in previous experiments and literature reading that the pancreatic ductal adenocarcinoma tissue and cell lines are highly expressed as c-FOXP3[6]., compared to normal tissue and cell lines. We designed the subject to further clarify the expression level, function effect, and regulatory mechanism of c-FOXP3 in pancreatic ductal adenocarcinoma tissue and cells, and the regulatory mechanism behind it. The purpose of this study is to explore the function and significance of c-FOXP3 in pancreatic duct adenocarcinoma from clinical patient level, cell function level, molecular biology level and animal experiment level. Method 1. FOXP3 immunohistochemical staining was used to analyze the expression level of c-FOXP3 protein in pancreatic ductal adenocarcinoma tissue by using paraffin section of pancreatic duct adenocarcinoma tissue, and the clinical data of the patients were collected and followed up to analyze the expression level of c-FOXP3 in pancreatic ductal adenocarcinoma and the clinicopathological indexes. Correlation.2. analysis of the expression of c-FOXP3 in pancreatic ductal adenocarcinoma tissue and the correlation of Treg cells to local enrichment of Treg cells, and the common influence of c-FOXP3 expression on the clinicopathological indexes and prognosis of pancreatic ductal adenocarcinoma by Treg cells.3. application Western blot test technique to detect 4 pancreatic cancer cell lines and positive The expression level of c-FOXP3 in the normal pancreatic duct cell line, and the construction of c-FOXP3 over expression and expression of stable transfection cell lines, and the level of c-FOXP3 expressed in the stable system constructed by Western blot. The detection of c-FOXP3 to the tumor cells through a variety of methods, such as cell apoptosis, cell cycle, Edu proliferation incorporation, and immunodeficiency animal models. Direct action; the effect of c-FOXP3 on the immune microenvironment of tumor by immune normal animal model.4. separation of human peripheral peripheral blood mononuclear cells (Peripheral Mononuclear Cells, PBMCs) and regulatory T cells (Regulatory T Cells, Treg cells). The system was used to simulate the recruitment process of Treg cells to the tumor microenvironment in vitro, to detect the chemotaxis level of Treg cells, and to construct an in situ tumor model of pancreatic cancer in nude mice, and by injecting human peripheral blood mononuclear cells (PBMCs) from the rat tail vein. In vivo, the chemotactic effect of c-FOXP3 promoting Treg cells to the microenvironment of pancreatic cancer was verified. 5. the RT-PCR method was used to screen the c-. FOXP3 promotes the chemokine spectrum secreted by tumor cells; uses Western blot, ELISA and immunohistochemical staining to verify the RT-PCR results. CHIP and double luciferase test detect the molecular mechanism of c-FOXP3 regulating the expression and secretion of chemokine,.6. through chemokine blocking test to determine c-FOXP3 promoting Treg cells to the tumor microenvironment. The mediating factor of the process is to construct a subcutaneous tumor model of C57/BL black mice. By blocking the chemokines, we further determine the pathway raised by c-FOXP3 to the microenvironment of the tumor and the biological function of the pathway, and determine the target of the targeted treatment of the pancreatic ductal adenocarcinoma to achieve the clinical transformation. The result of the expression of 1.c-FOXP3 and the aggregation of Treg cells. Immunohistochemical staining of 120 cases of pancreatic ductal adenocarcinoma found that most of the expression of c-FOXP3 in pancreatic ductal adenocarcinoma was positive, while the corresponding non expression or expression level of normal pancreatic tissue adjacent to the carcinoma was very weak. The data of the 120 cases were analyzed, and we found that c-FOXP3 The high expression of 63.3% (76 cases) and low expression of c-FOXP3 accounted for 36.6% (44 cases). Further study of the relationship between the expression level of c-FOXP3 protein and the clinicopathological index, we found that the total survival time of the pancreatic duct adenocarcinoma with high expression of c-FOXP3 (median: 24 vs 15 months) was significantly lower than that of the non recurrent survival period (median: 15 vs 9 months). The FOXP3 positive Treg cells were also expressed in different degrees in the pancreatic cancer tissues. The correlation between the expression of c-FOXP3 in pancreatic ductal adenocarcinoma cells and the degree of aggregation of FOXP3 positive Treg cells in the local microenvironment of the FOXP3 positive Treg cells was investigated. It was found that both of them showed a distinct positive phase. R=0.537 (p0.001**). After statistical analysis, we found that the total survival and non recurrent survival of patients with high expression of c-FOXP3 and high aggregation of Treg cells in the tumor were significantly shorter than those of c-FOXP3 with high expression of Treg low infiltration, while the size of the tumor in patients with high expression of c-FOXP3 and highly infiltrated Treg cells was also significantly larger than C -FOXP3 is highly expressed in patients with Treg low infiltration; these results suggest that the degree of Treg cell infiltration plays an important biological function in the impact of c-FOXP3 expression on the survival period of the patients, causing the direct and indirect effect of.2.c-FOXP3 on pancreatic duct adenocarcinoma cell lines. Western blot has been used to verify c-FOXP3 in 4 A pancreatic cancer cell line (PANC-1, MIA Pa Ca-2, As PC-1 and Bx PC-3) were expressed in varying degrees, but were very weak in the normal pancreatic cell line HPDE6C7, and 2 pancreatic cancer cell lines (PANC-1 and As) and 2 cell lines were successfully constructed. RN blot verified the changes in the expression of c-FOXP3. Through the cell apoptosis, the cell cycle, the proliferation of Edu and the immunodeficiency animal model, it was proved that c-FOXP3 had no obvious direct effect on the tumor cells, and the tumor cells with complete.3. immune system were subcutaneously tumorigenized in mice. The tumor size of the pancreatic adenocarcinoma cells expressing c-FOXP3 was significantly smaller than that of the control group. The Treg infiltration in the tumor block was also significantly less than that of the control group. At the same time, after the CD25 antibody specifically scavenged the immune system intact mouse Treg cells, there was no statistical difference between the tumor cells of the pancreatic cancer cells expressing c-FOXP3 and the control composition of the tumor, which showed that the Treg cells were involved in the indirect effect of c-FOXP3 on the biological function of the tumor cells by.4. utilization. In vitro co culture of tumor cells and Treg cells, and Edu proliferation assay found that c-FOXP3 did not promote the proliferation of Treg cells. By using in vitro Transwell chemotaxis test, the chemotactic capacity of pancreatic ductal adenocarcinoma cells was enhanced after c-FOXP3 expression, and c-FOXP3 was reduced to Treg after c-FOXP3. The chemotactic ability of the nude mice was decreased. The in situ tumor formation test in nude mice showed that the chemotaxis of Treg cells in human peripheral blood mononuclear cells by human peripheral blood mononuclear cells over expression of c-FOXP3 was stronger than that of the non overexpressed c-FOXP3 control composition.5. real-time quantitative PCR screening the expression level of chemokine CCL5 expression with c-FOXP3 expression. Western blot confirmed that the expression level of CCL5 in pancreatic cancer cells was regulated by c-FOXP3, and the ELISA experiment confirmed that the level of CCL5 release from pancreatic cancer cells was also regulated by c-FOXP3. At the same time, immunohistochemical staining showed that the expression of c-FOXP3 and CCL5 expressed in pancreatic cancer tissues, and there was a clear positive phase. R=0.681 (P0.001**).5. uses Panc-1, Pan02 and 293T cell lines. Our Ch IP experiment found that both human and mouse source transcription factor FOXP3 can be directly combined with the promoter region of the corresponding CCL5, and the double luciferase experiment confirmed that the transcriptional activity of the CCL5 promoter was enhanced after the overexpression was c-FOXP3, and the mutation was combined with the CCL5. After the loci, the enhancement of its transcriptional activity is reversed, which confirms that c-FOXP3 can directly regulate the expression of CCL5 expression in.6. Transwell chemotactic model. By neutralizing CCL5, the chemotactic effect of pancreatic cancer cells over the expression of c-FOXP3 can be obviously weakened; the pancreatic subcutaneous tumorigenesis experiment in the body found the hindrance of CCL5. It not only weakens the infiltration of Treg cells to tumor, but also inhibits the growth of tumor, and this phenomenon is more obvious in the tumor with high expression of c-FOXP3. Conclusion the expression of 1.c-FOXP3 protein in pancreatic ductal adenocarcinoma and cell lines is positive; the level of c-FOXP3 expression is positively related to the degree of Treg cell aggregation; the level of c-FOXP3 expression is positive. The patients with higher and higher Treg cells have a poor prognosis of.2.c-FOXP3 directly to the promoter region of CCL5, and promote their transcriptional translation process, up-regulation the expression of CCL5 in the cell and the secretion of.3. from the extracellular to CCL5, and c-FOXP3 promotes the chemotaxis of Treg cells to Treg cells. The high expression of c-FOXP3 and Treg cells The joint action of high infiltration in the tumor microenvironment promotes the growth of pancreatic ductal adenocarcinoma tumor and the neutralization of CCL5 in pancreatic ductal adenocarcinoma with high expression of c-FOXP3, which can inhibit the invasion of Treg and the growth of the tumor.

【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.9

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