宿主EGF介导的EGFR信号通路调控生发细胞周期及其影响泡球蚴生长发育的机制

Germinative cells, a population of cells that are totipotent and capable of extensively proliferating, are essential for the continuous larval growth and development of Echinococcus multilocularis in human. In our previous study, we found that host EGF can activate the EGFR signaling in the parasite and promote the proliferation/self-renewal of germinative cells and larval growth. Meanwhile, the transcriptional level of Cyclin D, a key regulator of G1/S phase, was significantly upregualted. We also found downregulated Cyclin D/E and impaired germinative cell proliferation and larval growth upon the inhibition of the parasite’s EGFR/ERK signaling. These results are suggestive of relevant cell-cycle-regulating signaling pathways involved in controlling the behaviors of germinative cells and the larval growth and development of E. multilocularis. In this project, we will study the mechanisms regulating the cell cycle of germinative cells in E. multilocularis. We will elucidate the role of EGFR-ERK-Cyclin D-CDK4/6 and Cyclin E-CDK2 signaling pathways in regulating larval development of the parasite as well as the influence of host EGF. We will also investigate in vivo the effects of CDK inhibitors on the development of the parasite in host using the animal model. This research will offer a new sight to study the mechanisms regulating the larval growth and development of E. multilocularis, and will be helpful in discovering new drug targets for the treatment of alveolar echinococcosis.

具有增殖能力和分化多能性的生发细胞是泡球蚴在宿主体内持续生长发育的基础。我们研究发现：宿主EGF可通过激活泡球蚴EGFR信号通路，促进生发细胞增殖/自我更新和泡球蚴生长发育，同时G1/S期关键分子Cyclin D显著上调；抑制EGFR/ERK信号通路则下调Cyclin D、E，生发细胞增殖和泡球蚴生长发育受到抑制，推测相关信号通路通过调控生发细胞周期继而影响泡球蚴生长发育。本项目为验证假说，拟开展：分析泡球蚴生发细胞周期调控特点；研究宿主EGF对生发细胞周期的影响；阐明EGFR-ERK-Cyclin D-CDK4/6、Cyclin E-CDK2信号通路在泡球蚴生长发育中的作用机制以及宿主因子的影响；动物实验探索靶向CDK激酶的小分子药物对宿主体内泡球蚴生长发育的影响。本项目从宿主因子影响生发细胞周期调控这一新角度出发，对泡球蚴生长发育的分子机理展开研究，亦有助于发现泡球蚴病的新药物靶标。

生发细胞是泡球蚴在人体组织器官内不断生长发育和侵袭转移的重要基础，也是泡球蚴病难以根治、极易复发的重要原因。生发细胞是泡球蚴内唯一具有增殖能力和分化潜能的成体干细胞，其功能正常运转需要对细胞周期进行精准调控。然而目前对生发细胞的细胞周期及其调控机制了解甚少。.本项目中我们鉴定和分析了泡球蚴细胞周期G1/S期中关键分子EmCyclinD、EmCyclinE、EmCDK4/6以及EmCDK2。EmCyclinD和EmCyclinE在生发细胞中特异高表达。EmCyclinD可与EmCDK4/6而非EmCDK2结合形成二聚体，进而磷酸化底物Rb蛋白，且EmCDK4/6的Thr187位点为其激酶活性所必需。Palbociclib可抑制EmCyclinD-EmCDK4/6复合体激酶活性，阻抑细胞周期，抑制生发细胞增殖以及S期相关分子的转录表达，同时抑制泡球蚴原头节发育和泡囊生长。宿主EGF可上调EmCyclinD表达水平，同时促进EmE2F和EmMCM2/6、EmH2B等S期相关分子转录表达，促进生发细胞增殖。抑制EGFR-ERK信号通路或是EmCDK4/6激酶活性则抑制了EGF的相关作用。这些结果表明宿主EGF通过泡球蚴EGFR-ERK信号通路参与EmCyclinD表达调控，进而影响EmCyclinD-EmCDK4/6复合物功能，影响生发细胞周期和泡球蚴生长发育。.通过对生发细胞各时相时长的分析，我们发现其一个完整周期约为26h，远快于之前的预期，而宿主EGF等因子可促进生发细胞的细胞周期进程。相关结果同时提示，生发细胞中可能存在G1进程较慢或者处于G0期静息态的细胞，宿主EGF在促进生发细胞的细胞周期进程的同时，也可能调控生发细胞分裂模式。.本项目从泡球蚴生发细胞周期的调控机制入手，分析了宿主EGF介导的EGFR/ERK信号通路调控Cyclin-CDK复合物活性，进而影响生发细胞增殖和泡球蚴生长发育的机理。这有助于加深我们对泡球蚴生发细胞功能调控机制的认识，以及理解在长期协同进化过程中形成的复杂的宿主-寄生虫相互作用关系。这些关于分子机制的基础研究也有助于发现治疗泡球蚴病的新药物靶标。