耐酸血链球菌拮抗变异链球菌作用机制的初步研究

发布时间：2018-05-01 07:28

本文选题：耐酸 + 血链球菌　；参考：《安徽医科大学》2017年硕士论文

【摘要】：1.背景与目的变异链球菌是口腔主要的致龋菌之一。药物杀灭变异链球菌一直是龋病防治工作的重要辅助手段,但细菌容易形成耐药性。随着生物工程的不断发展,运用益生菌替代疗法得到越来越多的关注。细菌替代疗法就是使用无害或者低毒力的菌株放入宿主的菌群中,防止特定病原体定植,从而起到预防、治疗疾病的目的。龋病替代疗法的候选菌种,除了变异链球菌本身外还有血链球菌。本研究通过逐渐降低培养基ph构建血链球菌耐酸株,研究血链球菌耐酸株的适应性耐酸能力及其相关生理变化。并通过平板实验、PCR等技术观察血链球菌耐酸株拮抗变异链球菌的能力,弄清血链球菌、变异链球菌在龋病发病过程中相互关系,为探索龋病替代疗法提供微生物学基础。2.方法2.1在逐渐降低培养液ph值的培养液中培养血链球菌,直至ph降至5.0。获得的菌株命名为血链球菌耐酸株(SN)。2.2比较血链球菌及其耐酸株在ph7、ph5培养液中的生长曲线。2.3测量血链球菌及其耐酸株、变异链球菌及其耐酸株的致死ph值。2.4测量氯霉素对血链球菌耐酸株适应性耐酸能力的影响。2.5通过平板实验检测血链球菌及其耐酸株与变异链球菌在ph7和ph5环境中的拮抗作用。2.6通过荧光定量PCR反应比较血链球菌及其耐酸株的耐酸基因DnaK、GroEL的变化。3.结果3.1经过亚致死ph培养的血链球菌耐酸株无论在正常环境还是在酸性环境生长均有优势。3.2血链球菌耐酸株较血链球菌致死ph降低了1.3,且有66.59%能在原致死ph存活。3.3当含有氯霉素时,血链球菌耐酸株不能在原致死ph存活。3.4血链球菌耐酸株对变异链球菌的竞争排斥作用更强。3.5血链球菌耐酸株相关耐酸基因GroEL、DnaK表达上调。4.结论4.1成功构建血链球菌耐酸株。4.2血链球菌耐酸株较血链球菌适应性耐酸能力明显提高。4.3血链球菌适应性耐酸能力的提高与应激蛋白的合成有关。
[Abstract]:1. Background & objective Streptococcus mutans is one of the major dental caries-causing bacteria. Drug killing Streptococcus mutans has always been an important aid in the prevention and treatment of caries, but bacteria are susceptible to drug resistance. With the development of bioengineering, more and more attention has been paid to the application of probiotic substitution therapy. Bacterial substitution therapy is the use of harmless or low virulent strains into the host flora to prevent the colonization of specific pathogens and thus to prevent and treat diseases. In addition to Streptococcus mutans, the candidate strains for alternative Caries Therapy are Streptococcus sanguis. In this study, Streptococcus sanguis acid resistant strain was constructed by decreasing the pH of culture medium gradually, and the adaptive acid tolerance ability of Streptococcus sanguis acid resistant strain and its related physiological changes were studied. The ability of Streptococcus sanguis acid resistant strain to antagonize Streptococcus mutans was observed by means of plate experiment and PCR, and the relationship between Streptococcus sanguis and Streptococcus mutans in the process of caries was clarified, which provided the microbiological basis for exploring alternative therapy of caries. Methods 2.1 Streptococcus sanguis was cultured in the medium that gradually decreased the ph value of the culture medium until the ph decreased to 5.0. Compared the growth curve of Streptococcus sanguis and its acid-resistant strain in ph7ph5 medium, the growth curve of Streptococcus sanguis and its acid-resistant strain were measured. The effect of chloramphenicol on adaptive acid-tolerant ability of Streptococcus sanguis and its acid-resistant strains. 2.5 Detection of Streptococcus sanguis, Streptococcus mutans and Streptococcus mutans in ph7 and ph5 environments By fluorescence quantitative PCR reaction, we compared the changes of acid-tolerant gene DnaKGlol of Streptococcus sanguis and its acid-resistant strain. 3. Results 3.1 Streptococcus sanguis acid-resistant strains cultured with sublethal ph had an advantage both in normal and acidic environments. 3.2 Streptococcus sanguis acid resistant strains decreased by 1.3 compared with Streptococcus sanguis lethal ph, and 66.59% could survive in primary lethal ph. .3.3 when chloramphenicol is present, Streptococcus sanguis acid resistant strain could not survive in the proto-lethal ph. 3.4 Streptococcus sanguis acid resistant strain had stronger competitive rejection to Streptococcus mutans. 3.5 the expression of GroELL DnaK gene associated with streptococcus sanguis acid resistance strain was up-regulated by .4. Conclusion 4.1 the successful construction of Streptococcus sanguis acid resistant strain. 4.2 Streptococcus sanguis acid resistant strain significantly improved the adaptive acid tolerance ability of Streptococcus sanguis. 4.3 the increase of adaptive acid tolerance ability of Streptococcus sanguis is related to the synthesis of stress protein.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R780.2

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