Th1/Th2漂移调控巨噬细胞活化在儿童创面愈合中的作用及机制研究

Impaired wound healing is one of the most common complications after pediatric surgery, however, its underlying mechanisms are not fully clarified. The macrophage is the most important immune cells involved in wound healing. Recent scientists have revealed that the pediatric immune system has the character of Th1/Th2 shift, and the Th1/Th2 cytokine are the activation inducers of caM/aaM. Our preliminary studies demonstrated that the wound healing speed of skin full-thickness defect model in young rats is slower than adult rats, and the expression of Th2 cytokines in young rats shows dominant position. We also found that there is caM (classical activation of macrophages) and aaM (alternative activation of macrophages) in wound healing of the adult rats, and abnormal activation could induce impaired wound healing. Thence we speculate that Th1/Th2 shift also exists in pediatric wound tissue and induce abnormal macrophage activation, which in turn lead to impaired wound repair. This project aims to simulate the pediatric wound healing through the skin full-thickness defect model in young rats with different ages , using laser scanning confocal technique to detect caM/aaM and Th1/Th2 cells in situ, detection of Th1/Th2 cytokines and macrophage activation markers in order to determinate its abnormal activation characteristics; to elucidate the effect of different Th1/Th2 cytokine proportion on macrophage activation function in cell biology experiments, and to clarify the effect and mechanism of pre-activated macrophages on the intrinsic repair cells through the co-culture system. This project will clarify the role and molecular mechanism of Th1/Th2-caM/aaM axis shift in the wound healing, and provide the theoretical basis and preliminary exploration for clinical intervention.

创面愈合不良是小儿外科常见并发症之一，其病生机制尚未明确。巨噬细胞是创面修复中最重要的免疫细胞。近期发现诱导巨噬细胞活化的Th1/Th2因子在儿童存在漂移现象。我们前期研究发现小周龄大鼠创面中Th2因子表达占优且其愈合慢于成年大鼠，巨噬细胞在成年大鼠创面中存在经典型活化（caM）和替代性活化（aaM），而活化异常可致创面难愈。我们推测儿童创面也存在Th1/Th2漂移，引发巨噬细胞活化异常致创面难愈。本项目拟通过不同周龄大鼠皮肤全层缺损伤模型模拟不同年龄儿童创面，激光扫描共聚焦技术原位检测caM/aaM和Th1/2细胞，并检测活化因子和标记物，明确异常活化特点；细胞生物学实验分析不同比例Th1/Th2因子对巨噬细胞活化的作用，共培养明确预活化巨噬细胞对固有修复细胞的影响及机制。本项目研究将阐明Th1/Th2-caM/aaM轴偏移在创面愈合中的作用及分子机制，为临床干预提供理论基础和前期探索。

创面愈合不良是小儿外科常见并发症之一，其病生机制尚未明确。巨噬细胞是创面修复中最重要的免疫细胞。文献发现诱导巨噬细胞活化的Th1/Th2因子在儿童存在漂移现象。本课题前期研究发现小周龄大鼠创面中Th2因子表达占优且其愈合慢于成年大鼠，巨噬细胞在成年大鼠创面中存在经典型活化（caM）和替代性活化（aaM），而活化异常可致创面难愈。.本课题研究中用原位检测证实不同周龄大鼠皮肤全层缺损伤模型中均存在caM和aaM。caM和aaM活化标记物表达水平在不同周龄动物创面愈合时相和Th1/Th2 因子，Th1/Th2 分化转录因子的表达水平有一致性趋势。从创面修复时相来说，所有周龄的大鼠皮肤创面修复过程中均表现出修复早期Th1-caM占优势，而修复中后期Th2-aaM占优势。然而在不同周龄却表现出在低周龄（1周龄和2 周龄）的大鼠在修复早期Th1-caM表达水平低于高周龄（4周龄和8 周龄），而修复中后期Th2-aaM的表达水平比高周龄大鼠要高，而且1周龄比2 周龄大鼠更要明显。这说明Th1/Th2-caM/aaM轴偏移现象同样存在于低周龄大鼠的皮肤创面修复过程中，而且周龄越低，Th1/Th2-caM/aaM轴偏移越明显。向Th2-aaM端偏倚的Th1/Th2 漂移体外环境提高了血管内皮细胞的血管化表达水平，也提高了成纤维细胞的胶原分泌表达水平。.本项目结果提示儿童创面愈合不良概率较高和巨噬细胞活化异常有关。在生理不成熟机体的创面修复中存在Th1/Th2-caM/aaM轴偏移，即向Th2-aaM端偏倚，年龄越低越明显。这导致创面愈合初期的应激炎症不足，而愈合中后期的修复时相延长。该研究结果从Th1/Th2-caM/aaM轴偏移的角度切入，为以后儿童创面愈合中的平衡好Th1/Th2-caM/aaM轴，使巨噬细胞接近正常成熟机体中有序的充分活化，从而降低儿童创面愈合不良概率提供了理论基础和干预方向。