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研究生:高怡婷
研究生(外文):Yi-Ting Kao
論文名稱:MUC1對口腔上皮細胞受細菌刺激之影響
論文名稱(外文):Influences of MUC1 on the epithelial responses to different oral bacteria
指導教授:吳靜宜吳靜宜引用關係
指導教授(外文):Ching-Yi Wu
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:口腔生物研究所
學門:醫藥衛生學門
學類:牙醫學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:中文
論文頁數:73
中文關鍵詞:牙周病黏蛋白口腔上皮角化細胞鏈球菌具核梭桿菌
外文關鍵詞:Periodontal diseaseMUCINOral keratinocyte cell lineStreptococcusFusobacterium nucleatum
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台灣40歲以上成年人,90﹪有輕重程度不一的牙周病。牙周病的形成有三大主因:環境、宿主健康情形及口腔菌叢失衡。當口腔常在菌減少,牙周致病菌成為優勢菌種,感染牙周組織時,會引發嚴重的發炎反應使齒槽骨吸收,最終造成牙齒的脫落。口腔細菌的黏附與黏蛋白有關係。黏蛋白(mucin, MUC)為醣蛋白,主要功能為使上皮維持濕潤、透過物理或化學屏障的方式抵抗致病菌以保護宿主。黏蛋白分為分泌型及穿膜型,在口腔中,分泌型蛋白的功能依種類有所不同,如MUC5B可調節口腔細菌貼附牙齒,MUC7凝集細菌增加清除效率,口腔上皮亦表現穿膜型蛋白MUC1,但其功能仍不清楚。在慢性發炎的胃中,MUC1的過度表現可幫助清除致病菌Helicobacter pylori,並且在胃及呼吸道上皮內皆發現MUC1能抑制NF-κB活化慢性發炎反應。為了解MUC1對口腔上皮細胞受細菌刺激之影響,本實驗假設在口腔中,早期菌種與中期菌種與MUC1之間的交互作用有差異,將利用早期菌種Streptococcus salivarius、Streptococcus sanguinus、中期Fusobacterium nucleatum刺激口腔上皮細胞株,除探討口腔牙周致病菌對MUC1表現的影響,並比較MUC1對常在菌及致病菌感染的影響。首先萃取細胞mRNA及蛋白質,分別利用即時聚合酶鏈鎖反應及西方點墨法,觀察MUC1在NOK、OC3及OECM1三株口腔上皮細胞中的表現,發現MUC1 β subunit在所有細胞株中皆有表現,NOK細胞受S. salivarius刺激後,MUC1 mRNA及蛋白質表現量隨感染劑量增加先降後(MOI 10)回復(MOI 100或1000);S. sanguinis刺激的細胞MUC1蛋白質表現量亦先降後回復,而mRNA則是下降;F. nucleatum則會增加細胞中MUC1表現量。受細菌刺激細胞之mRNA亦用於發炎相關分子: TLR-4、TLR-2、IL-8及TNF-α之表現分析:S. salivarius及S. sanguinis MOI 1、10、100、1000會刺激TLR-4、TLR-2及IL-8表現量上升,S. salivarius MOI 1000 TNF-α表現量則會上升。而S. salivarius MOI 1000,MUC1 mRNA表現量回復時,IL-8 mRNA表現量與MOI 1、10、100相比則有抑制的情形。F. nucleatum MOI 100時也會刺激TLR-4、IL-8及TNF-α表現。進一步利用MUC1的siRNA進行基因敲落(knock-down, KD),再以細菌刺激細胞,觀察細胞中NF-κB、JNK、ERK1/2的活化及TLR4、IL-8及TNF-α的表現,F. nucleatum刺激NOK細胞中NF-κB之入核;F. nucleatum刺激之MUC1 KD細胞,較轉染控制siRNA之細胞,入核有增加的趨勢。分別受三種細菌刺激之細胞JNK及ERK1/2皆會活化。未受細菌刺激的MUC1 KD細胞,磷酸化ERK1/2及JNK表現較控制細胞低,但受F. nucleatum細菌刺激之MUC1 KD細胞,其磷酸化JNK與受刺激之控制細胞相比增加,ERK1/2的活化或TLR4、IL-8及TNF-α表現無顯著差異。研究結果顯示屬早期感染的S. salivarius、S. sanguis與中期感染的F. nucleatum,對MUC1及細胞激素之表現影響有差異,且MUC1在口腔上皮細胞中,可能與免疫反應的負向調節有關。
Ninety percent of Taiwanese adults, who are older than 40 years old, have periodontal disease. Periodontal disease is caused by the interaction between environmental factors, host immune conditions and oral bacterial dysbiosis. When contents of subgingival biofilms shift from majority of commensal to pathogenic bacteria, severe inflammation will be induced and, eventually, leads to bone loss. The glycolprotein mucins can moisturize oral epithelium and protect host by forming physiological or chemical barriers against pathogen infection. Mucin is classified as secreted and transmembrane mucin. In the oral cavity, secreted mucin, such as MUC5B or MUC7, can modulate oral bacterial colonization or aggregate bacteria for clearance, respectively. The transmembrane mucin, MUC1, is expressed by oral epithelium. Its function is still unclear. In the chronic inflammatory gastric mucosa, overexpression of MUC1 increases the clearance of Helicobacter pylori. Modulation of immune responses via the controlling of NF-ĸB pathway by MUC1 is also reported in the gastric and alveolar epithelium. The purpose of the study was to determine the influences of MUC1 on the epithelial responses to different oral bacteria. It was proposed that the interaction between MUC1 and oral early or intermediate colonizers were different. Early colonizers, Streptococcus salivarius and Streptococcus sanguinus, or the intermediate colonizer, Fusobacterium nucleatum, were used to stimulate oral epithelial cell lines. Influences of oral bacterium on the expression of MUC1 were determined. Additionally, effects of MUC1 on commensal or pathogenic bacterial infection were compared. Messenger RNA and proteins were extracted from oral epithelial cells (NOK, OC3 and OECM1) to determine the expression of MUC1, using RT-PCR and western blot analyses, respectively. It was shown that the β subunit of MUC1 was expressed in these cell lines. S. sanguinus, MOI of which was less than 10, down-regulated MUC1 expression, which was recovered when MOI of S. salivarius was further increased to 100 and 1000 in NOK cells. The MUC1 protein in S. sanguinus-stimulated cells was down-regulated and then reversed in a dose-depend manner, too. However the mRNA level was decreased by S. sanguinus. F. nucleatum up-regulated MUC1 expression in NOK cells. Expression of inflammatory molecules, including TLR-2, TLR-4 IL-8 and TNF-α, were also examined in bacteria stimulated cells. Both S. salivarius and S. sanguinus, MOI of which was 1-1000, stimulated the expression of TLR-2, TLR-4 and IL-8. S. salivarius also stimulated the expression of TNF-α when MOI was 1000. Interestingly, in S. salivarius (MOI:1000) stimulated cells, when expression of MUC1 was recovered to the control level, levels of IL-8 were also less than the cells stimulated by S. salivarius of MOI 1-100. F. nucleatum (MOI 100) also stimulated the expression of TLR-4, IL-8, TNF-α. In some experiments, I used siRNA to knock down MUC1 in NOK cells, to observe the activation of NF-ĸB, ERK1/2 and JNK or the expression of TLR-4, IL-8 and TNF-α. After F. nucleatum infected NOK cells, NF-ĸB translocated from cytoplasm to nuclei. This translocation was more obviously in MUC1 knock-down cells. S. salivarius, S. sanguinus or F. nucleatum stimulated the phosphorylation of ERK or JNK. In mock-infected cells with MUC1 knockdown, the level of phosphorylated ERK or JNK was less than the level in the cells without MUC1 knockdown. However, in cells stimulated by F. nucleatum, only the level of phosphorylated JNK was increased in cells with MUC1 knockdown. Activation of ERK1/2 or expression TLR-4, IL-8 or TNF-α was unaffected by MUC1 knockdown. Data suggest that MUC1 and cytokine expression was differently regulated by early (S. salivarius, S. sanguinus) and intermediate (F. nucleatum) colonizers. Additionally, MUC1 might be a negative modulators of immune response in oral epithelial cells.
目錄
誌謝 i
中文摘要 ii
英文摘要 v
目錄 viii
圖目錄 x
表目錄 xi
緒論 1
牙周病與牙菌斑 1
口腔鏈球菌 2
牙菌斑中期菌種- Fusobacterium nucleatum 4
牙周組織的免疫反應 5
黏蛋白MUC1 7
MUC1在細菌感染扮演的角色 9
研究動機 11
材料與方法 12
細胞培養 12
細菌培養與製備 13
細菌處理NOK細胞 14
細胞蛋白質萃取物置備 14
蛋白質濃度測定 15
西方點墨法 15
細胞存活率分析 17
RNA之萃取 18
定量即時反轉錄錄酶鏈鎖反應 19
上皮細胞表現之MUC1 isoform鑑定 20
利用siRNA knockdown NOK細胞之MUC1 23
NF-κB的活化 24
統計 25
結果 26
MUC1在不同口腔上皮細胞之表現 26
MUC1 isoforms在口腔上皮細胞中的表現 26
細菌生長曲線之測定 28
S. salivarius、S. sanguinis及F. nucleatum對NOK細胞存活率之影響 28
口腔細菌對NOK細胞表現MUC1之影響 28
口腔細菌對NOK細胞表現NF-ĸB活化之影響 29
利用siRNA建立MUC1 knockdown之NOK 30
MUC1 knockdown對F. nucleatum刺激NF-ĸB活化之影響 30
口腔細菌對NOK細胞表現ERK、JNK磷酸化之影響 31
MUC1 knockdown對F. nucleatum刺激對細胞表現ERK、JNK磷酸化之影響 31
MUC1 knockdown對F. nucleatum刺激對細胞表現TLR-4、TNF-α、IL-8之影響 33
討論 35
參考文獻 41
附錄 72


圖目錄
圖一、不同上皮細胞MUC1蛋白之表現 46
圖二、口腔上皮細胞表現MUC1 isoforms 47
圖三、OECM1細胞isoforms之定序 48
圖四、細菌生長曲線之測定 49
圖五、失活菌對NOK細胞存活率之影響 50
圖六、S. salivarius對NOK細胞表現MUC1之影響 51
圖七、S. sanguinis對NOK細胞表現MUC1之影響 52
圖八、F. nucleatum對NOK細胞表現MUC1之影響 53
圖九、S. salivarius對NOK細胞p65入核之影響 55
圖十、S. sanguinis對NOK細胞之 p65之影響 57
圖十一、MUC1 siRNA抑制MUC1在NOK細胞中表現 58
圖十三、S. sanguinis、S. salivarius對NOK細胞ERK1/2 & JNK之影響 61
圖十四、F. nucleatum對NOK細胞ERK1/2 & JNK之影響 62
圖十五、F. nucleatum對siRNA MUC1 NOK細胞ERK1/2 & JNK之影響 63
圖十六、S. salivarius對NOK細胞表現TLR2、TLR4、IL-8、TNF-α之影響 64
圖十七、S. sanguinis對NOK細胞表現TLR-2、TLR-4、IL-8、TNF-α之影響 65
圖十八、F. nucleatum對NOK細胞表現TLR-4、IL-8、TNF-α之影響 66
圖十九、F. nucleatum對siRNA MUC1 NOK細胞之表現TLR-4、IL-8、TNF-α之影響 67


表目錄
表一 研究用一級抗體 68
表二 研究用二級抗體 69
表三 Taq probe 70
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