本文選題：變異鏈球菌　切入點：LuxS基因　出處：《天津醫(yī)科大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的：本實驗是通過采用常規(guī)生化鑒定、革蘭氏染色涂片鑒定和生長曲線測定等方法闡述了變鏈菌標準株與LuxS基因突變株在生物學(xué)性狀方面的異同。此外,建立LuxS基因缺失突變株的生物被膜體外模型,用結(jié)晶紫染色等方法觀察變鏈菌生物被膜結(jié)構(gòu),研究LuxS基因突變對其形成生物被膜的能力的影響,并借助于掃描電鏡觀察了LuxS基因缺失突變株和標準株所形成的生物被膜,為進一步研究LuxS信號系統(tǒng)對變鏈菌致齲毒力的調(diào)控機制奠定基礎(chǔ)。 方法：將變鏈菌標準株與LuxS突變株分別在含紅霉素及不含紅霉素的TSA固體培養(yǎng)基中作菌落培養(yǎng),觀察兩種菌落的生長情況并作常規(guī)生化檢測、革蘭氏染色涂片以比較兩種菌株的生長特性。將復(fù)蘇24h的標準株及LuxS突變株于紫外分光光度計600nm處制備成吸光度A=1.0的菌懸液備用。將上述菌懸液1000倍稀釋接種于BHI液體培養(yǎng)基,置于37℃恒溫搖床中孵育,定時取樣測量A值,觀察比較兩者在生長曲線上的差異。將LuxS基因突變株和標準株接種于BHI液體培養(yǎng)基中,培養(yǎng)48h后,取出相應(yīng)的微孔板,結(jié)晶紫染色生物被膜,觀察細菌生物被膜的形態(tài)變化。將提前制備好的無菌牙釉質(zhì)磨片置于6孔細胞培養(yǎng)板中,并依次編號第1-6孔,每孔放置4片,分別取變鏈菌標準株和LuxS突變株的A600=1.0的菌懸液向每孔中接種1ml(其中第1、4孔接種菌液為標準株,2、3、5、6四孔接種菌液為LuxS突變株),均勻滴至牙釉質(zhì)磨片表面,靜置約2min后向每孔中加入5mlTPY液體培養(yǎng)基(其中第1、2、3孔為含2%葡萄糖的TPY溶液,4、5、6孔為含2%蔗糖的TPY溶液),然后取提前制備好的標準株上清液各500μl加入至3、6兩孔。將上述菌液于37℃厭氧條件下培養(yǎng)24h,于掃描電鏡下觀察在無菌牙釉質(zhì)磨片上形成的生物被膜,并進行總體評價以對比兩種菌株的生物被膜的形成能力。 結(jié)果：在含紅霉素的TSA-Eymr固體培養(yǎng)基中,標準株基本不能生長,而突變株的生長狀況基本正常。在不含紅霉素的TSA固體培養(yǎng)基中培養(yǎng),兩種菌株的菌落形態(tài)沒有明顯差別,鏡下可見標準株菌體呈長鏈狀排列且相互纏繞,而突變株菌體多呈短鏈狀排列,形成長鏈的較少。通過對變鏈菌標準株與LuxS突變株測定生長曲線后發(fā)現(xiàn),兩者在生長模式上并無明顯差異,只是在進入生長的穩(wěn)定期后,兩者在細菌飽和度上有一定差異,標準株獲得了較群體感應(yīng)基因LuxS的突變株更多的細胞生長。在BHI中標準株和突變株均能夠形成生物被膜,但結(jié)構(gòu)存在差異：標準株形成光滑且均勻分布的生物被膜,突變株的生物被膜形態(tài)粗糙。于掃描電鏡下觀,與生長在補充了2%葡萄糖的TPY液體培養(yǎng)基中的變鏈菌標準株相比,LuxS基因突變株所形成的生物被膜表現(xiàn)型未見明顯差異。在補充了2%蔗糖的TPY液體培養(yǎng)基中生長時,LuxS基因突變株所形成的生物被膜的表現(xiàn)型在形態(tài)學(xué)上與標準株形成的生物被膜有明顯的不同,LuxS基因突變株形成較大的團簇狀菌落,生成生物被膜的能力下降。結(jié)構(gòu)相對粗糙,呈松散的蜂房狀,生物被膜基質(zhì)間有較大的間隙,而標準株生物被膜呈相對融合的外觀,分布更加均衡。當用標準株上清液來彌補突變株時,形成的聚集物要小的多,此時形成的生物被膜結(jié)構(gòu)介于標準株和突變株之間。 結(jié)論：本研究通過常規(guī)生化鑒定、革蘭氏染色涂片鑒定和生長曲線測定等方法證實了變鏈菌標準株與LuxS突變株在生長特性上有一定的差異性,LuxS基因突變可以抑制變鏈菌的生長,并闡述了兩菌株在生物學(xué)性狀方面的異同。此外,通過建立LuxS基因缺失突變株的生物被膜體外模型,用結(jié)晶紫染色等方法觀察了變鏈菌生物被膜結(jié)構(gòu),證實了LuxS基因突變對其形成生物被膜的能力有一定影響,掃描電鏡下分析,兩種菌株均表現(xiàn)出形成生物被膜的能力,但LuxS基因突變株形成生物被膜的能力有所減弱,結(jié)構(gòu)發(fā)生改變,標準株能彌補LuxS基因突變株的生物被膜的變化表型,依賴于LuxS的群體感應(yīng)系統(tǒng)會影響變鏈菌生物被膜的形成。
[Abstract]:Objective: the purpose of this study is through the use of conventional biochemical identification, gram elaborated between Streptococcus mutans standard strain strain in biological characters and LuxS mutation stain identification and determination of growth curve and other methods. In addition, the establishment of the LuxS gene deletion mutant biofilm model in vitro by crystal violet staining, variable chain the bacterial biofilm structure, mutation of LuxS gene on the biological effects of membrane formation by ability, and with the aid of scanning electron microscopy LuxS mutant strain and standard strain in the biofilm, and lay the foundation for the further study of the regulatory mechanism of LuxS signaling system on the cariogenicity of virulence.
Methods: the chain will become standard strain and LuxS mutant strain were cultured in culture medium for colony in contain erycin or not TSA solid, to observe the growth of two colonies and conventional biochemical test, Gram staining to compare the growth characteristics of two kinds of strains. The recovery of 24h standard strain and LuxS mutant on line ultraviolet spectrophotometer at 600nm prepared absorbance A=1.0 bacterial suspension. The suspension will reserve 1000 times dilution inoculated in BHI liquid medium, a constant temperature of 37 DEG C shaker in the incubation time sampling measurements of A were observed to compare the differences in the growth curve of the LuxS gene. Mutant strains and standard strains inoculated in BHI liquid medium, after 48h, remove the corresponding microplate, crystal violet staining of biological membrane, observe the morphological changes of bacterial biofilm. Early prepared sterile enamel slabs placed 6 Hole Fine Bao Pei A board, and numbered the 1-6 holes, each hole placed 4 tablets were taken S.mutan standard strain and LuxS mutant of A600=1.0 bacterial suspension inoculation to each hole 1ml (including 1,4 liquid fungus inoculation standard strain 2,3,5,6 four hole inoculated LuxS mutant). Uniform drop to the enamel surface of the disc, standing about 2min after adding 5mlTPY into each hole liquid medium (the 1,2,3 is TPY solution containing 2% 4,5,6 glucose is TPY solution containing 2% sucrose), and then advance the prepared standard strains of 500 mu l of the supernatant was added to 3,6 two holes. The bacteria liquid at 37 DEG C under anaerobic conditions in cultured 24h, scanning electron microscope in the formation of enamel slabs on the sterile biofilm, and overall evaluation to a comparison of two strains of biofilm formation ability.
Results: in TSA-Eymr solid medium containing erythromycin, basic standard strains can grow, and the growth status of the normal mutation strains. In the medium containing erythromycin TSA solid, there was no difference in colony morphology of two strains, microscopically standard strain was long chains and tangled, and the mutant bacteria in short chains, the formation of long chain less. By means of variable chain standard strain and LuxS mutant strain growth curves were found both in growth mode has no significant difference, but in stable growth, there are some differences in bacterial saturation are obtained with two standard strains the quorum sensing gene LuxS mutant growth. More cells in BHI standard strain and mutants were able to form biofilms, but there are differences in the structure: the standard strain to form a smooth and uniform distribution of biofilm process Strain biofilm morphology by scanning electron microscope. The rough concept, and growth in added 2% glucose TPY liquid culture medium of Streptococcus mutans standard strains compared with LuxS mutant in the biofilm phenotype. There is no significant difference in TPY added 2% sucrose liquid medium. When the LuxS gene mutation phenotype strains in the biofilm formation and standard strains in morphology of biofilm were significantly different, LuxS mutant formed clusters like the larger colonies, generating creature falling film ability. The structure is relatively rough, loose honeycomb, creature there is a large gap between the membrane matrix, and the standard strains of biofilm showed relatively fusion appearance, distribution is more balanced. When using standard strains of supernatant to compensate for the mutant, the formation of aggregates is much smaller, the formation of biofilm structure between standard strain and Among the mutant strains.
Conclusion: This study by conventional biochemical identification, Gram staining identification and determination of growth curve and other methods confirmed the Streptococcus mutans standard strain and LuxS mutant on growth characteristics have some differences, the mutation of LuxS gene can inhibit the growth of S.mutans, and expounds the similarities and differences between the two strains in biological characters. In addition, through the establishment of LuxS gene deletion mutant biofilm model in vitro, with the crystal violet staining method to observe the S.mutans biofilm structure, confirmed that LuxS gene mutation had certain influence on its ability to form biofilm, scanning electron microscope analysis, two strains showed the formation ability of creatures the film, but the LuxS mutant with the ability to form biofilm weakened, change the structure and standard strains can make up for the phenotypic change of LuxS gene mutation strains of biofilm, quorum sensing system relies on LuxS The association affects the formation of the biofilm of the Streptococcus mutans.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R378

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相關(guān)期刊論文 前1條

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本文編號：1585874