烟曲霉菌丝极性生长在其侵袭血管中作用的研究

Aspergillus fumigatus is very important pathogen in clinic. The study of aspergillus virulence was focus on the spores. There is not any virulence gene associated with hyphal growth to be comfirmed in the asperigllus invasive infection. The primary step of the invasive aspergillosis is the pathogen invading into vessel to be thrombosis. It is unclear whether the orientation of hyphal polarized growth play any roles in the aspergillus penetrating vascular endothelia in invasive infection. At present, the polarized growth is focus on the Aspergillus nidulans in vitro. Our project is bace on the screening of the different mRNA level of spores and hypha in vitro. Then the different genes probes are used to inspect infectional tissues express level of the genes to filtrate the key genes associated with polarized growth in vivo. Subsequently, the genes will be knockouted and the function of the genes was explored, including analysis of the hyphal growth, animal virulence, invasion of vessel, sensitivity to antifungal agents, and penetration of endothelia before and after the genes are KO. The project is to further explore the pathogenesis and the key genes of polarized growth in invasive aspergillus infection. The work will provide new target for anti-aspergillus study.

烟曲霉是临床上重要的致病真菌，对其毒性因子的研究多集中在孢子方面，目前还未发现与其菌丝生长密切相关的毒性基因。菌丝生长的方向性与其穿透血管是否有关尚未有研究报道。菌丝长生的方向性即极性生长，对其研究主要针对构巢曲霉的体外研究。由于侵犯血管是曲霉侵袭性感染的重要致病基础，因此研究菌丝极性生长与其穿透血管对了解侵袭性曲霉病的发病机制有着重要价值。本项目拟在体外筛选极性生长相关基因的基础上，采用动物模型组织Northern原位杂交的方法进一步筛选在组织内高达的极性生长相关基因。对所筛选到的基因进行功能研究，包括比较相关基因缺失前后菌丝本身的生长情况差别、对动物致病性的差异、对组织血管侵袭能力的差异、对体外抗真菌药物敏感性的差异、以及对血管内皮细胞穿透能力的差异等。通过以上研究来了解在侵袭性感染中的烟曲霉关键极性生长相关基因，进一步了解曲霉致病机制，并为研究抗真菌药物新靶位点提供理论基础。

烟曲霉是导致侵袭性肺曲霉病的主要致病菌，是一种严重危害人类健康的致病真菌。本课题主要针对烟曲霉极性生长与侵袭血管的能力进行研究。运用基因芯片和RNA-seq技术，我们得到了与极性生长相关的可信差异表达基因。通过对部分差异表达基因进行基因敲除，得到了极性生长改变的菌株，通过对比突变前后烟曲霉对动物致病性、对组织血管的侵袭能力、对体外培养血管内皮细胞的穿透能力以及对抗真菌药物敏感性的变化来进一步了解烟曲霉在侵袭组织中的致病机制。结果表明，敲除驱动蛋白kipA导致的烟曲霉极性生长发生变化，与HUVEC共孵育，突变株受内皮细胞内吞率下降，HUVEC的小管形成能力上升，内皮细胞损伤减轻，小鼠动物模型生存时间轻微上升，肺部负载下降。本项目的实施有利用深入了解烟曲霉与血管侵袭之间的作用机制，对阐明侵袭性肺曲霉病的发病机制、预防和治疗侵袭性肺曲霉病提供理论基础。