常压低温等离子体对小鼠伤口定植绿脓杆菌的作用研究

发布时间：2018-01-22 00:53

本文关键词： 常压低温等离子体伤口愈合 BalB/c小鼠细菌定植常压低温等离子体铜绿假单胞菌细菌定植毒力因子常压低温等离子体铜绿假单胞菌生物膜藻酸盐　出处：《华中科技大学》2011年博士论文　论文类型：学位论文

【摘要】：第一部分常压低温等离子体抑制小鼠伤口细菌定植并促进伤口愈合目的本实验用于评估常压低温等离子体在BalB/c小鼠伤口愈合中的作用。方法在每只小鼠(n=80)的后背钻取两个直径为6mm的皮肤伤口,并将小鼠随机平分为两组：第1组作为治疗组应用常压低温等离子体进行治疗,第2组作为对照组。在术后(POD)第4、7、10和14天从每组取5只小鼠,以伤口愈合百分数作为指标进行评估。在术后(POD)第1、4、7、10、14、21、28和35天从每组再取1只小鼠进行安乐死,取每只小鼠两个伤口的周围组织,石蜡包埋,苏木精伊红染色,应用改良评分系统评估伤口愈合情况。结果组织学检测结果证实,两组小鼠的伤口愈合情况有明显差异,在术后第7天急性炎症反应、术后第4天和第7天肉芽组织数量、术后第10天肉芽组织纤维母细胞形成、术后第7天和第10天表皮细胞再生以及术后第7天和第10天新生血管形成这些指标中,差异具有统计学意义(P0.05)。结论常压低温等离子体能够促进小鼠伤口愈合。进一步的体外试验发现,常压低温等离子体能抑制细菌增殖,提示其促进伤口愈合的作用可能与抑制细菌定植有关。第二部分常压低温等离子体对铜绿假单胞菌毒力因子表达的影响目的探讨常压低温等离子体对铜绿假单胞菌毒力因子表达的影响。方法分别将铜绿假单胞菌PAO1株和经常压低温等离子体照射后的铜绿假单胞菌PA0911、PA0912、PA0913加入到铜绿假单胞菌的液体培养基(OD600=0.05)中,应用刚果红弹性蛋白检测其弹性蛋白酶活性；通过氯仿、盐酸萃取法对绿脓菌素进行定量检测。结果与PAO1株相比,经常压低温等离子体照射后的铜绿假单胞菌PA0911、PA0912、PA0913株的生存能力明显减弱,弹性蛋白酶活性降低,绿脓菌素含量减少。结论低压等离子体能够抑制小鼠伤口定植铜绿假单胞菌毒力因子的表达,降低其生存能力。第三部分常压低温等离子体对铜绿假单胞菌生物膜形成的影响及机制目的探讨常压低温等离子体对铜绿假单胞菌生物膜形成的影响及其可能的机制。方法应用改良平板法分别建立铜绿假单胞菌PAO1株和经常压低温等离子体照射后的铜绿假单胞菌PA0912的生物膜模型,结晶紫染色后作生物膜定量分析；RT-PCR法测定两种菌株在生物膜形成阶段不同时间点藻酸盐合成基因algD的表达情况。结果铜绿假单胞菌PAO1株在第3天形成成熟生物膜,而铜绿假单胞菌PA0912株则丧失了形成生物膜的能力；在本实验的整个观察期中,铜绿假单胞菌PA0912株的algD基因表达水平都要明显低于PAO1株。结论常压低温等离子体能够阻止铜绿假单胞菌形成生物膜,其机制可能与抑制藻酸盐合成基因algD的表达有关。
[Abstract]:The purpose of this study was to evaluate the effect of atmospheric low temperature plasma on wound healing in BalB/c mice. Rats (. Two skin wounds with a diameter of 6 mm were drilled from the back of the nong 80). The mice were randomly divided into two groups: group 1 was treated with low temperature plasma under normal pressure and group 2 as control group. Five mice were taken from each group on 10 and 14 days to evaluate the wound healing percentage. On the 28th and 35th day, another mouse was taken from each group for euthanasia. The tissue around the wound was embedded in paraffin, stained with hematoxylin eosin, and the wound healing was evaluated by the improved scoring system. Results the results of histological examination confirmed that there was a significant difference in wound healing between the two groups. The acute inflammatory reaction was observed on the 7th day after operation and the number of granulation tissue on the 4th and 7th day after operation. On the 10th day after operation, fibroblast formation in granulation tissue, epidermal cell regeneration on the 7th and 10th day after operation, and angiogenesis on the 7th and 10th day after operation. The difference was statistically significant (P 0.05). Conclusion Atmospheric low temperature plasma can promote wound healing in mice. Further experiments in vitro showed that atmospheric low temperature plasma could inhibit the proliferation of bacteria. The results suggest that its effect on wound healing may be related to the inhibition of bacterial colonization. The second part: effect of low temperature plasma on virulence factor expression of Pseudomonas aeruginosa Objective to investigate the effect of low temperature plasma on virulence factor expression of Pseudomonas aeruginosa. Methods Pseudomonas aeruginosa PAO1 strain and P. aeruginosa PA0911 / PA0912 were irradiated by plasma under normal pressure and low temperature, respectively. PA0913 was added to the liquid culture medium of Pseudomonas aeruginosa OD600. 05) and the activity of elastase was detected by Congo erythroelastin. Pseudomonas aeruginosa was determined quantitatively by chloroform and hydrochloric acid extraction. Results compared with PAO1 strain, the survival ability of Pseudomonas aeruginosa strain PA0911 / PA0912 / PA0913 was significantly decreased after plasma irradiation under constant pressure and low temperature. The activity of elastase decreased and the content of pyocyanin decreased. Conclusion low pressure plasma can inhibit the expression of virulence factor of Pseudomonas aeruginosa and decrease its viability. The effect of low temperature plasma at atmospheric pressure on the biofilm formation of Pseudomonas aeruginosa and its mechanism Objective to investigate the effect of atmospheric low temperature plasma on the biofilm formation of Pseudomonas aeruginosa and its possible mechanism. Methods the biofilm models of Pseudomonas aeruginosa PAO1 strain and PA0912 were established by modified plate method. Quantitative analysis of biofilm was made after staining with crystal violet. The expression of alginate biosynthesis gene algD at different time points in biofilm formation of two strains was determined by RT-PCR method. Results Pseudomonas aeruginosa PAO1 strain formed mature biofilm on the 3rd day, while PA0912 strain lost the ability to form biofilm. During the whole observation period, the algD gene expression level of Pseudomonas aeruginosa PA0912 strain was significantly lower than that of PAO1 strain. Conclusion low temperature plasma can prevent Pseudomonas aeruginosa from forming biofilm, and its mechanism may be related to the inhibition of the expression of alginate synthesis gene algD.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R378

【共引文献】