巨噬细胞激活毛囊Lgr-5干细胞因子的发现研究

Stem cells with structural restoration capacity reside in the skin. Lgr-5 expressing stem cells residing in the hair follicle has been shown to have full regenerative potential of the hair follicle. However, hair follicles and other epidermal appendages lost to injury are not regenerated. Little has been understood how the stem cells respond to injury and what are the mediators controlling the stem cell behavior in the inflammatory niche after wounding. Our recent study (unpublished data) showed that wounding to the skin activated Lgr-5 stem cells in the hair follicle adjacent to the wound, and provoked hair follicle transition from the telogen (rest) phase to the anagen (growth) phase, resulting in the regeneration of new hairs. This process relied on the presence of Ly6C+ macrophages derived from the bone marrow, but not CX3CR1+ tissue resident macrophage, nor neutrophils in the injured tissue. Thus we propose that Ly6C+ macrophages in the wound secret certain molecules which activate Lgr-5 stem cells and enhance hair follicle regeneration, and aim to identify the mediator(s). To achieve this, we will perform comparative analysis of proteins secreted by Ly6C+ macrophages, CX3CR1+ tissue macrophages and neutrophils isolated from the wounded, and identify factors differentially expressed in higher levels in Ly6C+ macrophages, compared to the other two cell populations, as candidates for subsequent functional analysis in vitro and in vivo. We will conduct colony forming assay to test the candidate factors in stimulating colony formation of Lgr-5 stem cells which are isolated from the skin of Lgr5-EGFP transgenic mice in telogen hair follicle stage. We will test the functional involvement of the candidate factors in Lgr-5 hair follicle stem cell activation and promotion of follicle regeneration using mouse wounding model and hair follicle reconstitution assay, through gain and loss of the function of the candidate factors. Multiple approaches will be employed, which include local administration of candidate factor(s) or small molecule inhibitors targeting at the receptor or signaling pathway of the candidate factors; blockade of the signaling mediated by the candidate factors using functional blocking antibodies against the candidate factors or their corresponding receptors; gene knock-out mice with loss of candidate factors or their corresponding receptors. To further clarify the functional involvement of the candidate factors in activating Lgr-5 stem cells, mice with specific loss of the corresponding receptor(s) in Lgr-5 cells will be used; and to verify the factor responsible for macrophage mediated hair follicle activation, mice with specific loss of the factor in macrophages will be used. This study is of significance not only in the understanding of the biology underlying hair follicle stem cell response to injury, but also in developing novel therapies to enhance skin regeneration.

皮肤存在干细胞，Lgr-5毛囊干细胞具有再生毛囊的能力,但损伤后毛囊等结构不能再生；迄今对于损伤后皮肤干细胞的反应及影响因素所知甚少。我们的前期研究发现，损伤激活伤口周围组织的毛囊Lgr-5干细胞，使处于休止期的毛囊迅速进入生长期，而该过程依赖骨髓来源的Ly6C+巨噬细胞而非CX3CR1+的组织巨噬细胞或中性粒细胞。据此，我们假设皮肤受到损伤后，损伤组织中骨髓来源的巨噬细胞释放某些（种）因子，激活毛囊Lgr-5干细胞，促进毛囊再生。本研究的目标是发现该因子。我们将通过对皮肤损伤组织内中性粒细胞、Ly6C+炎性巨噬细胞和CX3CR1+组织巨噬细胞所分泌的因子进行对比分析，找出Ly6C+巨噬细胞高水平分泌的因子作为进行功能研究的候选因子，然后通过体外毛囊干细胞集落形成实验，和体内毛囊激活、毛囊再生等实验，包括增加或减少候选因子的功能水平，最终筛选出激活毛囊干细胞、促进损伤后毛囊再生的因子。

皮肤Lgr-5毛囊干细胞具有再生毛囊的能力，但皮肤损伤后缺失的毛囊等结构通常不能再生。我们的前期研究发现，小鼠皮肤损伤激活伤口周围组织的毛囊Lgr-5干细胞，使处于休止期的毛囊迅速进入生长期，而该过程依赖骨髓来源的Ly6C+巨噬细胞而非CX3CR1+的组织巨噬细胞或中性粒细胞。本项目我们对比分析了损伤皮肤组织中中性粒细胞、Ly6C+炎性巨噬细胞和CX3CR1+组织巨噬细胞差异表达的基因，尤其是Ly6C+巨噬细胞高水平分泌的因子，作为促进毛囊干细胞激活和毛囊再生的候选因子进行功能研究，通过体外皮肤干细胞增殖实验，体内毛囊激活、毛囊再生等实验，发现了TNF在小鼠毛囊再生中不可或缺的作用。研究进一步证明，TNF引起的干细胞增殖依赖AKT通路和β-catenin的激活；小鼠诱导性敲除Pten可导致休止期毛囊干细胞激活，毛囊进入生长期，并促进皮肤损伤后的毛囊再生。候选因子IL-1a对小鼠皮肤损伤后毛囊再生也具有一定的促进作用。这些研究结果为今后研发促进皮肤和毛囊再生的新方法奠定了基础。