本文選題：變異鏈球菌 + 報告株　； 參考：《第四軍醫(yī)大學》2010年碩士論文

【摘要】：齲病是常見的口腔感染性疾病之一,而變異鏈球菌是人類的主要致齲菌,它通過在牙齒表面的黏附和聚集形成生物膜,進而產(chǎn)酸耐酸致齲。因此研究其生長特性及生物膜結構能更好的研究齲病發(fā)病機理。近年來,隨著科學技術的不斷進步,對生物膜的研究也越來越深入。激光共聚焦顯微鏡與傳統(tǒng)的電鏡相比,前者不但可以保持生物膜的完整結構,而且可沿水平、垂直方向斷層掃描,進行三維重建,使人們可以從不同的角度來分析生物膜的結構特性。但是由于其樣本需經(jīng)熒光標記或染色,使實驗時間明顯延長,于是報告基因技術用于細菌形態(tài)學研究的優(yōu)勢便得以體現(xiàn)。細菌通過轉入的熒光蛋白自身發(fā)光,不需染色就可在鏡下動態(tài)實時的觀察。 本實驗比較研究變異鏈球菌熒光報告株與野生菌株的生長能力和生物膜形成能力,結果顯示二者無明顯差異。隨后研究在激光共聚焦顯微鏡下,報告株在樹脂,玻璃離子表面形成生物膜的差異,為臨床修復材料的選擇提供了依據(jù)。本研究共包括三個實驗。 實驗一變異鏈球菌熒光報告株與野生菌株生長能力的比較 將復蘇后的變異鏈球菌,變異鏈球菌熒光報告株調至相同的光密度值,37℃厭氧箱培養(yǎng)。于0,1.5,3,4,6,8,10,12,16,20,24h取培養(yǎng)液,酶標儀測OD600值。繪制生長曲線,結果兩者生長趨勢相近,提示兩者具有相同的生長狀態(tài)。 實驗二變異鏈球菌熒光報告株與野生菌株生物膜形成能力的比較 在96孔細胞培養(yǎng)板中培養(yǎng)變異鏈球菌熒光報告株及野生株,分別于培養(yǎng)后4h, 8h, 12h, 24h取樣,將生物膜用結晶紫染色,無水乙醇脫色,酶標儀測OD575。組間比較發(fā)現(xiàn)兩菌株生物膜形成量無統(tǒng)計學意義(p0.05)。 在蓋玻片上培養(yǎng)上述二菌,分別培養(yǎng)4h,8h, 12h,24h取樣,通過洗滌、固定、脫水、干燥和噴金處理后,SEM下觀察。結果發(fā)現(xiàn)不同時間段兩者的生物膜形態(tài)沒有明顯差異,都是由早期散在分布,少量交聯(lián);到后期成團、成膜分布、各個菌落融合并重疊,形成網(wǎng)狀結構。高倍鏡下細菌形態(tài)也未見改變。提示兩者生物膜形態(tài)相似。 實驗三變異鏈球菌熒光報告株在不同材料表面形成生物膜能力的研究 在已制作好的樹脂、玻璃離子片上培養(yǎng)變異鏈球菌熒光報告株,分別于培養(yǎng)后4h,8h, 12h,24h取樣、洗滌、晾干、封片,激光共聚焦顯微鏡下觀察,并在不同位置沿Z軸進行掃描,測量其厚度和組織結構。結果發(fā)現(xiàn)不同時間段玻璃離子表面形成的生物膜量及其厚度均少于同一時間段樹脂表面,提示變異鏈球菌熒光報告株在玻璃離子表面形成生物膜的能力低于樹脂表面。
[Abstract]:Dental caries is one of the common oral infectious diseases, and Streptococcus mutans is the main cariogenic bacteria. Therefore, the study of its growth characteristics and biofilm structure can better study the pathogenesis of caries. In recent years, with the development of science and technology, the study of biofilm is more and more in-depth. Compared with traditional electron microscope, laser confocal microscope can not only maintain the complete structure of biofilm, but also scan and reconstruct the biofilm in horizontal and vertical directions. It makes it possible to analyze the structure of biofilm from different angles. However, due to the fluorescence labeling or staining of the samples, the experimental time is obviously prolonged, so the advantage of the reporter gene technique in the study of bacterial morphology can be demonstrated. Bacteria can be observed dynamically and in real-time without staining through the fluorescent protein itself. The growth ability and biofilm forming ability of Streptococcus mutans fluorescent report strain and wild strain were studied in this experiment. The results showed that there was no significant difference between them. Then the difference of biofilm formation on the surface of resin and glass ion was studied under laser confocal microscope, which provided the basis for the selection of clinical repair materials. This study consists of three experiments. Experiment 1 comparison of growth ability between Streptococcus mutans fluorescent report strain and wild strain, the resuscitated Streptococcus mutans and Streptococcus mutans fluorescent report strain were adjusted to the same optical density value of 37 鈩，

本文編號：2076781