非平衡等离子体对粪肠球菌生物膜QS-fsr系统的作用研究

Quorum-sensing (QS) system controls the signal transduction among bacteria, and plays a key role in regulating bacterial biofilms formation, development and maturation during bacterial growing. The fsr quorum-sensing (QS-fsr) system is an important one in Enterococcus faecalis biofilms. In our previous studies, it has been revealed that nonequilibrium plasma has the antibacterial activity against Enterococcus faecalis biofilms effectively. However, the mechanism of nonequilibrium plasma killing biofilms cells is still unknown. We hypothesize that nonequilibrium plasma may destroy the regulation effect of QS-fsr system, and inhibit Enterococcus faecalis biofilms growing. To conform this hypothesis in this project, Enterococcus faecalis biofilms and infected dentinal tubules in vitro, and apical periodontitis in rats are established. The techniques of SEM, CLSM, flow cytometry, real-time PCR, western blot, and immunofluorescence are used to reveal the effect of nonequilibruim plasma killing Enterococcus faecalis biofilms cells, and indicate the mechanism of nonequilibrium plasma against QS-fsr system. This is a new viewpoint of preventing QS-fsr system in the present project. It is aimed to provide a wild theoretical basis for studying the mechanism of nonequilibrium plasma killing bacterial biofilms cells, and a novel choice in therapy of endodontic diseases.

群体感应（QS）系统能控制细菌间的信息传导，在细菌生长过程中扮演了重要角色，对调节生物膜形成、发展及成熟起着至关重要的作用。粪肠球菌生物膜的群体感应系统主要是fsr系统。我们的前期实验结果提示非平衡等离子体对粪肠球菌生物膜具有良好的灭活作用，但其作用机制还有待进一步探讨。为此，我们提出假说：非平衡等离子体可能通过干预QS-fsr系统的调控作用，从而抑制粪肠球菌生物膜的生长。为了验证这一假说，我们将通过建立粪肠球菌生物膜和感染牙本质小管体外模型以及牙髓根尖周病动物模型，采用SEM、CLSM、流式细胞术、real-time PCR、western blot、免疫荧光等手段，明确非平衡等离子体抑制粪肠球菌生物膜的重要作用，初步探讨其对QS-fsr系统的作用机制。本研究将从干预QS-fsr系统这个新视点为揭示非平衡等离子体抑制细菌生物膜的作用机制奠定基础，为牙髓根尖周病的治疗提供新的思路。

群体感应（QS）系统能控制细菌间的信息传导，在细菌生长过程中扮演了重要角色，对调节生物膜形成、发展及成熟起着至关重要的作用。粪肠球菌生物膜的群体感应系统主要是fsr系统。本项目建立了粪肠球菌生物膜的体外模型，证实了非平衡等离子体对感染根管粪肠球菌生物膜具有良好的杀灭细菌作用，并且利用扫描电镜（SEM）、流式细胞术和激光扫描共聚焦显微镜（CLSM）等连续观察非平衡等离子体对粪肠球菌生物膜的作用；其次，在体外培养了不同生长时间节点（1d、3d、7d和3w）的粪肠球菌生物膜，利用real-time PCR方法评估非平衡等离子体对QS-fsr系统调控作用的关键因子fsrD、fsrB、gelE和sprE表达的影响；最后，建立了粪肠球菌感染的比格犬根尖周病的动物模型，分别应用标准化的根管治疗及辅以非平衡等离子体处理，利用X线片、CBCT、HE染色及免疫组织化学染色等不同的方法观察控制细菌生物膜生长及根尖周组织愈合的情况。本研究的结果明确了非平衡等离子体控制细菌生物膜的作用机制，为防治牙髓根尖周病提供新的契机。