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

【摘要】：背景FOXP3是主要表達(dá)于CD4+CD25+調(diào)節(jié)性T細(xì)胞的標(biāo)志性轉(zhuǎn)錄因子[1]。它的主要功能是促進(jìn)T細(xì)胞向調(diào)節(jié)性T細(xì)胞的分化,促進(jìn)機(jī)體的免疫抑制功能[2]。然而,近期多項(xiàng)研究顯示,除外T細(xì)胞,FOXP3也被發(fā)現(xiàn)表達(dá)于多種腫瘤細(xì)胞,如乳腺癌細(xì)胞、腎癌細(xì)胞與黑色素瘤細(xì)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(dǎo)管腺癌的靶向治療提供新的靶點(diǎn)。我們在前期實(shí)驗(yàn)與文獻(xiàn)閱讀中發(fā)現(xiàn),相較于正常組織和細(xì)胞系,胰腺導(dǎo)管腺癌組織及細(xì)胞系高表達(dá)c-FOXP3[6]。因此,我們設(shè)計了該課題以進(jìn)一步明確c-FOXP3在胰腺導(dǎo)管腺癌組織及細(xì)胞中的表達(dá)水平、功能效應(yīng)、以及背后的調(diào)控機(jī)制;旨從臨床患者水平、細(xì)胞功能水平、分子生物學(xué)水平及動物實(shí)驗(yàn)水平深入探索胰腺導(dǎo)管腺癌中c-FOXP3的功能與意義。方法1.應(yīng)用胰腺導(dǎo)管腺癌組織的石蠟標(biāo)本切片進(jìn)行FOXP3的免疫組化染色,分析c-FOXP3蛋白在胰腺導(dǎo)管腺癌組織中的表達(dá)水平;同時收集整理患者的臨床病例資料并行預(yù)后隨訪,分析胰腺導(dǎo)管腺癌中c-FOXP3的表達(dá)水平與各項(xiàng)臨床病理指標(biāo)之間的關(guān)系。2.分析胰腺導(dǎo)管腺癌組織標(biāo)本中c-FOXP3表達(dá)水平與Treg細(xì)胞在腫瘤局部富集程度的相關(guān)性;并分析c-FOXP3表達(dá)于Treg細(xì)胞聚集對胰腺導(dǎo)管腺癌患者臨床病理指標(biāo)及預(yù)后的共同影響。3.應(yīng)用Western blot實(shí)驗(yàn)技術(shù)檢測4個胰腺癌細(xì)胞系及正常胰腺導(dǎo)管細(xì)胞系的c-FOXP3的表達(dá)水平,并構(gòu)建c-FOXP3過表達(dá)與降表達(dá)穩(wěn)定轉(zhuǎn)染細(xì)胞系;用Western blot驗(yàn)證構(gòu)建的穩(wěn)系所表達(dá)c-FOXP3的水平。通過細(xì)胞凋亡、細(xì)胞周期、Edu增殖摻入實(shí)驗(yàn)與免疫缺陷動物模型等多種方法檢測c-FOXP3對腫瘤細(xì)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(xì)胞(Peripheral Mononuclear Cells,PBMCs)與調(diào)節(jié)性T細(xì)胞(Regulatory T Cells,Treg cells)。利用共培養(yǎng)系統(tǒng)檢測c-FOXP3對Treg細(xì)胞的促增殖作用;利用Transwell系統(tǒng)在體外模擬Treg細(xì)胞向腫瘤微環(huán)境的募集過程,檢測Treg細(xì)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(xì)胞(PBMCs),體內(nèi)驗(yàn)證c-FOXP3促Treg細(xì)胞向胰腺癌微環(huán)境的趨化作用,5.應(yīng)用RT-PCR的方法篩選c-FOXP3促進(jìn)腫瘤細(xì)胞所分泌的趨化因子譜;應(yīng)用Western blot、ELISA及免疫組化染色等實(shí)驗(yàn)驗(yàn)證RT-PCR結(jié)果;應(yīng)用CHIP及雙熒光素酶實(shí)驗(yàn)檢測c-FOXP3調(diào)控趨化因子表達(dá)分泌的分子機(jī)制。6.通過趨化因子阻斷試驗(yàn)確定c-FOXP3促進(jìn)Treg細(xì)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(jìn)一步確定c-FOXP3引起Treg細(xì)胞向腫瘤微環(huán)境募集的通路及該通路的生物學(xué)功能,確定胰腺導(dǎo)管腺癌靶向治療目標(biāo),從而實(shí)現(xiàn)臨床轉(zhuǎn)化。結(jié)果1.c-FOXP3表達(dá)及Treg細(xì)胞聚集在胰腺導(dǎo)管腺癌組織中的意義。通過對120例胰腺導(dǎo)管腺癌組織標(biāo)本進(jìn)行免疫組織化學(xué)染色發(fā)現(xiàn),c-FOXP3在胰腺導(dǎo)管腺癌中多數(shù)表達(dá)陽性,而對應(yīng)的癌旁正常胰腺組織不表達(dá)或表達(dá)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(dá)的占63.3%(76例),c-FOXP3低表達(dá)的占36.6%(44例);進(jìn)一步研究c-FOXP3蛋白表達(dá)水平與臨床病理指標(biāo)之間的關(guān)系,我們發(fā)現(xiàn)高表達(dá)c-FOXP3的胰腺導(dǎo)管腺癌患者的總生存期(中位數(shù):24 vs 15個月)與無復(fù)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(dá)組的患者(p0.05*)。同時,FOXP3陽性的Treg細(xì)胞在胰腺癌組織中也有不同程度的表達(dá)。我們深入研究了胰腺導(dǎo)管腺癌腫瘤細(xì)胞中c-FOXP3的表達(dá)與FOXP3陽性Treg細(xì)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(dá)同時伴隨Treg細(xì)胞在腫瘤中高度聚集的患者的總生存期及無復(fù)發(fā)生存期明顯短于c-FOXP3高表達(dá)Treg低浸潤的患者,同時,c-FOXP3高表達(dá)同時Treg細(xì)胞高度浸潤的患者的腫瘤大小也明顯大于c-FOXP3高表達(dá)Treg低浸潤的患者;這些結(jié)果提示,Treg細(xì)胞浸潤程度在c-FOXP3高表達(dá)對患者生存期的影響中發(fā)揮了重要的生物學(xué)功能,引起胰腺導(dǎo)管腺癌的進(jìn)展。2.c-FOXP3對胰腺導(dǎo)管腺癌細(xì)胞系的直接與間接影響。利用Western blot驗(yàn)證了c-FOXP3在4個胰腺癌細(xì)胞系(PANC-1、MIA Pa Ca-2、As PC-1及Bx PC-3)均有不同程度的表達(dá),而在正常胰腺細(xì)胞系HPDE6C7中表達(dá)極弱;并且成功構(gòu)建了2個過表達(dá)c-FOXP3的胰腺癌細(xì)胞系(PANC-1及As PC-1)和2個降表達(dá)c-FOXP3的細(xì)胞系(MIA Pa Ca-2和Bx PC-3),構(gòu)建的穩(wěn)系經(jīng)Western blot驗(yàn)證了c-FOXP3表達(dá)的變化。通過細(xì)胞凋亡、細(xì)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(xì)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(shí)驗(yàn)證實(shí),降表達(dá)c-FOXP3的胰腺癌細(xì)胞成瘤大小明顯小于其對照組細(xì)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(xì)胞后,降表達(dá)c-FOXP3的胰腺癌細(xì)胞成瘤與對照組成瘤大小無統(tǒng)計學(xué)差別,證明了Treg細(xì)胞參與c-FOXP3對腫瘤細(xì)胞生物學(xué)功能的間接影響。4.利用體外共培養(yǎng)腫瘤細(xì)胞與Treg細(xì)胞實(shí)驗(yàn),通過Edu增殖摻入檢測發(fā)現(xiàn)c-FOXP3不能促進(jìn)Treg細(xì)胞自身增殖。利用體外Transwell趨化實(shí)驗(yàn),通過流式細(xì)胞術(shù)檢測發(fā)現(xiàn),胰腺導(dǎo)管腺癌細(xì)胞過表達(dá)c-FOXP3后對Treg細(xì)胞的趨化能力增強(qiáng);而降表達(dá)c-FOXP3后對Treg的趨化能力下降。裸鼠原位成瘤實(shí)驗(yàn)證實(shí),過表達(dá)c-FOXP3的人源胰腺癌細(xì)胞成瘤對經(jīng)由鼠尾注射的人外周血單個核細(xì)胞中Treg細(xì)胞的趨化能力明顯強(qiáng)于未過表達(dá)c-FOXP3的對照組成瘤。5.實(shí)時定量PCR篩選出趨化因子CCL5表達(dá)水平隨c-FOXP3表達(dá)變化而變化的程度最為顯著,Western blot證實(shí),胰腺癌細(xì)胞內(nèi)CCL5表達(dá)水平受c-FOXP3的調(diào)控;ELISA實(shí)驗(yàn)證實(shí),胰腺癌細(xì)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(dá)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(xì)胞系,我們行Ch IP實(shí)驗(yàn)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(yīng)種屬CCL5的啟動子區(qū);同時雙熒光素酶實(shí)驗(yàn)證實(shí),過表達(dá)c-FOXP3后CCL5啟動子轉(zhuǎn)錄活性增強(qiáng),而突變上述FOXP3與CCL5結(jié)合位點(diǎn)后,其轉(zhuǎn)錄活性的增強(qiáng)即被反轉(zhuǎn),從而證實(shí)c-FOXP3可以直接調(diào)控CCL5的表達(dá)。6.體外Transwell趨化模型阻斷實(shí)驗(yàn)發(fā)現(xiàn),通過對CCL5進(jìn)行中和阻斷可明顯減弱過表達(dá)c-FOXP3的胰腺癌細(xì)胞對Treg細(xì)胞的趨化作用;體內(nèi)胰腺皮下成瘤實(shí)驗(yàn)發(fā)現(xiàn),CCL5阻斷不僅減弱了Treg細(xì)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(dá)c-FOXP3的腫瘤中更為明顯。結(jié)論1.c-FOXP3蛋白在胰腺導(dǎo)管腺癌組織及細(xì)胞系中呈現(xiàn)陽性表達(dá);c-FOXP3表達(dá)水平與Treg細(xì)胞聚集程度正相關(guān);c-FOXP3表達(dá)水平較高且Treg細(xì)胞比例高的患者預(yù)后較差。2.c-FOXP3直接結(jié)合至CCL5的啟動子區(qū),并促進(jìn)其轉(zhuǎn)錄翻譯過程,上調(diào)CCL5在胞內(nèi)的表達(dá)及其向胞外的分泌。3.以CCL5為介導(dǎo),c-FOXP3促進(jìn)了胰腺導(dǎo)管腺癌細(xì)胞對Treg細(xì)胞的趨化能力。高表達(dá)的c-FOXP3與Treg細(xì)胞在腫瘤微環(huán)境中的高度浸潤共同作用,促進(jìn)胰腺導(dǎo)管腺癌腫瘤的生長。4.對高表達(dá)c-FOXP3的胰腺導(dǎo)管腺癌腫瘤進(jìn)行CCL5的中和阻斷,實(shí)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(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R735.9

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相關(guān)期刊論文 前2條

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