大鼠慢性延迟愈合创面与铜绿假单胞菌生物被膜相关性研究

Bacterial colonization and infection are key factors in compromised wound healing,particularly in chronic wounds.Reported in the literature refractory chronic skin ulcers and bedsores are closely related to Pseudomonas aeruginosa biofilm formation.Quorum sensing contributes much to biomembrane formation.In the support of National Natural Science Foundation, we investigated the regulation of Pseudomonas aeruginosa quorum sensing system on biomembrane formation．For the first time we found the Pseudomonas aeruginosa catabolite repression control protein( Crc) mutant has a higher fitness compared to the wild-type P.aeruginosa in vitro.The catabolite repression mutant strains with high metabolic activity show high resistantance to the treatment of antimicrobial peptides. P. aeruginosa Crc mutant is able to produce more pyocyanin and losts the movement ability associated with drug resistance. Moreover, the early stage biomembrane formation of P. aeruginosa Crc mutant is less than its wild-type strain. It can be inferred the Pseudomonas aeruginosa catabolite repression control protein（Crc）can affect the release of toxicity factor and affect the whole process of biofilm formation. We designed rat model of chronic skin ulcers in order to confirm this hypothesis. We discuss the influence of the Crc mutation for chronic wound biomembrane formation through the wild type and Crc gene mutation of pseudomonas aeruginosa.For improving treatment of chronic infection induced by biomembrane, we need to investigate the characteristics of the biomembrane in the animal model suffered from different types of chronic infection.However, the mechanism how Crc affects the P. aeruginosa pqs QS system in medium supplemented with different carbon sources is not clear, and the study of how Crc control long-term formation of biomembrane and host immunity. Further study coud reveral how different biomembrane affact would healing.and what is the difference between mutant and wild-type.Based on our previous study, the purpose of this work is to develop some drugs or treatments to control formation and collapse of biomembrane.

慢性难治性皮肤溃疡及褥疮与铜绿假单胞菌生物被膜形成密切相关，在生物被膜形成过程中密度感应系统发挥非常重要的作用，在自然基金资助下，我们系统的研究了铜绿假单胞菌密度感应信号系统对生物被膜形成的影响。在体外率先发现铜绿假单胞菌的分解代谢物阻遏调控蛋白（Crc）突变株比其野生型菌株有较高水平的的代谢活性，能够抵抗抗菌肽的作用，Crc突变菌可过量合成绿脓素，同时又丧失了与耐药性相关的涌动能力，影响早期生物被膜的形成。由此可以推断铜绿假单胞菌的分解代谢物阻遏调控蛋白能够影响细菌毒性因子的释放，且影响生物被膜形成的全过程。为了证实此假设我们设计制作大鼠慢性皮肤溃疡模型，播种野生型及Crc基因突变的铜绿假单胞菌，探讨Crc突变对慢性创面生物被膜形成的影响，同时明确不同生物被膜感染对创伤愈合的影响，深入了解野生株和突变株在细菌毒力上的差异，期待研发出控制生物膜形成或瓦解生物膜的药物及治疗方案。

细菌生物被膜的形成是创口迁延不愈形成慢性难愈性创面的最主要因素，铜绿假单胞菌是创面单细菌感染最主要菌种之一，形成的生物被膜复杂难控。有研究表明细胞外基质可影响细菌生长发育，大量的体外研究也证明透明质酸作为细胞外基质最主要的成分之一有抑制多种细菌生物被膜形成的作用，但具体机制不明确，可能与细菌定植密切相关，并且体外给予的透明质酸极易被细菌产生的透明质酸酶分解，难以达到理想的效果。本研究系统性研究了铜绿假单胞菌感染的大鼠慢性皮肤溃疡中细胞外基质的变化、人皮肤成纤维细胞中透明质酸合成的潜在分子机制，力求通过明确人体内透明质酸合成的具体调控分子机制，来调节人类宿主自身创面透明质酸的表达，从而干扰细菌定植，防止生物被膜形成。我们的研究结果显示：(1) 铜绿假单胞菌感染的大鼠创面皮肤组织中TGF-β1低表达，导致ECM的生物学改变与纤维化和再生有关，从而影响伤口愈合过程；表明铜绿假单胞菌感染导致细胞外基质降解可能是促使细菌成功定植的原因之一。(2) 异常修复瘢痕组织基因芯片分析结果中发现C-Fos、Rho、EGR-1等这些与成纤维细胞密切相关的基因表达均发生了异常该变；再次证明了成纤维细胞在创面修复中的重要作用。(2) UVA辐射诱导成纤维细胞损伤模型中透明质酸合成的减少与PcG蛋白关键分子EZH2的表观遗传学调控密切相关。(3) EZH2甲级转移酶抑制剂可修复UVA辐射所诱导的成纤维细胞损伤，影响透明质酸合成和分解相关酶类基因的表达；提示我们可以通过表观遗传学调控组蛋白H3K27me3水平，从而控制透明质酸的合成和分解。有望在体内通过调控透明质酸的含量，从而抑制创面细菌定植，防止细菌生物被膜的形成，促进慢性创面愈合。