药用壁虎免疫调节肽Gj-CATH5抗耐药菌感染的机制研究

The regulation of innate immune response against infection has become a hot topic in the field of anti-infection therapy, especially for the treatment of drug-resistant bacteria infection. Cathelicidin is one type of immunoregulatory factors existing in vertebrates, and plays important roles in the resistance of pathogens. However, no studies have been reported about the gecko cathelicidin. Recently, five novel cathelicidin genes (encoding Gj-CATH1-5) were firstly screened and characterized from Gekko japonicas. Compared with other cathelicidins, Gj-CATH5 exhibit stronger antimicrobial activities and can regulate immune response through NOD-like pathway, which urges us to further explore and study the anti-infection mechanism of Gj-CATH5. Then, two defective mouse models were established to determine the key anti-infection effector cells of Gj-CATH5 in vivo. Further, the effects and action mechanisms of Gj-CATH5 on phagocytic and bactericidal activities of immune cells were investigated by laser confocal microscopy (LSCM) and flow cytometry. In addition, the chemotaxis and action mechanisms of Gj-CATH5 on immune cells will be uncovered by Western Blotting, ELISA and qPCR. Our research will not only enrich the understanding of cathelicidin’s biological function, but be of great significance to provide theoretical basis for clinical application of gecko.

免疫疗法已成为抗感染治疗领域的研究热点，尤其是对耐药菌感染的治疗。Cathelicidin是脊椎动物体内的一类先天免疫调节因子，在抵御外界病原菌入侵的过程中扮演着重要的角色，但目前对壁虎科cathelicidin的研究还未见报道。我们前期筛选到5条多疣壁虎cathelicidins (Gj-CATH1-5)，发现Gj-CATH5相较于其他cathelicidins具有较强的抗耐药菌活性，且能通过NOD-like通路发挥作用，很有必要对其作用机制进一步研究。本课题以Gj-CATH5为中心，从调节先天免疫细胞的趋化和杀菌活性两个角度出发，通过体外细胞实验、构建特异性PMN或Mφ缺陷型鼠模型以及对动物模型指标的检测等深入探讨Gj-CATH5抗耐药菌感染机理。本课题的研究将有望为耐药菌感染的治疗提供新策略，为指导传统中药壁虎的临床应用提供理论依据，具有深远的科学意义。

调节先天免疫抗感染已成为抗耐药菌感染治疗领域的研究热点。Cathelicidin是脊椎动物体内的一类免疫调节因子，在宿主先天免疫防御过程中扮演着重要角色。我们前期从多疣壁虎体内筛选到5条结构各异的cathelicidin家族免疫调节肽（Gj-CATH1-5）。研究发现，Gj-CATH5具有很强的调节先天免疫抗铜绿假单胞菌感染活性，能够显著降低小鼠腹腔液和脾脏中的细菌总负荷。抗感染机制研究表明，Gj-CATH5具有两亲性的α螺旋结构，可破坏细菌细胞膜发挥in vitro杀菌活性；此外，Gj-CATH5可通过对中性粒细胞和单核/巨噬细胞的诱导趋化和功能调控实现in vivo抗感染活性。Gj-CATH5可以浓度依赖性的方式促进中性粒细胞和巨噬细胞呼吸爆发，且同等剂量下中性粒细胞被诱导产生的活性氧量远多于巨噬细胞。Gj-CATH5处理不仅有助于巨噬细胞和中性粒细胞的吞噬杀菌活性，高浓度下还对中性粒细胞脱颗粒活性和NETs诱捕病原菌的能力具有促进作用。与Gj-CATH5不同，Gj-CATH2在抗龋齿领域具有一定的应用潜力。研究发现，Gj-CATH2可通过靶标细菌粘附和生物膜成熟阶段抑制变形链球菌生物膜形成。Gj-CATH2能够通过抑制变形链球菌毒力因子相关基因的表达进而显著抑制变形链球菌酸的产生、细胞聚集及EPS的合成。与Gj-CATH5和Gj-CATH2不同，Gj-CATH3虽不具有抗感染活性，但在皮肤创伤修复治疗领域具有很大的应用前景。本项目的研究丰富了我们对cathelicidin家族肽生物学功能的认知，对指导中药壁虎的临床应用具有深远的科学意义。