siRNA干扰ffh、htrA基因对变形链球菌耐氟菌株毒力因子的影响

发布时间：2018-05-12 08:43

本文选题：siRNA + htrA基因　；参考：《吉林大学》2014年硕士论文

【摘要】：目的：龋病是一种非常常见的口腔疾病，是由多种因素共同作用所导致的牙齿硬组织的进行性病损。其中细菌感染是龋病发生的重要因素之一[1.2.3]，尤其多发于儿童[4]。在对龋病微生物学特性研究结果显示，变形链球菌[5]是致龋能力最强的龋病致病菌[6-7]。耐酸性和产酸性是变形链球菌在口腔环境中尤其是牙的生物膜上生存的一个重要因素[8]。研究[3]表明：ffh基因的表达和htrA基因的表达与变形链球菌的产酸，耐酸及生物膜的形成密切相关。本研究通过对比siRNA[9]靶向分别沉默ffh基因[10-11]和htrA[12]基因后变形链球菌在强酸弱酸及各种糖的作用下，研究其耐氟菌株的产酸性、耐酸性及生物膜[13]的形成等生物学性状的影响。更深刻的揭示变形链球菌的耐酸机制，也为龋病的防治提供新的思路和理论指导。方法：体外诱导变形链球菌耐氟菌株根据本实验组常规方法将鉴定后的变形链球菌放入以50ug/ml NaF递加直到在还有1000ug/ml NaF的BHI固体培养基中获得耐氟菌落。电穿孔转染将鉴定后的UA159-FR悬液与siRNA oligo按2:1比例混合加入电转杯中电极选择适合电极。用real time-PCR方法检测转染效果并筛选出条件较好的siRNA序列。功能检测将转染后的UA159-FR培养在以0.5为间隔，配置pH从3.5至7.0的培养液中进行耐酸检测收集数据并分析；培养在含有5%不同糖类的BHI培养液中进行产酸检测收集数据并分析；培养在放有盖玻片[14]的孔板中观察生物膜的形成。结果： siRNA oligo在变形链球菌耐氟菌株中成功干扰ffh基因和htrA基因表达。应用real-time PCR技术对四组siRNA序列进行筛选，得出干扰较好的一组siRNA序列进行后续的实验。对干扰后的UA159-FR进行产耐酸检测，干扰htrA基因的数据优于干扰ffh基因的数据。证实siRNA对变形链球菌的干扰是有效的。检测效果时间均规定为24小时以便对比数据及分析。在生物膜构建过程中，干扰htrA基因的UA159-FR没能观察到完整的类似生物膜结构；干扰ffh基因的UA159-FR可观察到类似生物膜结构，，但是结构上有大小不等的孔隙。结论：成功将siRNA序列电穿孔转染进入变形链球菌耐氟菌株当中，成功分别干扰变形链球菌耐氟菌株的ffh基因和htrA基因的表达，并成功检测干扰后功能，得出产酸耐酸和生物膜的样本及相关检测数据并分析。
[Abstract]:Objective: Dental caries is a very common oral disease. It is a progressive lesion of hard tissue caused by various factors. Bacterial infection is one of the important factors in the occurrence of caries [1.2.3], especially in children [4]. The microbiological characteristics of dental caries showed that Streptococcus mutans [5] was the most potent cariogenic pathogen [6-7]. Acid tolerance and acidogenesis are important factors for the survival of Streptococcus mutans in oral environment, especially on dental biofilm [8]. The study [3] showed that the expression of the htrA gene and the expression of the htrA gene were closely related to the production of acid, acid tolerance and biofilm formation of Streptococcus mutans. In this study, we compared the effects of siRNA [9] targeted silencing ffh gene [10-11] and htrA [12] gene on the production of acid, acid tolerance and biofilm formation of fluorine-tolerant strains under the action of strong acid and weak acid and various sugars. The mechanism of acid tolerance of Streptococcus mutans is revealed more deeply, and it also provides new ideas and theoretical guidance for the prevention and treatment of caries. Methods: Fluoride-resistant strains of Streptococcus mutans were induced in vitro. The identified Streptococcus mutans were added with 50ug/ml NaF until they were obtained in BHI solid medium with 1000ug/ml NaF. Electroporation transfection mixed the identified UA159-FR suspension and siRNA oligo at 2:1 ratio to the electrode in the electrorotary cup to select the suitable electrode. The transfection effect was detected by real time-PCR method and the siRNA sequence with better conditions was screened out. After transfection, the UA159-FR were cultured at 0.5 intervals and the pH ranged from 3.5 to 7.0. The acid-resistance data were collected and analyzed in the culture medium containing 5% different sugars, and the acid-producing data were collected and analyzed in the culture medium containing 5% different sugars. The biofilm formation is observed in a hole plate with a cover glass [14]. Results: SiRNA oligo successfully interfered with the expression of ffh and htrA genes in Streptococcus mutans. Four groups of siRNA sequences were screened by real-time PCR technique, and a group of siRNA sequences with good interference was obtained for subsequent experiments. The data of interfering htrA gene was better than that of interfering ffh gene. It is proved that siRNA is effective in interfering with Streptococcus mutans. The effect time is 24 hours to compare data and analysis. During the process of biofilm construction, UA159-FR which interfered with htrA gene could not observe the complete biofilm-like structure, while UA159-FR which interfered with ffh gene could observe biofilm-like structure, but there were some holes in the structure. Conclusion: The siRNA sequence was successfully transfected into Streptococcus mutans fluoride resistant strain, and the expression of ffh gene and htrA gene were interfered respectively, and the function after interference was detected successfully. Acid-resistant and biofilm samples were obtained and analyzed.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R781.1

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