【作者】 顾永耀；

【导师】 施焕中；

【作者基本信息】 广西医科大学 ， 病理学与病理生理学， 2010， 博士

【摘要】 肺癌是当前世界上最常见的人类恶性肿瘤,其死亡率居癌症相关死因的首位。根据组织学特征及临床特点,肺癌可分为非小细胞肺癌(non-small cell lung cancer, NSCLC)和小细胞肺癌(small cell lung cancer, SCLC)两大类,其中前者约占80%。虽然临床上采用了手术、放疗、化疗等综合性治疗方案,但晚期肺癌患者的预后仍然很差,5年生存率往往不足15%；即使是诊断为早期肺癌的患者,仍有高达50%的几率在手术后复发,而传统化疗对复发患者的有效率不到25%。因此,探寻更为有效的肺癌治疗策略具有重要意义。CD4+CD25+调节性T淋巴细胞是特征性表达白细胞介素-2(interleukin-2, IL-2)受体α链(CD25)的独特CD4+T细胞亚群,具有低反应性和免疫抑制性,属于“职业抑制性T细胞”。CD4+CD25+调节性T淋巴细胞通过其T细胞受体(T cell receptor, TCR)激活发挥抑制活性,一旦激活后其抑制活性是非特异的,可通过细胞间直接接触、分泌抑制性细胞因子等方式抑制CD4+和CD8+等T淋巴细胞的活化、增殖和细胞因子释放。近期研究发现,叉头／翼状螺旋核转录因子FOXP3对调节性T细胞发生及抑制功能发挥极为重要,并被当做最特异的调节性T细胞标记而用于当前研究。已有研究表明,CD4+CD25+FOXP3+调节性T淋巴细胞能削弱抗肿瘤免疫促进肿瘤进展。本研究将探讨非小细胞肺癌中CD4+CD25+FOXP3+调节性T淋巴细胞的数量变化及其临床意义,并研究CD4+CD25+FOXP3+调节性T淋巴细胞局部上调的可能机制,为非小细胞肺癌免疫学治疗提供新的切入点。第一部分非小细胞肺癌CD4+CD25+FOXP3+调节性T淋巴细胞检测及其意义目的：了解非小细胞肺癌患者CD4+CD25+FOXP3+调节性T淋巴细胞的数量变化、免疫表型特征、功能特性及其与临床病理参数间的关系方法：同步采集25例非小细胞肺癌患者肿瘤组织、瘤旁组织和外周血,另采集15例健康对照组外周血。用四色流式细胞术检测CD4+CD25+FOXP3+调节性T细胞的百分比及CD45R0、CTLA4、GITR、CD39、CCR4的表达情况,并结合临床病理参数进行分析。分选肿瘤组织和外周血中CD4+CD25highT细胞和CD4+CD25-T细胞,体外培养并观察CD4+CD25highT细胞对CD4+CD25-T细胞增殖反应的影响。结果：肿瘤组织内CD4+CD25+FOXP3+调节性T细胞占CD4+T细胞的百分比(13.05%±7.16%)显著高于瘤旁组织(3.86%±2.35%)(P<0.001)：患者外周血CD4+CD25+FOXP3+调节性T细胞占CD4+T细胞的百分比(3.43%±2.14%)高于健康人外周血(1.49%±0.80%)(P<0.05)。该细胞亚群高表达CD45R0、CTLA4、GITR、CD39、CCR4分子,并能强烈抑制CD4+CD25-T细胞的增殖反应。CD4+D25+FOXP3+调节性T细胞上调与临床分期、肿瘤大小、组织学类型有关,与年龄、性别、组织学分级、淋巴结转移无关。结论：非小细胞肺癌患者体内CD4+CD25+FOXP3+调节性T细胞上调,能削弱抗肿瘤免疫,促进肿瘤生长和演进。第二部分非小细胞肺癌患者肿瘤局部FOXP3+调节性T细胞上调与诱导转化机制的研究目的：了解非小细胞肺癌患者肿瘤局部CD4+CD25+FOXP3+调节性T淋巴细胞上调是否与诱导转化机制有关。方法：用四色流式细胞术检测11例健康对照外周血和17例非小细胞肺癌患者外周血、肿瘤组织、瘤旁组织中,CD4+CD25+T细胞TGFβ、CD4+CD25-T细胞GFRβⅡ的表达情况。用免疫组织化学方法检测肿瘤组织和瘤旁组织TGFβ的表达。分选肿瘤组织和外周血中CD4+CD25-T细胞,以不同浓度TGFβ重组蛋白体外培养并观察CD4+CD25-T细胞的转变。结果：肿瘤组织CD4+CD25+T细胞膜TGFβ分子表达率高于患者外周血和健康对照外周血(分别为69.50%±22.13%,45.63%±25.10%和22.20%±8.77%)。患者肿瘤组织和外周血CD4+CD25-T细胞TGFβRⅡ分子表达率高于健康对照外周血(分别为11.24%±4.33%,9.74%±4.56%,3.03%±1.65%)。TGFβ在肿瘤组织内表达高于瘤旁组织,主要由间质细胞产生。人重组TGFβ能在较高浓度组有效诱导CD4+CD25-T细胞转化为CD4+CD25+FOXP3+T细胞。结论：肿瘤局部上调的CD4+CD25+FOXP3+调节性T细胞,部分可能是由肿瘤组织来源的TGFβ与高表达TGFβRⅡ的CD4+CD25-T细胞结合,诱导CD4+CD25-T细胞表达FOXP3后转化而来。第三部分非小细胞肺癌患者肿瘤局部CD4+CD25+FOXP3+调节性T细胞上调与趋化机制的研究目的：了解非小细胞肺癌患者肿瘤局部CD4+CD25+FOXP3+调节性T淋巴细胞上调是否与趋化机制有关。方法：流式细胞术分别检测10例健康对照外周血和10例非小细胞肺癌患者外周血、肿瘤组织中,CD4+CD25highT细胞CCR4的表达情况；以及10例患者肿瘤组织中CCL22+CD14+细胞比例和CD4+CD25+FOXP3+T细胞比例。分选肿瘤组织和外周血中CD4+CD25highT细胞,以肿瘤组织培养上清或恶性胸腔积液进行体外趋化实验。结果：肿瘤组织CD4+CD25highT细胞CCR4表达率高于患者外周血和健康对照外周血(分别为69.50%±22.13%,45.63%±25.10%和22.20%±8.77%)。肿瘤组织CCL22+CD14+细胞比例与CD4+CD25+FOXP3+T细胞比例呈正相关关系。肿瘤培养上清和恶性胸腔积液对CD4+CD25highT细胞具有趋化活性,CCL22单克隆抗体可阻断这一趋化活性。结论：患者外周血CD4+CD25+FOXP3+T细胞可通过CCL22-CCR4轴趋化募集到肿瘤局部。第四部分FOXP3+细胞、CD8+细胞与非小细胞肺癌患者预后关系的研究目的：了解非小细胞肺癌患者肿瘤局部FOXP3+细胞、CD8+细胞是否影响预后。方法：运用免疫组织化学方法,标记FOXP3+和CD8+细胞并进行计数。结合患者临床病理参数以及随访情况,分析FOXP3+和CD8+细胞对预后的影响。结果：肿瘤组织FOXP3+细胞、CD8+细胞均高于瘤旁组织。间质内FOXP3+细胞数量与肿瘤分化程度、临床分期有关,但不影响患者累积生存率。间质内CD8+细胞与淋巴结状况、临床分期有关；高CD8+组患者较低CD8+组具有更好的预后。CD8+细胞／FOXP3+细胞比值与预后无关。结论：间质内CD8+细胞而非FOXP3+细胞是非小细胞肺癌患者正向预后指标。更多还原

【Abstract】 Lung cancer is the most common malignant tumour and the leading cause of cancer death in the world. The major histologic subtypes of lung cancer are non-small cell lung cancer (non-small cell lung cancer, NSCLC,80%) and small cell lung cancer (small cell lung cancer, SCLC,20%). Despite medical advances, the overall 5-year survival rate is a dismal 15%. Even when diagnosed at an early stage, patients relapse at a rate as high as 50% after surgical resection. Conventional chemothery are only effective for 25% relapse ptients. These facts make clear the need for new therapeutic strategy that will allow better prognosis for this malignancy. CD4+CD25+regulatory T cells is a distinct population of CD4+T cells that constitutively express the interleukin-2 (IL-2) receptor (R)α-chain (CD25). These cells are both hyporesponsive and suppressive. It was defined as "professional" regulatory/suppressor T cells. Following T cell receptor (TCR) engagement, CD4+CD25+ T cells can suppress the activation and proliferation of other CD4+ and CD8+ T cells in an antigen-nonspecific manner. CD4+CD25+ T cells mediate the suppression of effectors T cell function both in vitro and in vivo via several mechanisms requiring either cell-cell contact or the production of immunosuppressive cytokines. Recent studies have suggested that the transcription factor FOXP3 is critical for the development and function of regulatory T cells in mice and humans. Several studies have suggested that the CD4+CD25+FOXP3+ regulatory T cells may impaire the antitumour immunity and subsequently contribute to malignancy progression. Based on the theory above, our study will focus on the quantity and clinical significance of CD4+CD25+FOXP3+ regulatory T cells in non-small cell lung cancr, and investigate the potential mechanisms about which CD4+CD25+FOXP3+ regulatory T cells increased in tumour microenviroment. The purpose is to help to find a new strategy for the immune treatment of non-small cell lung cancr.PART I:DETECTION AND SIGNIFICANCE OF CD4+CD25+FOXP3+REGULATORY T CELL IN NON-SMALL CELL LUNG CANCEROBJECTIVES:To detect the proportion, phenotypic profile, function of CD4+CD25+FOXP3+ regulatory T lymphocyte in non-small cell lung cancer and to determine whether the proportion is associated with clinicopathological parameters.METHODS:The percentages of CD4+CD25+FOXP+T lymphocytes in tumour tissue, non-tumour tissue and peripheral blood from patients with non-small cell lung cancer, and peripheral blood from healthy control were determined by four-color flow cytometry. The expressions of CD45R0, CTLA4, GITR, CD39, CCR4 were also examined. Meanwhile, the relationship between clinicopathological parameters and the percentages of CD4+CD25+FOXP+T lymphocytes was determined. CD4+CD25high and CD4+CD25-T cells from tumour tissue and peripheral blood were isolated, and were cultured to investigate the effects of CD4+CD25high cells on proliferation response of CD4+CD25-T cells in vitro.RESULTS:There were increased percentages of CD4+CD25+FOXP3+T cells in tumour tissue (13.05%±7.16%) compared with non-tumour tissue (3.86%±2.35%) from patients with non-small lung cancer, in peripheral blood from patients (3.43%±2.14%) compared with peripheral blood from healthy control (1.49%±0.80%), and that these cells have constitutive high-level expression of CD45RO、CTLA4、GITR、CD39、CCR4. CD4+CD25high T cells mediate potent inhibition of proliferation response of CD4+CD25- T cells in vitro. The proportion of CD4+CD25+FOXP3+ T cells is related to TNM stage, tumour size, and histologic type.CONCLUSIONS:The proportion of CD4+CD25+FOXP3+ T cells was increased in patients with non-small cell lung cancer, which consequently resulted in the impairment of anti-tumour immunity and advancement of tumor progression.PARTⅡ:RESERCH OF INDUCED-CONVERSION MACHENISM AND UP-REGULATION OF CD4+CD25+FOXP3+ REGULATORY T LYMPHOCYTE IN PATIENTS WITH NON-SMALL CELL LUNG CANCEROBJECTIVES:To investigate whether the induced-conversion mechanism is concerned with the up-regulation of CD4+CD25+FOXP3+ T cells in tumour microinviroment.METHODS:Lymphocytes isolated from peripheral blood, tumor tissue and non-tumor tissue of lung cancer patiens(n=17) were analyzed for expression of TGFβon CD4+CD25+T lymphocytes and of TGF βRⅡon CD4+CD25-T lymphocytes by flow cytometry. Lymphocytes from health adults(n=11) were served as control.The expression of TGFβin tumor and nontumor tissue was determined by immunohistochem-istry. CD4+CD25-T cells isolated from tumour tissue and peripheral blood were cocultured with hrTGFβor monoclonal TGFβantibody to investigate whether hrTGFβcan convert CD4+CD25-T cells into CD4+CD25+FOXP3+T cells in vitro.RESULTS:The expression of membrane TGF 0 on CD4+CD25+T lymphocytes from tumour tissue was significantly higher than peripheral blood. The expression of membrane TGFβRⅡon CD4+CD25-T lymphocytes from tumour tissue and peripheral blood was also significantly higher than peripheral blood from healthy donors. TGFβ, mainly produced by stromal cells, was prominent in tumour tissues. hrTGFβcan actually convert CD4+CD25-T cells into CD4+CD25+FOXP3+T cells at higher concertration in vitro.CONCLUSIONS:Up-regulation of CD4+CD25+FOXP3+T cells in tumour microenviroment may be partially due to TGFβinduced-conversion. Both TGFβ,produced by stromal cells, and higher expression of TGFβRⅡon CD4+CD25-T lymphocytes contribute to this process.PARTⅢ:RESERCH OF CHMOTATIC MACHENISM AND UP-REGULATION OF CD4+CD25+FOXP3+ REGULATORY T LYMPHOCYTE IN PATIENTS WITH NON-SMALL CELL LUNG CANCEROBJECTIVES:To investigate whether the chemotatic mechanism is concerned with the up-regulation of CD4+CD25+FOXP3+ T cells in tumour microinviroment. METHODS:Lymphocytes isolated from peripheral blood, tumor tissue of lung cancer patiens were analyzed by flow cytometry for expression of CCR4 on CD4+CD25highT lymphocytes. Lymphocytes from health adults were served as control. The proportion of CCL22+ CD14+cells and CD4+CD25+FOXP3+T cells in tumor tissue were also analyzed by flow cytometry. CD4+CD25highT cells isolated from peripheral blood, tumour supernatants and malignant pleural effusion were used for in vitro chemotatic experiments.RESULTS:The expression of CCR4 on CD4+CD25+T lymphocytes from tumour tissue was significantly higher than peripheral blood. The proportion of CCL22+CD14+cells was positively related to that of CD4+CD25+FOXP3+T cells in tumor tissue. CD4+CD25high T cells were attracted by tumour supernatants and malignant pleural effusion in vitro, and monoclonal antibody to CCL22 can block this chemotactic activity.CONCLUSIONS:CD4+CD25+FOXP3+T cells in peripheral blood can be selectively attracted into tumour microenviroment by CCL22-CCR4 axis.PART IV:RESERCH OF FOXP3+CELLS, CD8+CELLS AND THE PROGNOSIS OF PATIENTS WITH NON-SMALL CELL LUNG CANCEROBJECTIVES:To investigate whether the numbers of FOXP3+cells CD8+ cells in tumour tissue impact the prognosis of patients with non-small cell lung cancer.METHODS:Immunohistochemistry was used to evaluate the epithelial and stromal FOXP3+, CD8+ cells. Combined with clinicopathological parameters and follow up data, the clinical significances of FOXP3+ cells and CD8+ cells were determined.RESULTS:The numbers of FOXP3+ cells and CD8+ cells from tumour tissue were significantly higher than non-tumour tissue. Stromal FOXP3+ cells related to differentiation and TNM stage, but had no effect on survival. Stromal CD8+ cells related to nodal status and TNM stage. Increasing numbers of stromal CD8+ cells significantly correlated to an improved survival. The ratio of CD8+ cells to FOXP3+ cells had no effect on prognosis.CONCLUSIONS:Stromal CD8+ cells, not FOXP3+ cells were positive prognostic indicators for resected NSCLC patients.更多还原