【作者】 程万民；

【导师】 郑春泉； 王纾宜； 藏朝平； 余洪猛；

【作者基本信息】 复旦大学 ， 耳鼻咽喉科学， 2007， 博士

【摘要】 第一部分：鼻息肉组织Th细胞亚群的流式细胞术测定背景鼻息肉是临床常见病，手术后极易复发，尚无根治性手段。尚无确定的临床病理分型和围手术期综合治疗原则。鼻息肉的发生机制也不确定，关于鼻息肉的发生机制，主要存在两种学说之争，即金黄色葡萄球菌超抗原学说和真菌变态反应学说。细菌、病毒、真菌及变应原等可能作为最初的触发因素引起鼻腔外侧壁的炎症而发展形成鼻息肉。鼻息肉组织含大量炎症细胞如嗜酸性粒细胞和淋巴细胞，这些细胞可能在鼻息肉发病中起重要作用。T淋巴细胞按表面分子不同分为CD4+T淋巴细胞（又称辅助T淋巴细胞，T help cell，Th细胞）和CD8+T淋巴细胞（又称细胞毒性T细胞，CTL，或杀伤性T细胞，TC）。Th细胞按产生细胞因子和功能的不同又分为Th1细胞和Th2细胞。功能不同的辅助T淋巴细胞（Th1细胞、Th2细胞）存在的证据对基础和临床免疫学产生了重大影响。Th1和Th2细胞在免疫应答的平衡中起关键性作用。Th1细胞主要产生干扰素-γ（IFN-γ），促进细胞免疫，增强吞噬细胞的抗感染机制；Th2细胞主要产生白介素-4（IL-4），促进体液免疫，在变态反应中起作用。这些基础免疫学发现结合鼻息肉的发生机制，可以推测在鼻息肉组织中，Th1细胞可能与金葡菌感染引起的免疫应答有关，Th2细胞可能与真菌及变应原引起的变态反应有关。关于鼻息肉组织T细胞亚群及细胞因子的研究已有报道，但对Th细胞亚群的深入研究尚少，尚无关于合并与不合并AR的鼻息肉在Th细胞亚群方面的研究报道。本实验以流式细胞术测定合并与不合并AR的鼻息肉组织的Th1和Th2细胞百分率，以明确不同类型鼻息肉组织在Th细胞亚群和免疫应答类型方面的差异，评价金葡菌感染和真菌变态反应因素在不同类型鼻息肉病因学中的作用，评价金黄色葡萄球菌超抗原学说和真菌变态反应学说对不同类型鼻息肉发生形成的意义，指导鼻息肉分型和综合治疗。方法32例鼻息肉病例分为两组，其中包括不合并变应性鼻炎（AR）的鼻息肉组16例，合并变应性鼻炎的鼻息肉组16例，依据变应性鼻炎病史和临床表现，以及变应原点刺试验区分不合并AR的鼻息肉与合并AR的鼻息肉。新鲜手术标本制备单细胞悬液，流式细胞仪测定Th1和Th2细胞百分率，在CD4+细胞中，细胞内产生IFN-γ的为Th1细胞，产生IL-4的为Th2细胞，同时产生IFN-γ和IL-4的为Th0细胞。测得的Th细胞百分率用（?）±s表示，用SPSS for Windows Ver．11.5软件进行统计分析，对两鼻息肉组的Th1、Th2细胞百分率进行正态性检验和独立样本t检验。结果鼻息肉组织含有大量Th细胞并且以Th1细胞为主；不合并AR的鼻息肉组Th1和Th2细胞平均百分率分别为46.28％±14.95％和0.63％±0.31％；合并AR的鼻息肉组Th1和Th2细胞平均百分率分别为38.25％±9.16％和7.34％±2.54％；统计学分析显示不合并AR的鼻息肉组与合并AR的鼻息肉组相比Th1细胞平均百分率无显著差异，t=1.83，P＞0.05；合并AR的鼻息肉组Th2细胞平均百分率显著高于不合并AR的鼻息肉组，t=10.48，P＜0.01。结论鼻息肉组织以Th1细胞亚群为主；合并AR的鼻息肉Th2细胞数显著高于不合并AR的鼻息肉，合并与不合并AR的鼻息肉在Th细胞亚群和免疫应答类型方面存在差异；金葡菌感染可能在不合并AR的鼻息肉的发生形成中起关键作用，金葡菌感染和真菌变态反应因素可能在合并AR的鼻息肉的发生形成中均有一定作用。第二部分：鼻息肉组织嗜酸性粒细胞浸润与Th2细胞的关系背景鼻息肉组织含大量炎症细胞如嗜酸性粒细胞和淋巴细胞，这些细胞可能在鼻息肉的发生和形成中起重要作用。在过去的二十年中，对嗜酸性粒细胞在某些病理生理过程中的作用有了逐步认识。嗜酸性粒细胞的前体细胞从骨髓释放进入血液循环，在化学趋化性因子的作用下进入作用部位，嗜酸性粒细胞的发育和成熟可发生在外周炎症部位。活化嗜酸性粒细胞在哮喘、寄生虫病、肉芽肿病变、纤维化、许多恶性肿瘤及鼻腔鼻窦疾病中起作用。在变应性鼻炎（AR），嗜酸性粒细胞从外周血浸润到鼻腔粘膜组织后主要参与迟发相反应，Th-2细胞分泌的细胞因子可促使嗜酸性粒细胞的集聚和活化。在Th-2类细胞因子中，IL-4起中心性作用，它可促使IgE的合成，通过上调粘附分子的表达引起嗜酸性粒细胞集聚，增加粘液的生成。嗜酸性粒细胞的颗粒中含有许多细胞毒性蛋白包括嗜酸性细胞阳离子蛋白（ECP），主要碱性蛋白（MBP），嗜酸性粒细胞过氧化物酶（EPO）和嗜酸性细胞来源神经毒素。嗜酸性粒细胞的细胞毒性作用可能在针对变应原和对抗真菌的免疫反应中起关键性作用。关于鼻息肉组织嗜酸性粒细胞浸润的研究已有报道，但对合并与不合并AR的鼻息肉组织嗜酸性粒细胞浸润程度差异的研究尚少，尚无鼻息肉组织嗜酸性粒细胞浸润与Th2细胞关系的研究报道。本实验对合并与不合并AR的鼻息肉组织石蜡标本HE切片进行嗜酸性粒细胞计数，并分析嗜酸性粒细胞数与与Th2细胞百分率的相关性，以明确合并与不合并AR的鼻息肉组织在嗜酸性粒细胞浸润数量上的差别，明确嗜酸性粒细胞浸润与Th2细胞的关系，探讨鼻息肉组织嗜酸性粒细胞浸润的发生机制。方法对两组鼻息肉病例常规病理标本HE切片进行嗜酸性粒细胞浸润程度的定量，计数每高倍视野平均嗜酸性粒细胞数。嗜酸性粒细胞数用均数±标准差表示。使用SPSS for Windows Ver. 11.5软件对两鼻息肉组的嗜酸性粒细胞数进行正态性检验和独立样本t检验，并对全部病例的Th2细胞百分率与嗜酸性粒细胞数进行相关性分析。结果不合并AR的鼻息肉组嗜酸性粒细胞数为14.38±5.6，合并AR的鼻息肉组嗜酸性粒细胞数为54.5±15.76；合并AR的鼻息肉组织嗜酸性粒细胞数明显高于不合并AR的鼻息肉组织，差异有统计学意义，P＜0.02；Th2细胞平均百分率与嗜酸性粒细胞数存在正相关，r=0.80，P＜0.01。结论合并AR的鼻息肉组织嗜酸性粒细胞数显著高于不合并AR的鼻息肉组织；鼻息肉组织的Th2细胞与嗜酸性粒细胞数相关，Th2细胞可能通过释放Th2细胞因子促使鼻息肉组织嗜酸性粒细胞浸润。第三部分：鼻息肉组织Th细胞分化相关转录因子表达背景鼻息肉的发生机制尚不确定，关于鼻息肉的发生机制，主要存在两种学说之争，即金黄色葡萄球菌超抗原学说和真菌变态反应学说。细菌、病毒、真菌及变应原等可能作为最初的触发因素引起鼻腔外侧壁的炎症而发展形成鼻息肉。幼稚CD4细胞分化为成熟的Th细胞亚群是机体防御机制和免疫介导疾病发病机制的关键过程，Th细胞分化决定免疫应答的性质。Th1细胞主要分泌IL-2、IFN-γ、TNF，功能是促进细胞免疫，增强吞噬细胞介导的抗感染机制；Th2细胞主要分泌IL-4、IL-5、IL-10、IL-13，功能是促进B细胞增殖、分化和产生抗体，增强B细胞介导的体液免疫应答，在变态反应和机体抗寄生虫免疫中发挥作用。局部环境中的细胞因子以及细胞内关键转录因子在Th分化中起主要作用。T细胞表达T-box（T-box expressed in T cells，T-bet）和GATA结合蛋白3（GATA-binding protein 3，GATA3）是Th细胞分化相关的关键转录因子，T-bet促使Th0细胞向Th1细胞方向分化；GATA3促使Th0细胞向Th2细胞方向分化，并且GATA3与T-bet存在相互拮抗的作用。这些基础免疫学发现结合鼻息肉的发生机制，可以推测在鼻息肉组织中，Th1细胞及其分化相关的转录因子T-bet可能与金葡菌感染引起的免疫应答有关，Th2细胞及其分化相关的转录因子GATA3可能与真菌及变应原引起的变态反应有关。目前尚无关于合并与不合并AR的鼻息肉组织在Th细胞分化相关转录因子表达方面的研究报道。本实验对合并与不合并AR的鼻息肉及对照下鼻甲液氮冷冻标本采用实时荧光定量PCR法测定Th1细胞分化相关转录因子T-bet和Th2细胞分化相关转录因子GATA-3 mRNA表达，以进一步明确合并与不合并AR的鼻息肉组织在Th细胞分化和免疫应答类型方面的差异，评价金葡菌感染和真菌变态反应因素在不同类型鼻息肉病因学中的作用，评价金黄色葡萄球菌超抗原学说和真菌变态反应学说对鼻息肉不同类型发生形成的意义，指导鼻息肉分型和综合治疗。方法标本为液氮冷冻标本，包括对照下鼻甲组织10例，不合并AR的鼻息肉16例及合并AR的鼻息肉16例。根据试剂使用说明用TRIzol Reagent从液氮冷冻下鼻甲和鼻息肉组织提取总RNA：DNA酶Ⅰ去除基因组DNA；使用逆转录酶及2μg提取的总RNA合成cDNA；实时荧光定量PCR在30μl容积中进行：其中包含27.5μl TaqMan Universal PCR Master Mix，0.6μl正向引物，0.6μl反向引物，0.3μl探针和1μl模板（100ng）。反应条件为：95℃3 min预变性，然后（95℃变性20s，60℃的退火／延伸20s）×60循环。磷酸甘油醛脱氢酶（GAPDH）基因表达做内参。实时荧光定量PCR在实时荧光定量PCR仪BIO RAD Icycler 5中进行，用荧光定量PCR分析软件Icycler version3.1.7050进行分析。结果鼻息肉组织和对照下鼻甲组织的GATA-3 mRNA及T-bet mRNA表达的相对量用2^—ΔCT值表示。单因素方差分析结果显示三组标本GATA-3及T-bet mRNA表达差异有统计学意义，F=83.26，P＜0.01；F=17.6，P＜0.01。两两比较结果显示不合并AR的鼻息肉及合并AR的鼻息肉组织GATA-3、T-bet mRNA表达分别显著高于下鼻甲组织，P＜0.05或P＜0.01；两鼻息肉组之间T-bet mRNA表达无显著性差异，P＞0.05；合并AR的鼻息肉GATA3 mRNA表达显著高于另两组，P值均小于0.01。结论鼻息肉组织GATA-3 mRNA及T-bet mRNA表达高于下鼻甲组织，鼻息肉的发生和形成是长期鼻腔慢性持续性炎症的结果；合并与不合并AR的鼻息肉组织在Th2细胞分化相关转录因子GATA-3 mRNA表达方面存在差异，合并与不合并AR的鼻息肉在Th细胞分化和免疫应答类型方面存在差异；金葡菌感染可能在不合并AR的鼻息肉的发生形成中起关键作用，金葡菌感染和真菌变态反应因素可能在合并AR的鼻息肉的发生形成中均有一定作用。更多还原

【Abstract】 Th Cell Population, Eosinophilia and Related Transcription Factors in Nasal PolypsPARTⅠTh1 and Th2 Cell Population in Nasal Polyps, A flow cytometry AssayBackground The nasal polyposis (NP) remains a common clinical entity. Classification of nasal polyp is a controversial topic. Its etiology and pathogenesis is still not entirely known. There exist two theories concerning the pathogenesis of nasal polyps, the staphylococcus aureus superantigen theory and the fungul allergy theory. The NPs is the ultimate manifestation of chronic inflammation. Bacterial infection, viral infection, fungal infection, allergy, and environmental pollution have all been suggested as possible initial triggers that may upregulate inflammation of the lateral wall of the nose to develop NPs. In most cases, the lamina propria of NPs demonstrates large numbers of eosinophils and lymphocytes. These infiltrated cells may play a part in pathogenesis of the polyp.Demonstration of the existence and functions of T help cells (Th1 and Th2 cells) has been an enormous impact on basic and applied immunology. Th1 and Th2 cells have a crucial role in balancing the immune response. Th1 cells produce interferon-gamma (IFN-γ), promoting cell-mediated immunity and control of intracellular pathogens. It related with the immune response induced by infectious agents. In contrast, Th2 cells produce interleukin-4 (IL-4), which promotes allergic responses. It related with the immune response induced by allergy.Previously, the study about T cell in chronic rhinosinusitis (CRS) and nasal polyps (NP) was limited to the T cell subpopulation and cytokines, but lacking deep study on Th cell. There was no reports concerning the Th cell in NPs with and without allergy. To analyze the immunological pattern of NP in patients with and without allergy, and evaluate the role of infectious agents and allergy in the etiology and pathogeneses of NPs, the percentages of CD4+ cells expressing intracellular IFN-γand IL-4 (Th1 and Th2 cells) were measured by flow cytometry. Methods 16 cases of NPs without allergy and 16 cases of NPs with allergy were involved in this study. Based on medical history of allergy and skin prick test, the NPs with allergy or the NPs without allergy were differentiated. The fresh NP samples were prepared into single cell suspension for flow cytometric analysis. Th1 cells were defined as CD4+ lymphocytes with intracellular IFN-γbut without intracellular IL-4. Th2 cells were defined as CD4+ lymphocytes with intracellular IL-4 but without intracellular IFN-γ.Results Nasal polyp possesses both Th1 and Th2 cells, but Th1 cells presented the majority in all the NPs. There was no significant difference in the mean percentages of IFN-γ-producing Th1 cells between the NPs without allergy group (Mean＝46.28, SD＝4.95) and the NPs with allergy group (Mean＝38.25, SD＝9.16; P＞0.05); The mean percentages of IL-4-producing Th2 cells in the NPs with allergy group(Mean＝7.34, SD＝2.54) were significantly higher than that in the NPs without allergy group(Mean＝0.63, SD＝0.31, P＜0.01 ).Conclusions Th1 cells were predominant in NPs; The NPs with allergy possesses more Th2 cells than the NPs without allergy did; The NPs without allergy and the NPs with allergy have some difference in immunological pattern.PARTⅡEosinophilia and Its correlation with Th2 Cells in Nasal PolypsBackground During the last two decades, there has been an increased awareness regarding the role of the eosinophil in several physiologic and pathologic processes. Eosinophil progenitors are released from the blood marrow into the circulation and are chemically attracted to the site of action by chemotactic factors. Development and maturation of eosinophils can also occur in situ in peripheral sites of inflammation containing pre-existing increased tissue eosinophils. The eosinophil is involved in physiologic and pathologic processes, such as asthma, parasitic diseases, granulomatous disorders, fibrosis, malignant tumors and several sino-nasal diseases. In AR, eosinophils are mainly involved in the late-phase reaction after infiltration from the peripheral blood into the tissue. Cytokines secreted by Th-2 cells account for recruiting and activating the eosinophils in the nose. Among them, IL-4 is considered to be pivotal, since it promotes IgE synthesis, up-regulates adhesion molecules selective for eosinophil recruitment, and causes increased mucus production.The role of the eosinophil in the pathogenesis of CRS and NP has been particularly emphasized in the past few years. The lamina propria of NP demonstrates large numbers of eosinophils. Eosinophilic granulocytes are known to carry a variety of cytotoxic proteins in their granula, which can be released by degranulation. These proteins include the eosinophilic cationic protein(ECP), major basic protein(MBP), eosinophilic peroxidase(EPO) and eosinophil-derived neurotoxin(EDN). The cytotoxicity of the eosinophils may be a crucial element of an immunological reaction against allergen and fungi, which are present in the nasal mucus. Histopathologic studies of the CRS and NP demonstrate eosinophilic infiltration was a characteristic of NP, but there was less study concerning the eosinophilic infiltration difference in NPs with and without allergy, and there was no reports concerning the relation of the eosinophilic infiltration with the Th2 cells population. In these experiment, to define the eosinophilia in NPs with and without allergy and explore the eosinophilic infiltration mechanism in NPs, eosinophil counting were carried out on the H&E staining sections of all the NP samples. The correlation between the mean percentages of the Th2 cells and the eosinophilia were analyzed.Methods Eosinophil counting were carried out on the H&E staining sections of all the NP samples. The mean percentages of IL-4-producing Th2 cells were measured by flow cytometric analysis. Using SPSS for Windows Ver.11.5 Statistics Package, the average eosinophils number of the two patient group were compared by Independent Samples T-test. The correlation between the mean percentages of the Th2 cells and average eosinophils number of all cases were analysed by Bivariate Correlaions.Results Eosinophilia was significantly upregulated in the NPs with allergy group (Mean＝54.5, SD＝15.76) compared with it did in the NPs without allergy group(Mean＝14.38, SD＝5.6, P＜0.01 ). Additionally, significant positive relationships were found between the mean percentages of IL-4-producing Th2 cells and the average eosinophil numbers in all the polyps (r＝0.80, P＜0.01).Conclusions Eosinophilia was significantly upregulated in the NPs with allergy. The cytotoxicity of the eosinophils may be a crucial element of an immunological reaction against allergen and fungi, which are present in the nasal mucus. Eosinophilia may induced by Th2 cells in the NPs.PARTⅢTh Cell Differentiation Related Transcription Factors in Nasal PolypsBackground The differentiation of naive CD4+ T cells into Thl or Th2 effector cells is a critical process during immune responses. Th1 cells produce interferon-gamma (IFN-γ), promoting cell-mediated immunity and control of intracellular pathogens. It related with the immune response induced by infectious agents. In contrast, Th2 cells produce interleukin-4 (IL-4), which promotes allergic responses. It related with the immune response induced by allergy. T-bet and GATA3 are two important transcription factor in regulating Th cell differentiation. T-bet promotes Th1 cell differentiation, and GATA3 promotes Th2 cell differentiation. T-bet and GATA3 have antagonistic effect on each other. There was no reports concerning the Th cell differentiation related transcription factor in NPs with and without allergy. To analyze the immunological pattern of NPs in patients with and without allergy, and evaluate the role of infectious agents and allergy in the etiology and pathogeneses of NPs, the mRNA expression of T-bet and GATA-3 were analyzed by real-time quantitative PCR in NPs with allergy and the NPs without allergy.Methods The samples were devided into three groups, which include 16 cases of NPs with allergy, 16 cases of NPs without allergy and 10 cases of inferior turbinate mucosa samples. Liquid nitrogen stored samples were analyzed by real-time quantitative PCR for mRNA expression of T-bet and GATA-3. Total RNA was extracted from tissue using TRIzol Reagent, Using reverse transcriptase, cDNA was synthesized. Real-time PCR analyses were performed in BIO RAD Icycler 5 and analyzed by Icycler version3.1.7050 software.Results The three groups have significant difference in GATA3 and T-bet mRNA expression (F＝83.26, P＜0.01; F＝17.6, P＜0.01). Multiple Comparison in ANOVA analysis demonstrated that the expression of T-bet and GATA3 mRNA was significantly higher in the two NPs groups than that in the inferior turbinate mucosa (P＜0.01, or P＜0.05). The NPs with allergy had significantly higher expression of GATA3 mRNA than that in the NPs without allergy (P＜0.01). The NPs with allergy and the NPs without allergy have no significant difference in T-bet mRNA expression (p＞0.05).Conclusions NPs expressed higher level of T-bet and GATA3 mRNA than that in the inferior turbinate mucosa. NPs with allergy expressed higher level of GATA3 mRNA than the NPs without allergy did. NPs without allergy and NPs with allergy have some difference in immune response and pathogenesis.更多还原