应用CRISPR/Cas9构建多基因敲入荧光报告小鼠研究巨噬细胞极性化在皮肤修复与再生中的作用与机制

Macrophages are credited with an essential core in regulating skin repair and regeneration while the molecular mechanisms remain elusive. Our previous studies suggest that at different stages of skin repair and regeneration, dynamic expression of inflammatory cytokines may affect the macrophage M1/ M2 polarization. M1 and M2-type macrophages may play different roles in skin repair and regeneration, in consistence with their dynamic number and proportion diversity. With the generation and application of a macrophage reporter mouse strain via CRISPR/Cas9 mediated multiple gene knock-in strategy, this study will focus on macrophage polarization in skin repair and regeneration by demonstrating the macrophage polarity induced by microenvironmental inflammatory cytokines, revealing the particular effect on skin repair of M1 and M2 macrophages. Interaction with inflammatory cytokines such as interferon-γ and interleukins is likely an important component of the M1/M2 types of macrophages in skin regeneration and remodelling. These properties of cell-cytokine crosstalk and new animal model may present usful tools in many fields of academic research as well as clinical practice.

巨噬细胞是调控皮肤修复与再生的核心细胞之一，调控途径和分子机制有待进一步解析。我们前期实验数据提示皮肤修复与再生中，M1和M2型巨噬细胞的数量和比例存在动态变化，且可能与创面微环境中炎症因子的表达水平有关。本研究将应用新型基因操作技术CRISPR/Cas9构建能同时标记巨噬细胞及其亚型的多基因、内源性、荧光报告基因敲入小鼠，可通过活体荧光成像技术连续观测巨噬细胞的迁移和分化，绘制皮肤修复与再生中M1和M2型巨噬细胞极性转化的动力学图谱，并借助已有的基因敲除小鼠研究炎症因子和巨噬细胞极性在皮肤修复与再生中所扮演的角色，加深对组织修复与再生这一基础生物学事件中“炎症因子--免疫细胞”调控系统的理解，可能为提高机体创伤耐受、改进创伤治疗、预防难愈创伤、避免过度修复提供新的思路和治疗靶点。项目拟构建的新型基因修饰动物模型可能在免疫学、肿瘤学、药物新靶点探索及临床前药效学评价等多个领域得到应用。

巨噬细胞是调控皮肤修复与再生的核心细胞之一，在体内外不同因子刺激下可极化为不同表型， M1和M2 型巨噬细胞的数量和比例的动态变化参与皮肤的创伤愈合过程的调控。本研究成功构建了M2 型巨噬细胞极化模型，首次发现JNK和Herpud1均能够促进巨噬细胞的极化过程，通过药理学和遗传学干预实验证明JNK主要通过上调c-Myc的表达影响M2 型巨噬细胞的极化和迁移过程，而Herpud1则可能是通过增强内质网应激反应而直接促进巨噬细胞的极化和迁移。综上，我们的研究加深了对巨噬细胞极化过程的认识，为进一步研究巨噬细胞极化调控皮肤修复与再生奠定了基础，为提高机体创伤耐受、改进创伤治疗、预防难愈创伤、避免过度修复提供新的思路和治疗靶点。