臺灣博碩士論文加值系統

在耳鼻喉感染症包括急慢性腺樣體炎、扁桃腺炎、咽喉炎、鼻竇炎、中耳炎等。上述急性細菌性感染症在抗生素治療無效之後，往往需要以外科手術治療，常見的手術方式有腺樣體、扁桃腺切除，舌扁桃體切除手術、功能性鼻竇內視鏡手術、及各類中耳手術等。在耳鼻喉感染症以內科抗生素治療無效之病患中我們發現他們感染部位的組織中多有細菌生物膜的形成。細菌生物膜的形成與抗生素抗藥性有相當關聯，也是藥物治療失敗後需要以手術治療以移除這些感染組織的細菌生物膜主要原因。本研究之第一部份在我們耳鼻喉腺樣體及扁桃腺感染症大型資料庫裡的研究發現，在腺樣體及扁桃腺感染的患者中，若是我們將腺樣體及扁桃腺切除後因為可能形成的細菌生物膜被移除，病患因而減少因上呼吸道感染症就醫的次數。在第一部份的腺樣體及扁桃腺細菌抗藥性及細菌生物膜形成之研究發現，多重抗藥性之金黃色葡萄球菌 (MRSA) 是容易形成細菌生物膜的菌種，而多重抗藥性之金黃色葡萄球菌造成腺樣體感染中多易併發中耳腔的感染。回顧文獻，細菌生物膜形成與本體免疫所分泌之SPLUNC1蛋白不足或功能缺損有不可分的關聯性。SPLUNC1是分布在人類軟顎、肺部、懸雍垂、鼻腔、等上呼吸道之蛋白，被發現主要分布在上呼吸道之上皮及腺體組織中，至於它在表面上皮或皮下的腺體組織之分布有因組織不同而有消長之差異，它被認為是人類本體免疫之重要物質，可以在後天免疫作用之前，第一時間將感染原作初步之阻隔。在第二部份的研究中我們以鼻竇炎手術患者作進一步研究，鼻竇炎為耳鼻喉常見之感染症為困擾病患生活與工作之相當普遍之疾病，也成為醫療資源耗費之主要疾病，常有許多是用抗生素效果仍不佳而需借助手術治療的情況，我們針對SPLUNC1表現高低與耳鼻喉鼻竇感染症的關聯性作分析，我們也發現低表現之SPLUNC1組織有顯著高的比例感染綠膿桿菌及金黃色葡萄球菌感染，並且低表現之SPLUNC1組織有顯著感染多菌的可能，因此恢復組織中較低表現之SPLUNC1實為要務。然而並非所有慢性鼻竇炎以手術治療的患者，都能得到最佳的治療效果，有些甚至在開刀後仍反覆感染而需重複開刀之狀況，在這些情況中控制手術技術及方法的情況下，我們發現病患本身之免疫功能也是一重要影響預後之因子，在研究結果中，我們證明了SPLUNC1表現量之差異與疾病預後有相關性，與治療結果及疾病變化有關，另外特別在綠膿桿菌與金黃色葡萄球菌等易生成細菌生物膜等研究中，發現SPLUNC1有抑制細菌生物膜形成之能力，尤其是在鼻竇炎及腺樣體炎的組織中最明顯。此外，我們也發現在人類鼻腔上皮細胞中，過敏性鼻炎患者分泌之IL-13會抑制SPLUNC1表現，進一步使得細胞在LPS之內毒素影響之下存活率下降。而人類呼吸道上皮在急性細菌之內毒素刺激之下會藉由活化MAPK傳遞路徑來增加細胞核分泌SPLUNC1之表現，進一步增加細胞對抑制細菌生物膜形成及增強嗜中性球對細菌之毒殺能力，不過在感染超過48小時之後SPLUNC1在上皮之表現量會下降，是被LPS毒素消耗掉或其他原因我們仍需進一步研究。另外當前處理JNK-c-Jun路徑之抑制劑後，SPLUNC1在上皮之表現量會明顯下降。在第三部份的研究中我們發現乳鐵蛋白是我們可用來保持組織中SPLUNC1在感染後不被消耗的重要蛋白。乳鐵蛋白也是一個有效抑制上呼吸消化道感染症之物質，對人類本體免疫功能有加強之效果。發現乳鐵蛋白與SPLUNC1結合後會增強細胞對細菌生物膜形成之抑制能力並大大減少綠膿桿菌及金黃色葡萄球菌之感染，因此在未來治療耳鼻喉感染症方面，類SPLUNC1及乳鐵蛋白也許能在將來當作一個治癒疾病之新方向。

Infection diseases in the ear nose and throat (ENT) field includes the acute or chronic adenoiditis, tonsillitis, pharyngitis, sinusitis and otitis media etc. The infection become chronic when the patients met the antibiotics treatment failure and the consequent ear nose and throat surgery for treating the chronic infection diseases is warranted for these patients. The common and often surgical methods we use for treating the ENT infections are including adenoidectomy, tonsillectomy, lingual tonsillectomy, functional endoscopic sinus surgery, and various kinds of middle ear surgeries. The antibiotics resistant condition is correlated to biofilm formation in the infection tissue surface. The biofilm formation is also correlated to the demands of ENT surgeries. In the first part, we found the upper airway infection rate decreases after tonsillectomy and adenoidectomy in the national health insurance databank (cohort of adenoid, tonsils and upper airway infections) survey. We also found the biofilm formation significantly higher in the surgical specimens of adenoid tissue by MRSA infection compared to MSSA adenoid colonization. MRSA have higher ability to form the biofilm not only because of its resistance to methicillin but also because of host innate immune deficiency. The deficiency of SPLUNC1 is correlated to bacteria biofilm formation.
The short palate, lung, and nasal epithelium clone 1 (SPLUNC1) is an innate immune material and distributed in the human soft palate, lung, uvula, nasal cavity. It is a secreted protein and it is found at nasal fluid and bronchial fluid. The distribution of SPLUNC1 is mainly in the submucosa glandular area and epithelial area in the upper aerodigestric tract. It is an important innate material for protecting the upper airway. The Ear-Nose-Throat (ENT) infections is also a commonly affect patient’s life quality and even life threating diseases and waste a lot of portion in the national health insurance fees. It is a leading 4th disease in the Taiwan national health insurance cost.
The ENT infections are commonly including nasal sinusitis, oral and gum infections, adenoiditis, pharyngitis, tonsillitis, bronchitis and even lead to further pneumonia. The all above diseases are often managed first by the ENT doctors and sometimes they are very difficult to treat and eventually need surgical intervention. However, the surgical outcome is different in each patient even in a stable surgical technique by an experienced physician. The patient’s immune condition is also a factor that affects the treatment outcome in our clinical survey. In our second part studies, we found the lower SPLUNC1 associated biofilm formation and this is an import outcome predictor in our study. We found SPLUNC1 has an anti-bacterial function and anti-biofilm function which are easily formed by methicillin-resistant Staphylococcus aureus (MRSA) and pseudomonas infections. Among the chronic sinusitis with nasal polyps patients, the nasal allergy is often comorbid or co-existing. We found it is more difficult to treat the combine condition in a CRSwNP patient. The patient with allergic rhinitis who had higher Interleukin-13 (IL-13) expression leads to suppression of the SPLUNC1 expression, that will had a higher infection rate, but the further mechanism is showed in the third part of dissertation. In the final part of this dissertation, the lactoferrin (LF) is found to recover SPLUNC1 expression in the upper airway infection. We also found by adding LF in LPS induced nasal epithelial inflammation model, the decreased SPLUNC1 after treating LPS 48 hours (mimicking human chronic sinusitis) could be recovered and decreased the further tissue inflammation. The initial mechanism is showed at the end of this dissertation. However, the detail mechanism and further study is still warranting.

中文摘要 i
Abstract iii
Abbreviation List v
Contents viii
List of Tables xii
List of Figures xiii

Chapter 1. Literature reviews: The role of SPLUNC1 in airway infection and correlated diseases 1

1.1. Introduction 2
1.2. Identification 2
1.3. Family 3
1.4. Distribution 4
1.5. Detection marker in carcinomas 5
1.6. Function 6
1.7. Diseases 6
1.8. Mechanism and therapy 8
1.9. Specific aims of this thesis 9

Chapter 2. Adenotonsillectomy reducing clinic visits of pediatric upper respiratory tract infections is associated with adenoid hyperplasia and bacterial biofilm formation in children with adenoiditis in Taiwan 10

2.1. Introduction 11
2.2. Materials and methods 12
2.2.1. Adenotonsillectomy reduces clinic visits for pediatric upper
respiratory tract infections in Taiwan 12
2.2.1.1. Data source 12
2.2.1.2. Study subjects 13
2.2.1.3. Study subjects in database statistical analysis 13
2.2.2. Association of adenoid hyperplasia and bacterial biofilm
formation in children with adenoiditis in Taiwan 14
2.2.2.1. Patient selection and sample collection 14
2.2.2.2. Lateral cephalometric analysis and scoring of maxillary
sinusitis severity 15
2.2.2.3. Scanning electron microscopy (SEM) 15
2.2.2.4. Biofilm formation assay 16
2.2.2.5. Confocal laser scanning microscopy (CLSM) 16
2.2.2.6. Image analysis of biofilm criteria 17
2.2.2.7. Statistical analysis 17
2.3. Results 18
2.3.1. Adenotonsillectomy reduces clinic visits for pediatric upper
respiratory tract infections in Taiwan 18
2.3.1.1. Population Characteristics 18
2.3.1.2. Number of outpatient visits for URTI during the study
period 18
2.3.1.3. Effects of surgery on URTI outpatient visit 18
2.3.2. Association of adenoid hyperplasia and bacterial biofilm
formation in children with adenoiditis in Taiwan 18
2.3.2.1. Association of maxillary sinusitis severity and antibiotic
resistance in S. aureus 19
2.3.2.2. Biofilm formation on adenoid tissues 19
2.3.2.3. Quantification of bacterial biofilm formation 20
2.4. Discussion 21

Chapter 3. Decreased SPLUNC1 expression is associated with multibacterial infection including pseudomonas in patients with chronic rhinosinusitis that may require repeated sinus surgery 36

3.1. Introduction 37
3.2. Materials and methods 39
3.2.1. Decreased SPLUNC1 expression is associated with
pseudomonas infection in surgical treated chronic
rhinosinusitis patients who may require repeated sinus
surgery 39
3.2.1.1. Patient selection and collection of clinical data 39
3.2.1.2. Bacterial culture 40
3.2.1.3. Phadiatop test 40
3.2.1.4. Tissue immunohistochemical (IHC) staining 40
3.2.1.5. Tissue RNA preparation 40
3.2.1.6. Gene expression analysis using Quantitative PCR (Q-
PCR) 41
3.2.1.7. Statistical analysis 41
3.2.2. Decreased SPLUNC1 expression in nasal polyps is
associated with multibacterial colonization in chronic
rhinosinusitis patients after sinus surgery 41
3.2.2.1. Patient selection and sample collection 41
3.2.2.2. Immunohistochemical (IHC) staining for SPLUNC1 42
3.2.2.3. Preparation of tissue RNA and quantitative RT-PCR 43
3.2.2.4. Statistical analysis 43
3.3. Results 44
3.3.1. Decreased SPLUNC1 expression is associated with
pseudomonas infection in surgical treated chronic
rhinosinusitis patients who may require repeated sinus
surgery 44
3.3.1.1. Bacteriology results and clinical parameters 44
3.3.1.2. SPLUNC1 mRNA level, IHC staining intensity, and clinical
outcome 44
3.3.2. Decreased SPLUNC1 expression in nasal polyps is
associated with multibacterial colonization in chronic
rhinosinusitis patients after sinus surgery 45
3.3.2.1. Association of SPLUNC1 expression levels and various
clinical factors 45
3.3.2.2. Bacterial infection reduced SPLUNC1 expression in
patients with sinusitis 46
3.4. Discussion 47

Chapter 4. The effect of IL-13 on inhibiting LPS-induced SPLUNC1 expression is attenuated by the interaction between SPLUNC1 and Lactoferrin via down-regulated MEK1/2-MAPK signaling 61

4.1. Introduction 62
4.2. Materials and methods 64
4.2.1. IL-13 inhibits lipopolysaccharide-induced SPLUNC1
expression in nasal epithelial cells 64
4.2.1.1. Antibodies and reagents 64
4.2.1.2. Cell culture and treatment 64
4.2.1.3. Western blot analysis 65
4.2.1.4. Reverse transcription and real-time quantitative PCR 65
4.2.1.5. Reporter activity assay 65
4.2.1.6. Immunofluorescence staining 66
4.2.1.7. Patient selection and ethics statement 66
4.2.1.8. Immunohistochemistry (IHC) analysis 66
4.2.1.9. Statistical analysis 67
4.2.2. Interaction between SPLUNC1 and Lactotransferrin in LPS
induced inflammation 67
4.2.2.1. Cell culture and drug 67
4.2.2.2. MTT assay 67
4.2.2.3. Antibodies 67
4.2.2.4. Western blot 68
4.2.2.5. Indirect immunofluorescence and fluorescence
microscopy 68
4.2.2.6. Statistical analysis 69
4.3. Results 70
4.3.1. IL-13 inhibits lipopolysaccharide-induced SPLUNC1
expression in nasal epithelial cells 70
4.3.1.1. LPS-induced SPLUNC1 expression in nasal epithelial
cells 70
4.3.1.2. Involvement of JNK/c-Jun in LPS-mediated SPLUNC1
up-regulation 70
4.3.1.3. Involvement of the transcription factor AP-1 in LPS-
induced SPLUNC1 expression 71
4.3.1.4. Attenuation of LPS-induced SPLUNC1 expression by IL-
13 71
4.3.1.5. Decreased SPLUNC1 expression was associated with IL-
13 secretion in eosinophilic CRSwNP patients with
bacterial colonization 72
4.3.2. Interaction between SPLUNC1 and LF in LPS induced
inflammation 72
4.3.2.1. LF rescued SPLUNC1 expression in LPS induction 72
4.3.2.2. LF reduced inflammation-related protein MEK1/2 and
p42/44 MAPK activation 73
4.4. Discussion 74

Chapter 5. General conclusion 93
References 96

Appendix 1. Curriculum vitae 118
Appendix 2. Ph.D. dissertation-related papers 132

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