本文選題：變異鏈球菌 + 報(bào)告株　； 參考：《第四軍醫(yī)大學(xué)》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(jìn)而產(chǎn)酸耐酸致齲。因此研究其生長(zhǎng)特性及生物膜結(jié)構(gòu)能更好的研究齲病發(fā)病機(jī)理。近年來,隨著科學(xué)技術(shù)的不斷進(jìn)步,對(duì)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(jié)構(gòu),而且可沿水平、垂直方向斷層掃描,進(jìn)行三維重建,使人們可以從不同的角度來分析生物膜的結(jié)構(gòu)特性。但是由于其樣本需經(jīng)熒光標(biāo)記或染色,使實(shí)驗(yàn)時(shí)間明顯延長(zhǎng),于是報(bào)告基因技術(shù)用于細(xì)菌形態(tài)學(xué)研究的優(yōu)勢(shì)便得以體現(xiàn)。細(xì)菌通過轉(zhuǎn)入的熒光蛋白自身發(fā)光,不需染色就可在鏡下動(dòng)態(tài)實(shí)時(shí)的觀察。 本實(shí)驗(yàn)比較研究變異鏈球菌熒光報(bào)告株與野生菌株的生長(zhǎng)能力和生物膜形成能力,結(jié)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(bào)告株在樹脂,玻璃離子表面形成生物膜的差異,為臨床修復(fù)材料的選擇提供了依據(jù)。本研究共包括三個(gè)實(shí)驗(yàn)。 實(shí)驗(yàn)一變異鏈球菌熒光報(bào)告株與野生菌株生長(zhǎng)能力的比較 將復(fù)蘇后的變異鏈球菌,變異鏈球菌熒光報(bào)告株調(diào)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(biāo)儀測(cè)OD600值。繪制生長(zhǎng)曲線,結(jié)果兩者生長(zhǎng)趨勢(shì)相近,提示兩者具有相同的生長(zhǎng)狀態(tài)。 實(shí)驗(yàn)二變異鏈球菌熒光報(bào)告株與野生菌株生物膜形成能力的比較 在96孔細(xì)胞培養(yǎng)板中培養(yǎng)變異鏈球菌熒光報(bào)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(jié)晶紫染色,無水乙醇脫色,酶標(biāo)儀測(cè)OD575。組間比較發(fā)現(xiàn)兩菌株生物膜形成量無統(tǒng)計(jì)學(xué)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(jié)果發(fā)現(xiàn)不同時(shí)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(tuán)、成膜分布、各個(gè)菌落融合并重疊,形成網(wǎng)狀結(jié)構(gòu)。高倍鏡下細(xì)菌形態(tài)也未見改變。提示兩者生物膜形態(tài)相似。 實(shí)驗(yàn)三變異鏈球菌熒光報(bào)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(bào)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(jìn)行掃描,測(cè)量其厚度和組織結(jié)構(gòu)。結(jié)果發(fā)現(xiàn)不同時(shí)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(shí)間段樹脂表面,提示變異鏈球菌熒光報(bào)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，

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