臺灣博碩士論文加值系統

齲齒(caries) 在台灣是盛行率相當高的口腔疾病，先前研究指出轉糖鏈球菌(Streptococcus mutans)為主要病原菌，能貼附於牙齒表面形成生物膜(biofilm)，代謝醣類後產生有機酸造成牙齒的去礦化。目前預防齲齒的方法皆有一定效果，但並非每個人都適合，使得開發新的齲齒預防策略，變得十分重要。綠茶與益生菌目前被認為具有預防齲齒的潛力：兒茶素(catechin)在綠茶的含量最高：其中的表沒食子酸沒食子(epigallocatechin-3-gallate, EGCG)，已知有抗菌的功效。許多含有乳酸菌的發酵製品，也被發現具有抑制S. mutans的能力。台灣人十分喜愛飲用手搖飲料，部分飲品會將茶跟乳酸菌發酵乳混合，但這兩種對齲齒可能具保護效果的物質混合時，對S. mutans的影響仍不清楚。實驗室之前觀察到EGCG與乾酪乳酸桿代田菌 (Lactobacillus casei strain Shirota, LcS)，可影響S. mutans生物膜中死菌與胞外多醣的含量，但如何影響之機制以及是否影響齲齒形成則尚未清楚。本研究假設EGCG及LcS會影響S. mutans生物膜的致齲力，並改變與生物膜形成的毒力因子，希望探討EGCG及LcS同時存在時，對S. mutans生物膜強度、產酸能力、致齲能力及毒力基因表現的影響。研究結果發現，隨著EGCG濃度增加至125 μg/ml，超音波震盪移除S. mutans生物膜的移除率顯著增加，在有LcS時則有增加的趨勢但無統計差異。EGCG濃度增加至250 μg/ml，生物膜在有LcS的組別移除率較無LcS的組別高，但此現象可能與有LcS的組別生物膜質量在移除前較高有關。利用C-SNARF染色觀察生物膜酸鹼值分布，發現，EGCG濃度250 μg/ml時，無論有無LcS，生物膜產酸能力都有被抑制的趨勢。在人類牙塊表面形成生物膜，在沒有LcS的組別，EGCG 250 μg/ml或500 μg/ml組的培養液pH值維持在中性，125 μg/ml組在5.5左右，0 μg/ml或62.5 μg/ml則是4.5左右；有LcS的組別則是僅EGCG 250 μg/ml、500 μg/ml的組別培養液pH值維持中性，其餘EGCG濃度的組別pH值小於5.5。蔗糖間歇性處理，第一日的隔夜培養液，僅EGCG 500 μg/ml可抑制S. mutans生物膜產酸，有LcS的組別，EGCG 500 μg/ml的抑制效果較差，pH值為5.5；經過九天的間歇性蔗糖處理後，所有的組別的隔夜培養液pH值皆低於5.5。利用光學相干斷層掃描觀察牙塊表面，發現S. mutans或S. mutans及LcS生物膜的組別，無論使用何種濃度EGCG，皆出現符合牙塊表面脫鈣的結構改變。抽取生物膜的RNA反轉錄cDNA後進行定量PCR，發現當EGCG為250 μg/ml具有抑制S. mutans生物膜表現gtfB、gtfC及grfD的趨勢，但在有LcS的生物膜中250 μg/ml EGCG抑制gtfB、gtfC表現的效果有變差的趨勢。EGCG可能透過抑制gtf表現，進而影響S. mutans生物膜的質量及強度，使其變得容易被移除，但有LcS時，EGCG 250 μg/ml抑制gtf基因表現的能力較差。無論有無LcS，EGCG會使生物膜的酸化程度較低；但有LcS時，需要較高劑量的EGCG才能避免培養液的酸化。而無論有沒有LcS，EGCG都無法避免牙釉質表面的脫鈣。

Caries is a highly prevalent oral infectious disease in Taiwan. Streptococcus mutans, the cariogenic pathogen, adheres on the tooth surface to form biofilms, metabolizing carbohydrates, producing organic acids and causing tooth demineralization. Current preventive methods against caries are effective but with some limitation. Thus, it is important to develop new preventive strategies against caries. Green tea and probiotics are considered to be potent anti-caries reagents. Catechin is abundant in green tea. Among tea catechin, epigallocatechin-3-gallate (EGCG) is known to inhibit bacterial growth. Several fermented products containing probiotics are known to inhibit S. mutans. Tea cocktails are popular in Taiwan. When green tea and fermented milk are mix to make tea cocktails, the effect of combining these two inhibitors remains to be calrified. Previously, studies conducted in our lab showed that EGCG and Lactobacillus casei strain Shirota (LcS) affected the amount of dead cell and extracellular polysaccharides in biofilms; however, the mechanism and their effect on the cariogenic ability of biofilms are still unclear. Thus, in this study, it was assumed that EGCG and LcS affected the cariogenic ability of S. mutans biofilms and altered the virulence factors associated with biofilm formation. The effects of EGCG and LcS on the biofilm strength, acid production, the cariogenic ability and virulence gene expression by S. mutans were investigated. Data showed that the removal efficiency of S. mutans biofilms by sonication was significantly increased, as the EGCG concentration was increase of to 125 μg/ml. In the presence of LcS, the trend of increases in removal efficiency remained, but without statistical difference. When EGCG was further increased to 250 μg/ml, the removal efficiency for biofilm was higher in the group with LcS, compared to the S. mutans only group. This phenomenon might be related to that more biomass was present in the biofilm with LcS before sonication. In situ acid production in biofilms was also observed, using C-SNARF staining. EGCG of 250 μg/ml inhibited acid production, whether LcS was present or not. Culture medium collected from biofilms, which were formed by S. mutans only on the human enamel slabs, remained neutral when EGCG of 250 or 500 μg/ml were added. pH value of the culture medium was reduced to less than 5.5 when no EGCG or EGCG less than 125 μg/ml was added. After the feast-and-famine cycles began, the pH value of the culture medium collected from the biofilm of S. mutans only treated by 500 μg/ml EGCG after the first over-night incubation was neutral. The pH values of all the other groups were less than 5.5. After 9 days of feast-and-famine cycles, pH values of none of the over-night culture medium were higher than 5.5. Using OCT, in vitro caries formation on the tooth slabs caused by the single-species biofilm or dual-species biofilms grown in the presence of EGCG was observed. The result of QPCR showed the expression of gtfB, gtfC and gtfD in S. mutans biofilms was reduced by EGCG of 250 μg/ml However, the inhibitory ability of EGCG tended to be reduced by LcS. In conclusion, inhibition of gtf expression by EGCG led to the reduction of biomass and strength of biofilms, which tended to be removed by sonication. LcS decreased the inhibitory ability of 250 μg/ml EGCG against gtf gene expression. EGCG reduced the acidification caused by S. mutans; however, more EGCG was required to prevent the acidification of culture medium when LcS was present. Whether LcS was present or not, demineralization of enamel surfaces was not prevented by EGCG.

目錄
誌謝 I
中文摘要 II
Abstract V
目錄 VII
圖目錄 VIII
表目錄 IX
緒論 1
齲齒流行病學 1
齲齒與生物膜 2
轉糖鏈球菌 3
齲齒預防 4
益生菌 5
益生菌對口腔的影響 6
綠茶多酚對口腔的影響 7
光學相干斷層掃描技術(Optical Coherence Tomography, OCT) 9
研究動機 10
材料與方法 11
細菌培養及製備 11
超音波移除生物膜 12
結晶紫染色定量生物質量 12
牙塊製備及唾液貼附 13
齲齒誘導 13
生物膜之pH分布 14
RNA純化 15
反轉錄聚合酶鏈鎖反應 16
結果 18
EGCG對S. mutans或S. mutans及LcS混合生物膜抗外力之影響 18
EGCG對S. mutans或 S. mutans及LcS混合生物膜酸蝕人類牙塊之影響 21
EGCG對人類牙齒上S. mutans單菌或S. mutans及LcS混合生物膜產酸能力之影響 22
EGCG對S. mutans或S. mutans及LcS混合生物膜S. mutans毒力基因表現之影響 23
討論 25
參考文獻 30
附錄 51

圖目錄
圖 一、EGCG對生長中S. mutans生物膜或S. mutans及LcS混合生物膜質量的影響及超音波震盪後S. mutans生物膜或S. mutans及LcS混合生物膜生物質量減少百分比 36
圖 二、EGCG對S. mutans生物膜或S. mutans及LcS混合生物膜產酸能力之影響 38
圖 三、EGCG對S. mutans生物膜或S. mutans及LcS混合生物膜產酸能力之影響之平均、最大或最小pH值 41
圖 四、EGCG對S. mutans生物膜酸蝕人類牙塊之影響 43
圖 五、EGCG對S. mutans及LcS混合生物膜酸蝕人類牙塊之影響 45
圖 六、EGCG對人類牙齒上S. mutans生物膜產酸能力之影響 46
圖 七、 EGCG對人類牙齒上S. mutans及LcS混合生物膜酸蝕產酸能力之影響 47
圖 八、EGCG對S. mutans或S. mutans及LcS混合生物膜基因表現之影響 48

表目錄
表 一 研究使用之專一性引子 50

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