本文選題：胰腺導管腺癌 + c-FOXP3��； 參考：《天津醫(yī)科大學》2016年博士論文

【摘要】：背景FOXP3是主要表達于CD4+CD25+調(diào)節(jié)性T細胞的標志性轉(zhuǎn)錄因子[1]。它的主要功能是促進T細胞向調(diào)節(jié)性T細胞的分化,促進機體的免疫抑制功能[2]。然而,近期多項研究顯示,除外T細胞,FOXP3也被發(fā)現(xiàn)表達于多種腫瘤細胞,如乳腺癌細胞、腎癌細胞與黑色素瘤細胞等等[3-5]。研究腫瘤源性FOXP3(cancer-FOXP3,c-FOXP3)的功能,可能為胰腺導管腺癌的靶向治療提供新的靶點。我們在前期實驗與文獻閱讀中發(fā)現(xiàn),相較于正常組織和細胞系,胰腺導管腺癌組織及細胞系高表達c-FOXP3[6]。因此,我們設(shè)計了該課題以進一步明確c-FOXP3在胰腺導管腺癌組織及細胞中的表達水平、功能效應、以及背后的調(diào)控機制;旨從臨床患者水平、細胞功能水平、分子生物學水平及動物實驗水平深入探索胰腺導管腺癌中c-FOXP3的功能與意義。方法1.應用胰腺導管腺癌組織的石蠟標本切片進行FOXP3的免疫組化染色,分析c-FOXP3蛋白在胰腺導管腺癌組織中的表達水平;同時收集整理患者的臨床病例資料并行預后隨訪,分析胰腺導管腺癌中c-FOXP3的表達水平與各項臨床病理指標之間的關(guān)系。2.分析胰腺導管腺癌組織標本中c-FOXP3表達水平與Treg細胞在腫瘤局部富集程度的相關(guān)性;并分析c-FOXP3表達于Treg細胞聚集對胰腺導管腺癌患者臨床病理指標及預后的共同影響。3.應用Western blot實驗技術(shù)檢測4個胰腺癌細胞系及正常胰腺導管細胞系的c-FOXP3的表達水平,并構(gòu)建c-FOXP3過表達與降表達穩(wěn)定轉(zhuǎn)染細胞系;用Western blot驗證構(gòu)建的穩(wěn)系所表達c-FOXP3的水平。通過細胞凋亡、細胞周期、Edu增殖摻入實驗與免疫缺陷動物模型等多種方法檢測c-FOXP3對腫瘤細胞的直接作用;通過免疫正常的動物模型檢測c-FOXP3對腫瘤免疫微環(huán)境的作用。4.分離人源外周血單個核細胞(Peripheral Mononuclear Cells,PBMCs)與調(diào)節(jié)性T細胞(Regulatory T Cells,Treg cells)。利用共培養(yǎng)系統(tǒng)檢測c-FOXP3對Treg細胞的促增殖作用;利用Transwell系統(tǒng)在體外模擬Treg細胞向腫瘤微環(huán)境的募集過程,檢測Treg細胞的趨化水平。構(gòu)建裸鼠胰腺癌原位成瘤模型,同時由鼠尾靜脈注射人外周血單個核細胞(PBMCs),體內(nèi)驗證c-FOXP3促Treg細胞向胰腺癌微環(huán)境的趨化作用,5.應用RT-PCR的方法篩選c-FOXP3促進腫瘤細胞所分泌的趨化因子譜;應用Western blot、ELISA及免疫組化染色等實驗驗證RT-PCR結(jié)果;應用CHIP及雙熒光素酶實驗檢測c-FOXP3調(diào)控趨化因子表達分泌的分子機制。6.通過趨化因子阻斷試驗確定c-FOXP3促進Treg細胞向腫瘤微環(huán)境趨化過程的中介因子。構(gòu)建C57/BL黑鼠皮下成瘤動物模型,通過阻斷趨化因子進一步確定c-FOXP3引起Treg細胞向腫瘤微環(huán)境募集的通路及該通路的生物學功能,確定胰腺導管腺癌靶向治療目標,從而實現(xiàn)臨床轉(zhuǎn)化。結(jié)果1.c-FOXP3表達及Treg細胞聚集在胰腺導管腺癌組織中的意義。通過對120例胰腺導管腺癌組織標本進行免疫組織化學染色發(fā)現(xiàn),c-FOXP3在胰腺導管腺癌中多數(shù)表達陽性,而對應的癌旁正常胰腺組織不表達或表達程度極弱;分析該120例患者的病例資料,我們發(fā)現(xiàn)c-FOXP3高表達的占63.3%(76例),c-FOXP3低表達的占36.6%(44例);進一步研究c-FOXP3蛋白表達水平與臨床病理指標之間的關(guān)系,我們發(fā)現(xiàn)高表達c-FOXP3的胰腺導管腺癌患者的總生存期(中位數(shù):24 vs 15個月)與無復發(fā)生存期(中位數(shù):15 vs 9個月)均明顯低于短于c-FOXP3低表達組的患者(p0.05*)。同時,FOXP3陽性的Treg細胞在胰腺癌組織中也有不同程度的表達。我們深入研究了胰腺導管腺癌腫瘤細胞中c-FOXP3的表達與FOXP3陽性Treg細胞在腫瘤局部微環(huán)境中聚集程度的相關(guān)性,發(fā)現(xiàn)兩者呈明顯的正相關(guān)(r=0.537,p0.001**)。統(tǒng)計分析后我們發(fā)現(xiàn),c-FOXP3高表達同時伴隨Treg細胞在腫瘤中高度聚集的患者的總生存期及無復發(fā)生存期明顯短于c-FOXP3高表達Treg低浸潤的患者,同時,c-FOXP3高表達同時Treg細胞高度浸潤的患者的腫瘤大小也明顯大于c-FOXP3高表達Treg低浸潤的患者;這些結(jié)果提示,Treg細胞浸潤程度在c-FOXP3高表達對患者生存期的影響中發(fā)揮了重要的生物學功能,引起胰腺導管腺癌的進展。2.c-FOXP3對胰腺導管腺癌細胞系的直接與間接影響。利用Western blot驗證了c-FOXP3在4個胰腺癌細胞系(PANC-1、MIA Pa Ca-2、As PC-1及Bx PC-3)均有不同程度的表達,而在正常胰腺細胞系HPDE6C7中表達極弱;并且成功構(gòu)建了2個過表達c-FOXP3的胰腺癌細胞系(PANC-1及As PC-1)和2個降表達c-FOXP3的細胞系(MIA Pa Ca-2和Bx PC-3),構(gòu)建的穩(wěn)系經(jīng)Western blot驗證了c-FOXP3表達的變化。通過細胞凋亡、細胞周期、Edu增殖摻入與免疫缺陷動物模型證明c-FOXP3對腫瘤細胞無明顯的直接影響。3.免疫系統(tǒng)完整的小鼠體內(nèi)皮下成瘤實驗證實,降表達c-FOXP3的胰腺癌細胞成瘤大小明顯小于其對照組細胞成瘤,并且其瘤塊中Treg浸潤也明顯少于對照組;同時,CD25抗體特異清除免疫系統(tǒng)完整小鼠Treg細胞后,降表達c-FOXP3的胰腺癌細胞成瘤與對照組成瘤大小無統(tǒng)計學差別,證明了Treg細胞參與c-FOXP3對腫瘤細胞生物學功能的間接影響。4.利用體外共培養(yǎng)腫瘤細胞與Treg細胞實驗,通過Edu增殖摻入檢測發(fā)現(xiàn)c-FOXP3不能促進Treg細胞自身增殖。利用體外Transwell趨化實驗,通過流式細胞術(shù)檢測發(fā)現(xiàn),胰腺導管腺癌細胞過表達c-FOXP3后對Treg細胞的趨化能力增強;而降表達c-FOXP3后對Treg的趨化能力下降。裸鼠原位成瘤實驗證實,過表達c-FOXP3的人源胰腺癌細胞成瘤對經(jīng)由鼠尾注射的人外周血單個核細胞中Treg細胞的趨化能力明顯強于未過表達c-FOXP3的對照組成瘤。5.實時定量PCR篩選出趨化因子CCL5表達水平隨c-FOXP3表達變化而變化的程度最為顯著,Western blot證實,胰腺癌細胞內(nèi)CCL5表達水平受c-FOXP3的調(diào)控;ELISA實驗證實,胰腺癌細胞向外釋放CCL5的水平也受c-FOXP3調(diào)控;同時免疫組化染色發(fā)現(xiàn),c-FOXP3與CCL5的表達胰腺癌組織中呈現(xiàn)共定位,并有明顯的正相關(guān)性(r=0.681,P0.001**)。5.利用Panc-1、Pan02和293T細胞系,我們行Ch IP實驗發(fā)現(xiàn)人源與鼠源轉(zhuǎn)錄因子FOXP3均可分別直接結(jié)合于相應種屬CCL5的啟動子區(qū);同時雙熒光素酶實驗證實,過表達c-FOXP3后CCL5啟動子轉(zhuǎn)錄活性增強,而突變上述FOXP3與CCL5結(jié)合位點后,其轉(zhuǎn)錄活性的增強即被反轉(zhuǎn),從而證實c-FOXP3可以直接調(diào)控CCL5的表達。6.體外Transwell趨化模型阻斷實驗發(fā)現(xiàn),通過對CCL5進行中和阻斷可明顯減弱過表達c-FOXP3的胰腺癌細胞對Treg細胞的趨化作用;體內(nèi)胰腺皮下成瘤實驗發(fā)現(xiàn),CCL5阻斷不僅減弱了Treg細胞向腫瘤局部的浸潤,同時抑制了腫瘤的生長,并且該現(xiàn)象在高表達c-FOXP3的腫瘤中更為明顯。結(jié)論1.c-FOXP3蛋白在胰腺導管腺癌組織及細胞系中呈現(xiàn)陽性表達;c-FOXP3表達水平與Treg細胞聚集程度正相關(guān);c-FOXP3表達水平較高且Treg細胞比例高的患者預后較差。2.c-FOXP3直接結(jié)合至CCL5的啟動子區(qū),并促進其轉(zhuǎn)錄翻譯過程,上調(diào)CCL5在胞內(nèi)的表達及其向胞外的分泌。3.以CCL5為介導,c-FOXP3促進了胰腺導管腺癌細胞對Treg細胞的趨化能力。高表達的c-FOXP3與Treg細胞在腫瘤微環(huán)境中的高度浸潤共同作用,促進胰腺導管腺癌腫瘤的生長。4.對高表達c-FOXP3的胰腺導管腺癌腫瘤進行CCL5的中和阻斷,實現(xiàn)了對Treg浸潤和腫瘤生長的抑制作用。
[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.

【學位授予單位】：天津醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R735.9

【參考文獻】

相關(guān)期刊論文 前2條

1 Rosella Spadi;Federica Brusa;Agostino Ponzetti;Isabella Chiappino;Nadia Birocco;Libero Ciuffreda;Maria Antonietta Satolli;;Current therapeutic strategies for advanced pancreatic cancer: A review for clinicians[J];World Journal of Clinical Oncology;2016年01期

2 張峰;呂凌;浦立勇;李相成;姚愛華;張偉;俞悅;王學浩;;CD4~+CD25~+Tr細胞與大鼠肝移植自發(fā)免疫耐受關(guān)系的研究[J];中華外科雜志;2006年21期

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