【作者】 王锋；

【导师】 王德辉；

【作者基本信息】 复旦大学 ， 耳鼻喉科学， 2009， 博士

【摘要】 慢性鼻窦炎是影响人类健康的常见疾病,长期大量流脓涕是困扰患者的一大主要症状。传统的治疗方法如应用抗生素、血管收缩剂、穿刺冲洗等均不能有效改善其长期流脓涕的症状。即使通过鼻内镜手术,改善了鼻窦的引流问题,术后流脓涕的问题仍不能得到有效解决。慢性鼻窦炎长期流脓涕与鼻窦黏膜中粘蛋白(mucin, MUC)的大量分泌有着密切关系,使其具有“粘液高分泌性疾病”的特点。近年来研究发现,在人呼吸道上皮中钙离子激活的氯离子通道(calcium-activatied chloride channell CaCCl或HCLCAl)参与促进上皮杯状细胞增生,增加MUC5AC的合成。在许多具有“粘液高分泌性疾病”特点的下呼吸道疾病中,如慢性阻塞性肺疾病(chronic obstructive pulmonary disease, COPD)、哮喘、慢性支气管炎等,已发现CaCCl均有特异性的高表达,通过应用CaCCl抑制剂尼弗灭酸(niflumic acid NFA),可以有效抑制呼吸道上皮杯状细胞再生颗粒的产生和MUC5AC的分泌,对这类疾病具有潜在的治疗作用。因此我们设想在慢性鼻窦炎的病理机制中,CaCCl可能同样发挥重要作用,通过对CaCCl的干预,观察能否改善其“高黏液素”的病理环境,从而从新的角度探讨解决其长期流脓涕的问题。因此,对CaCCl在慢性鼻窦炎中对黏蛋白MUC5AC表达的调控作用的关系进行了实验研究,全文由三部分组成：第一部分钙离子激活的氯离子通道与慢性鼻窦炎关系的研究目的：观察CaCCl在慢性鼻窦炎和/伴有鼻息肉黏膜中的表达,探讨其在慢性鼻窦炎发病机制中的作用。方法：取Ⅰ型和Ⅱ型慢性鼻窦炎患者筛窦黏膜各10例,另取10例正常筛窦黏膜作为对照。应用Real一time PCR方法检测各组鼻窦黏膜中CaCCl mRNA表达,应用免疫荧光方法观察CaCCl蛋白在各组鼻窦黏膜中的表达情况,比较CaCCl蛋白在各组中平均阳性细胞面积比。结果：CaCClmRNA在Ⅰ型和Ⅱ型慢性鼻窦炎鼻窦黏膜中的表达明显高于对照组(P<0.01),而在两组慢性鼻窦炎组之间差异无统计学意义(P>0.05)。Ⅰ型和Ⅱ型慢性鼻窦炎鼻窦黏膜均见杯状细胞增生。CaCCl在两组鼻窦黏膜中均主要表达于上皮杯状细胞,平均阳性细胞面积比在Ⅰ型和Ⅱ型慢性鼻窦炎鼻窦黏膜中差异无统计学意义(P>0.05),而与对照组相比,差异均有统计学意义(P<0.01)。结论：在基因和蛋白水平上,CaCCl在慢性鼻窦炎和/伴有鼻息肉的鼻窦黏膜中均有明显表达,其在慢性鼻窦炎的发病机制中具有重要作用,可能与上皮杯状细胞的增生及粘液高分泌有关。第二部分黏蛋白5AC与慢性鼻窦炎关系的实验研究目的：观察MUC5AC在慢性鼻窦炎和/伴有鼻息肉鼻窦黏膜中的表达,探讨其在慢性鼻窦炎病理机制中的作用。方法：实验分组同第一部分,应用real-time PCR方法检测各组鼻窦黏膜MUC5ACmRNA表达,免疫荧光方法观察MUC5AC蛋白在各组鼻窦黏膜中的表达,比较MUC5AC蛋白在各组中平均阳性细胞面积比。结果:MUC5ACmRNA在Ⅰ型和Ⅱ型慢性鼻窦炎鼻窦黏膜中的表达明显高于对照组(P<0.01),而在两组慢性鼻窦炎组之间差异无统计学意义(P>0.05)MUC5AC在两组鼻窦黏膜中均主要表达于上皮杯状细胞,平均阳性细胞面积比在Ⅰ型和Ⅱ型慢性鼻窦炎鼻窦黏膜中差异无统计学意义(P>0.05),而与对照组相比,差异均有统计学意义(P<0.01)。结论：MUC5AC在慢性鼻窦炎和/伴有鼻息肉的鼻窦黏膜中表达明显均增高,MUC5AC在慢性鼻窦炎的发病过程中可能发挥重要作用。第三部分CaCCl在鼻窦粘膜组织培养中对MUC5AC调控作用的实验研究目的：通过对CaCCl不同方式的干预,观察体外培养的鼻窦粘膜组织中CaCCl及MUC5AC蛋白表达的变化。方法：取鼻内镜手术中切除的筛窦黏膜组织5例,采用组织块培养法培养24小时,每例组织分为5组,A组:培养液不加任何干预；B组：加入人重组TNF-a 10ng/mL；C组：加入NFA(CaCCl抑制刹)10umol/L30分钟后,加入人重组TNF-a10ng/mL;D组:加入NFAl00umol/L30分钟后加入人重组TNF-a 10ng/mL;E组：加入NFA 500umol/L 30分钟后加入人重组TNF-a 10ng/ml。以上5组培养24小时后进行western Blotting实验,观察CaCCl及MUC5AC蛋白在各组织中的表达。结果：TNF-上调CaCCl、MUC5AC蛋白表达水平(p<0.01).CaCCl抑制剂NFA下调CaCCl及MUC5AC蛋白表达(p<0.01),呈剂量依赖效应关系。结论：从蛋白水平上发现,TNF-a诱导的MUC5AC表达在体外培养的筛窦黏膜组织中被CaCCl抑制剂NFA下调。证实了在鼻窦粘膜组织培养中CaCCl对MUC5AC的分泌具有调节作用。更多还原

【Abstract】 Part I Experimental Studies Of The Relationship Between Calcium-Activatied Chloride Channel 1 And Chronic RhinosinusitisObjective:To exmatine the expression of calcium-activatied chloride channe 1 messenger RNA(mRNA) and proteins in human sinus mucosa of chronic rhinosinusitis without nasal polyposis (CRS) and chronic rhinosinusitis with nasal polyposis(CRS/NP). Methods: Ethmoid sinus mucosa was harvested from patients undergoing endoscopic sinus surgery for CRS with and without nasal polyposis and non-CRS pathologies (control).Then sinus mucosa was analyzed using real-time polymerase chain reaction (real-time PCR) to detect the mRNA of calcium-activatied chloride channe 1. Immunofluorescent staining was used to evaluate the expression of calcium -activatied chloride channe 1 proteins in the sinus mucosa. Area ratio of positive cells in the epithelia was compared among the CRS,CRS/NP and the control groups. Results:the mRNA of calcium -activatied chloride channe 1 in the sinus mucosa of CRS and CRS/NP significantly increased compared with that in normal sinus mucosa (p< 0.01). and no significant difference was found between the mucosa of CRS and that of CRS/NP (p>0.05). Calcium-activatied chloride channel 1 proteins were expressed mainly in the goblet cells, hyperplasia of goblet cells were observed in all cases of CRS and CRS/NP. Area ratio of positive cells in the epithelia was found no different between the CRS group and the CRS/NP group (p> 0.05), whereas it was significantly lower in the normal group compared with the other two groups, respectively (p 0.01).Conclusions:This study showed that calcium -activatied chloride channel 1 gene was up-regulated in sinus mucosa of CRS and CRS/NP. Calcium -activatied chloride channel 1 may play an important role in the pathogenesis of CRS and CRS/NP for the over-production of MUC.Part II Experimental Investagation On The Relationship Between MUC5AC And Chronic RhinosinusitisObjective:To investagate the expression of MUC5AC messenger RNA(mRNA) and protein in human sinus mucosa of chronic rhinosinusitis with and without nasal polyposis (CRS and CRS/NP). Methods:Ethmoid sinus mucosa was obtained from patients with CRS with and without nasal polyposis undergoing endoscopic sinus surgery and non-CRS pathologies (control).Then sinus mucosa was analyzed using real-time polymerase chain reaction (real-time PCR) to examine the mRNA of MUC5AC. Immunofluorescent staining was used to evaluate the expression of MUC5AC protein in the sinus mucosa. Area tatios of positive cells in the epithelia were compared among the CRS, CRS/NP and normal groups. Results:Expressions of mRNA of MUC5AC in the sinus mucosa of CRS and CRS/NP significantly increased compared with that in normal sinus mucosa (p< 0.01), and no significant difference was found between the mucosa of CRS and that of CRS/NP (p> 0.05). MUC5AC protein expressed mainly in the goblet cells, hyperplasia of goblet cells was observed in all cases of CRS and CRS/NP. There was no difference of area tatios of positive cells in the epithelia founded between the CRS group and the CRS/NPgroup (p> 0.05), whereas it was significantly lower in the normal group compared with the other two groups, respectively (p< 0.01).Conclusions:These results suggested that MUC5AC gene up-regulatation in sinus mucosa of CRS and CRS/NP may play an important role in the pathogenesis of CRS and NP associated with the hyperplasia of goblet cells and mucin overproduction.Part III Study Of The Regulation Of Calcium-Activatied Chloride Channel L On MUC5AC Expression In Sinus Mucosa Tissue CultureObjective:To investigate the regulation of CaCClon MUC5AC expression in sinus mucosa tissue culture. Methods:mucosa (sinus) tissures obtained from patients with chronic rninosinusitis undergoing sinus surgery were cultured for 24 hours.every specimen was divided into 5 groups, A group:maintained in medium without adding any stimulators; group B:added with recombinant human (rh)TNF-a (10 ng/mL); group C:added with CaCC1 inhibitor niflumic acid lOumol/L followed by TNF-a (10 ng/mL) 30 minutes later. group D:added with CaCC1 inhibitor niflumic acid 100 umol/L followed by TNF-a (10 ng/mL) 30 minutes later:group E:added with CaCC1 inhibitor niflumic acid 500 umol/L followed by TNF-a (10ng/mL) 30 minutes later. CaCC1 and MUC5AC protein expressions were quantified by using Western blotting. Results:TNF-a significantly increased CaCCl and MUC5AC proteins expressions in the mucosal explant tissue (P<0.01). Inhibition of CaCC1 with niflumic acid showed a significant dose-dependent reduction of CaCCl and MUC5AC proteins in the mucosal explant tissue (P<.05). Conclusions:TNF-a induced MUC5AC protein expression could be decreased by niflumic acid in explant sinus mucosa tissue which certificated that CaCC1 regulated MUC5AC expression in humann sinus mucosa tissure culture.更多还原